Articles de revues : « Hearing Loss,illumina,Agilent »

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Liu, Xiao-Wen, Su-Yang Wang, Zhan-Kui Xing, Yi-Ming Zhu, Wen-Juan Ding, Lei Duan, Xiao Cui, Bai-Cheng Xu, Shu-Juan Li et Yu-Fen Guo. « Targeted next-generation sequencing identified a novel variant of SOX10 in a Chinese family with Waardenburg syndrome type 2 ». Journal of International Medical Research 48, n^o 11 (novembre 2020) : 030006052096754. http://dx.doi.org/10.1177/0300060520967540.

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Objective Waardenburg syndrome type 2 (WS2) is an autosomal dominant syndrome, characterized by bright blue eyes, hearing loss, and depigmented patches of hair and skin. It exhibits high phenotypic and genetic heterogeneity. We explored the molecular etiology in a Chinese family with WS2. Methods We recruited a three-generation family with three affected members. Medical history was obtained from all family members who underwent detailed physical examinations and audiology tests. Genomic DNA was extracted from peripheral blood of each individual, and 139 candidate genes associated with hearing loss were sequenced using Illumina HiSeq 2000 (Illumina Inc., San Diego, CA, USA) and verified by Sanger sequencing. Results Genetic evaluation revealed a novel nonsense heterozygous variant, NM_006941.4: c.342G>A (p.Trp114Ter) in exon 2 of the SOX10 gene in the three affected patients; no unaffected family member carried the variation. We did not detect the variation in 500 Chinese individuals with normal hearing or in 122 unrelated Chinese families with hearing loss, suggesting that it was specific to our patients. Conclusions We identified a novel heterozygous nonsense variation in a family with syndromic hearing loss and WS2. Our findings expand the pathogenic spectrum and strengthen the clinical diagnostic role of SOX10 in patients with WS2.

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Gunnarsson, Rebeqa, Johan Staaf, Mattias Jansson, Anne Marie Ottesen, Hanna Göransson, Ulrika Liljedahl, Ulrik Ralfkiaer et al. « Screening for Copy Number Alterations and Loss of Heterozygosity in Chronic Lymphocytic Leukemia - A Comparative Study of Four Differently Designed, High Resolution Microarray Platforms. » Blood 110, n^o 11 (16 novembre 2007) : 2084. http://dx.doi.org/10.1182/blood.v110.11.2084.2084.

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Abstract Screening for copy number alterations (CNA) has improved by applying genome wide microarrays, where SNP-arrays also allow analysis of loss of heterozygosity (LOH). Currently, comparisons of high resolution microarray platforms are few, thus we performed a study to evaluate the power of differently designed microarrays for copy number analysis and LOH. We here analyzed 10 diagnostic chronic lymphocytic leukemia (CLL) samples (five IGVH mutated and five IGVH unmutated) using four different high-resolution platforms: BAC-arrays (32K), oligonucleotide-arrays (185K, Agilent), and two SNP-arrays (250K, Affymetrix and 317K, Illumina). Comparison of copy number data showed that the platforms are concordant in terms of detecting large CNA, including the known recurrent alterations. Mono-allelic and bi-allelic loss of 13q14 (3 and 1 sample, respectively), mono-allelic loss of 11q (1 sample), trisomy 12 (2 samples) and mono-allelic loss of 17p (2 samples) were concordant in all platforms. These aberrations were validated with FISH, which in addition identified subclones with mono-allelic loss of 13q14 in two cases, only detected with the BAC platform, rendering a cut-off for the power of detecting subclones to approximately 25% of investigated cells. As expected, all poor prognostic aberrations were detected in patients carrying unmutated IGHV genes whereas four of five mutated samples were detected with mono-allelic loss of 13q14, as the only recurrent alteration. Furthermore, detection of small CNA were in many cases discordant between platforms. Therefore, we defined alterations identified by at least two platforms and identified 47 losses and 31 gains using this criterion. We are currently validating the presence of a number of these alterations using other techniques. Evaluation of LOH showed concordance for 86 regions between the Illumina and Affymetrix platforms. Of these regions 12 LOH coincided with CNA, leaving the remaining 74 as copy-neutral LOH. In conclusion, all platforms investigated are powerful tools for screening of CNA, however, since non-overlapping CNA were detected by individual platforms, we emphasize the importance of validating findings. Also, there is a cut-off for detecting subclones, here estimated to 25%. Genomic arrays will improve the detection of new recurrent aberrations, which may potentially refine the prognostic hierarchy established by FISH.

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Hosono, Katsuhiro, Yuko Harada, Kentaro Kurata, Akiko Hikoya, Miho Sato, Shinsei Minoshima et Yoshihiro Hotta. « NovelGUCY2DGene Mutations in Japanese Male Twins with Leber Congenital Amaurosis ». Journal of Ophthalmology 2015 (2015) : 1–10. http://dx.doi.org/10.1155/2015/693468.

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Purpose. Leber congenital amaurosis (LCA), a genetically and clinically heterogeneous disease, is the earliest onset retinitis pigmentosa (RP) and is the most severe of hereditary retinal dystrophies. This study was conducted to investigate genetic and clinical features of LCA in a set of Japanese male twins with LCA.Methods. To identify causative mutations, 74 genes known to cause RP or LCA were examined by targeted-next generation sequencing (NGS). Targeted-NGS was performed using a custom designed Agilent HaloPlex target enrichment kit with Illumina Miseq sequencer. Identified potential pathogenic mutations were confirmed using Sanger sequencing. Clinical analyses were based on ophthalmic examination, fundus photography, and electroretinography (ERG).Results. Compound heterozygousGUCY2Dmutations of novel splicing mutation c.2113+2_2113+3insT and novel missense mutation p.L905P were detected in both twins. Their father and mother were heterozygous for c.2113+2_2113+3insT and p.L905P, respectively. The twins had phenotypic features similar to those previously reported in patients withGUCY2Dmutations. This included early childhood onset of visual loss, nystagmus, unrecordable ERG, photophobia, and hyperopia.Conclusions. To the best of our knowledge, this is the first report of genetic and clinical features of Japanese LCA twins withGUCY2Dmutation, which were detected using targeted-NGS.

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Hagleitner, Melanie M., Marieke J. H. Coenen, Hans Gelderblom, Peter Hoogerbrugge, Henk-jan Guchelaar et Dunja Maroeslea W. M. Te Loo. « Association of the genetic variants in the nucleotide excision repair genes XPA and XPC with cisplatin-induced hearing loss in patients with osteosarcoma. » Journal of Clinical Oncology 30, n^o 15_suppl (20 mai 2012) : 10077. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.10077.

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10077 Background: Cisplatin is a widely used and effective chemotherapeutic agent in the treatment of osteosarcoma. However, cisplatin-induced ototoxicity is a serious problem, affecting more than 60% of patients and compromising language and cognitive development. Unfortunately, individuals at risk to develop ototoxicity cannot be identified upfront. Genetic variants in genes involved in the metabolism of cisplatin may predispose to cisplatin-induced hearing loss and help to identify patients at risk. Methods: In a candidate gene pathway approach, we selected 224 SNPs in 30 candidate genes related to Platinum-DNA repair pathways and genotyped for a discovery group of 105 patients with osteosarcoma for these variants. Cisplatin-induced ototoxicity (n = 47), defined as the development of grade 2–4 hearing impairment using Common Terminology Criteria for Adverse Events (CTCAE version 3), showing a hearing loss of >25 dB at frequencies of 4–8 kHz, was associated with genetic variation. A replication study was performed in a independent cohort of 51 patients with osteosarcoma. Genotyping was performed using the Illumina GoldenGate assay. Association analysis and meta-analysis were performed using the whole genome association analysis toolset PLINK. Results: In the discovery cohort a total of 13 SNPs were significantly (p value < 0.05) associated with ototoxicity. Upon meta-analysis, addition of the replication set resulted in lower p-values for 2 SNPs. The two SNPs showing a strong association with hearing loss in patients with osteosarcoma were rs2805835 in the gene XPA (p-value 0.01, OR=2.7 (95%CI: 1.20-6.15) and rs2227999 in XPC (p-value 0.02; OR=3.2 (95% CI:1.19-8.80). Conclusions: The Nucleotide Excision Repair (NER) genes XPA and XPC form an important molecular mechanism by which cisplatin DNA adducts can be repaired and it has recently been shown that these genes have a high expression in the cochlea. Our data suggest that genetic variants in these genes, may contribute to cisplatin ototoxicity. This study should be viewed as the first step in the development of genetic markers to predict cisplatin-induced ototoxicity in individual patients.

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Riza, Anca-Lelia, Camelia Alkhzouz, Marius Farcaș, Andrei Pîrvu, Diana Miclea, Gheorghe Mihuț, Răzvan-Mihail Pleșea et al. « Non-Syndromic Hearing Loss in a Romanian Population : Carrier Status and Frequent Variants in the GJB2 Gene ». Genes 14, n^o 1 (26 décembre 2022) : 69. http://dx.doi.org/10.3390/genes14010069.

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The genetic causes of autosomal recessive nonsyndromic hearing loss (ARNSHL) are heterogeneous and highly ethnic-specific. We describe GJB2 (connexin 26) variants and carrier frequencies as part of our study and summarize previously reported ones for the Romanian population. In total, 284 unrelated children with bilateral congenital NSHL were enrolled between 2009 and 2018 in northwestern Romania. A tiered diagnostic approach was used: all subjects were tested for c.35delG, c.71G>A and deletions in GJB6 (connexin 30) using PCR-based methods. Furthermore, 124 cases undiagnosed at this stage were analyzed by multiplex-ligation-dependent probe amplifications (MLPA), probe mix P163, and sequencing of GJB2 exon 2. Targeted allele-specific PCR/restriction fragment length polymorphism (RFLP) established definite ethio-pathogenical diagnosis for 72/284 (25.35%) of the cohort. Out of the 124 further analyzed, in 12 cases (9.67%), we found compound heterozygous point mutations in GJB2. We identified one case of deletion of exon 1 of the WFS1 (wolframin) gene. Carrier status evaluation used Illumina Infinium Global Screening Array (GSA) genotyping: the HINT cohort-416 individuals in northwest Romania, and the FUSE cohort-472 individuals in southwest Romania. GSA variants yielded a cumulated risk allele presence of 0.0284. A tiered diagnostic approach may be efficient in diagnosing ARNSHL. The summarized contributions to Romanian descriptive epidemiology of ARNSHL shows that pathogenic variants in the GJB2 gene are frequent among NSHL cases and have high carrier rates, especially for c.35delG and c.71G>A. These findings may serve in health strategy development.

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Armstrong, Andrew J., Jing Li, Joshua Beaver, Rhonda Lynn Bitting et Simon Gregory. « Genomic analysis of circulating tumor cells (CTCs) from men with metastatic castration resistant prostate cancer (mCRPC) in the context of enzalutamide therapy. » Journal of Clinical Oncology 32, n^o 4_suppl (1 février 2014) : 65. http://dx.doi.org/10.1200/jco.2014.32.4_suppl.65.

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65 Background: Given the evolving treatments available in metastatic castration resistant prostate cancer (mCRPC), predictive biomarkers are desirable that maximize benefit and minimize harms and costs.The goal of this study was to determine the feasibility of DNA copy number and whole exome sequencing (WES) analysis of circulating tumor cells (CTCs) from men with mCRPC receiving enzalutamide. Methods: We collected CTCs from men with mCRPC in the context of enzalutamide therapy. CTCs were isolated from EDTA blood through red cell lysis, CD45 depletion, and flow sorting on EpCAM/CD45 expression. Whole genomic amplification and array based comparative genomic hybridization (CGH) was performed using Qiagen Repli-Gene Single Cell kit, multiple displacement amplification, and Agilent microarray analysis. CTC copy number changes were compared with patient leukocyte DNA and reference metastatic PC datasets. CTC AR amplification and PTEN loss was confirmed with FISH. WES on REPLI-g amplified CTC and leukocyte DNA was performed using GeneWiz and TruSeq Exome Capture Kit, and sequenced with Illumina HiSeq 2000 (20x). Results: A novel method for CTC array CGH was developed that reproducibly identified genomic lesions previously reported in metastatic CRPC including: AR amplification or focal deletions, deletions of CHD1, Rb, PHLLP, FGFR2, FOXA1, and NCOA2, and amplifications of EZH2 and MYC. AR amplification was noted in a man with mCRPC who subsequently responded to enzalutamide, with loss of AR amplification and gain of MYCN and c-MET amplification noted at progression. CGH analysis was feasible down to 10 to 20 cells using spiked cell lines. Interpatient tumor specific genomic heterogeneity was observed. FISH confirmed AR changes and PTEN loss heterogeneity. WES demonstrated acquired PTEN, MAGI1, SMAD4, and RB1 mutations in a patient who progressed through enzalutamide therapy, in addition to AR region deletion detected by CGH. Conclusions: Whole genome DNA copy number and exome sequencing analysis from CTCs in men with mCRPC is feasible in men with high CTCs and identified previously validated and novel genomic lesions and suggest the potential to identify predictive biomarkers of enzalutamide efficacy and resistance in the clinic.

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O’Halloran, Katrina, Moiz Bootwalla, Daria Merkurjev, Kristiyana Kaneva, Alex Ryutov, Jennifer Cotter, Jianling Ji, Dejerianne Ostrow, Jaclyn A. Biegel et Xiaowu Gai. « RARE-57. PEDIATRIC CHORDOMA : WHOLE EXOME SEQUENCING OF 11 PEDIATRIC CHORDOMA SAMPLES ». Neuro-Oncology 22, Supplement_3 (1 décembre 2020) : iii454. http://dx.doi.org/10.1093/neuonc/noaa222.767.

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Abstract Chordoma is a rare tumor and while SMARCB1 alterations have been observed in poorly differentiated chordomas, conventional chordomas are not well understood. We interrogated nuclear and mitochondrial genomes of 11 chordoma samples from 7 children. Frozen tumor tissue DNA was extracted and whole exome libraries generated using Agilent SureSelect Human All Exon V6 kit plus mtDNA genome capture kit. Libraries were sequenced using Illumina Nextseq 500. MuTect2, VarDict and LUBA variant callers were used with allele frequency cutoff 2%. Potential germline variants were filtered bioinformatically. In total, 656±74 high-confidence somatic variants, including 368±43 nonsynonymous variants per sample were detected. Of 2,607 combined unique nonsynonymous variants, 95% were missense. Remaining high impact variants were frameshift (37%), stop gain (39%), splice acceptor/donor (22%), start and stop loss (2%). Of the unique nonsynonymous variants, 137 fall within Cosmic Cancer Census Genes, including high impact variants in SETD2, MLLT4. No previously reported TBXT, CDKN2A, PI3K, LYST mutations identified. Tumor Mutation Burden/Megabase was 10±1. The mitochondrial analysis revealed heteroplasmic m.11727C>T MT-ND4 missense variants in three tumors resected at different time points from the same patient, and another heteroplasmic m.1023C>T rRNA mutation from the primary and recurrent tumors of another patient. Intriguingly, two Children’s Brain Tumor Tissue Consortium patients with chordoma had identical heteroplasmic m.10971G>A MT-ND4 nonsense mutations. Pediatric chordomas appear to lack somatic nuclear mutations. Observing recurrent mitochondrial mutations across multiple tumors from the same and/or different patients is striking, suggesting they may be implicated in tumorigenesis and be potential diagnostic markers.

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Li, Qian, Yuxiao Zou et Sentai Liao. « Mulberry Leaf Polyphenols and Fiber Induce Synergistic Antiobesity and Display a Modulation Effect on Gut Microbiota and Metabolites ». Current Developments in Nutrition 4, Supplement_2 (29 mai 2020) : 1654. http://dx.doi.org/10.1093/cdn/nzaa063_052.

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Abstract Objectives In this study we compared the antiobesity effects of mulberry leaf powder, dietary fiber, polyphenols, and a fiber/polyphenols mixture.Combining intestinal community modulation and metabolite analysis, we investigated the antiobesity effects and mechanisms of mulberry leaf components, detecting the interaction between mulberry leaf dietary fiber and polyphenol. Methods An obesity model was established by feeding rats with a high-calorie diet. Rats were divided into seven groups: the obesity model control (MC), positive control (PC), mulberry leaf powder (MLP), mulberry leaf fiber (MLF), mulberry leaf polyphenols (MLPS), mulberry leaf fiber and polyphenols mixture (MLM), and normal control (NC), and fed daily for 6 consecutive weeks.the 16S rRNA gene was sequenced using Illumina MiSeq.UPLC Triple TOF MS/MS system and Agilent 6890 N GC-MS were used to profile the urinary/fecal metabolites. Results The synergistic interaction between mulberry dietary fiber and polyphenols (MLM) in antiobesity was reported for the first time. The content of Firmicutes in the MC group was increased significantly. Except for the MLPS group, other test groups regulated the Firmicutes content to a normal level. Our study demonstrated that different components of mulberry leaves might achieve weight loss by reducing the amount of Lachnespiraceae. At the same time, the reduction Lactobacillus_vaginalis and Lactobacillus_gasseri species was closely related to the improvement of lipid metabolism profiles. In addition, the high energy diet induced feces and urine metabolic disorders in MC group with significant difference. The amino acid and oligopeptide metabolites were regulated to the NC level under the regulation of mulberry leaf components. Conclusions MLM group had the best efficiency on weight loss, indicating synergistic interactions between MLPS and MLF. The reduction of Firmicutes abundance, and the downstream Clostridiales, Lachnespiraceae, was a key pathway for the antiobesity effects. The increased abundances of Lactobacillus vaginalis and Lactobacillus gasseri might result in lipid metabolism disorder. The test groups regulated the amino acid and oligopeptides metabolic disorder tents to normal levels compared with the MC and NC groups. Funding Sources The Science & Technology Projects of Guangdong Province No.2017A050501022/No.2017A030310416.

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Toomey, Sinead, Aoife Carr, Jillian Rebecca Gunther, Joanna Fay, Anthony O'Grady, David Weksberg, Scott W. Piraino et al. « Clonal evolution in locally advanced rectal cancers in response to neoadjuvant chemoradiotherapy. » Journal of Clinical Oncology 35, n^o 15_suppl (20 mai 2017) : 3616. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.3616.

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3616 Background: Locally advanced rectal cancer, LARC (T3/4 and/or N+) is currently treated with neoadjuvant chemoradiotherapy (NACRT), however clinicopathological response is variable. Monitoring clonal evolution in response to NACRT may identify mutations driving therapeutic resistance or tumor growth after treatment. Methods: Fresh-frozen pre- and post-NACRT tumor and matched normal tissue from LARC patients were stratified into good (RCPath A), intermediate (RCPath B) and poor (RCPath C) responders. Following histological review, targeted exome capture was performed using an Agilent SureSelect Human all Exome V3 kit. Samples were sequenced to a minimum of 100X coverage on an Illumina HiSeq2000, and clonal evolution was assessed in matched pre- and post-NACRT tumor samples. Results: The median somatic mutation burden in pre-treatment samples was 114 (IQR 19-207). Two tumors were microsatellite (MSI) unstable and had elevated mutational burdens. The least evolution occurred in the poor responders, where there was little change in clonal composition after treatment, and driver mutations in genes including TP53 and APC were retained. On average 79% of pre-treatment mutations were retained post-treatment in poor responders and 33% of mutations were retained in intermediate responders. Many of the intermediate responders had loss of driver mutations including TP53 from the pre-treatment sample, but also shared a number of mutations in genes including PIK3CA and BRAF between pre- and post-treatment samples. There was also increased frequency in the post-treatment samples of clones that were not present in the pre-treatment samples. In one intermediate responder, all 47 mutations that were present in the pre-treatment sample including the driver mutations TP53 and APC were absent in the post-treatment sample, while 10 completely new mutations were identified. Conclusions: Dynamic mutational processes occur in LARC following selective pressures of exposure to NACRT, including changes in somatic mutation presence or frequency after treatment, owing to persistence or loss of sub-clones. As NACRT can profoundly affect the LARC genome, monitoring molecular changes during treatment may be clinically useful.

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Teule, Alex, Francisco Quiles, Rafael Valdes-Mas, Miguel Angel Pujana, Monica Salinas, Lidia Feliubadalo, Gabriel Capella et al. « Searching for new genes responsible for unexplained hereditary breast and ovarian cancer patients. » Journal of Clinical Oncology 31, n^o 15_suppl (20 mai 2013) : e12516-e12516. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e12516.

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e12516 Background: About 10-20% of breast and ovarian cancer patients show family history of the disease. Germline mutations in the BRCA1 and BRCA2 genes are usually found in 20-25% of these cases, while the remaining ones are commonly known as BRCAX. Next generation sequencing (NGS) studies have the promise to expedite the identification of the genetic basis underlying BRCAX cases. Aim: To identify gene mutations associated with BRCAX cases by means of whole exome sequencing. Methods: Five selected BRCAX families with more than three affected individuals across at least three generations, and with an apparent dominant pattern of inheritance of the disease were selected in this study. All type of potential alterations of BRCA1/2and linkage to the corresponding loci were excluded in these families. For sequencing, libraries representing the whole exome of multiple affected individuals were obtained using the SureSelect Human All Exon 50 Mb Kit (Agilent) together with the Paired-End Sample Preparation Kit (Illumina) following manufacturers' protocols. Bioinformatics analysis to identify candidate variants present in each family was performed as previously described (PubMed ID: 21549337). Selected rare variants have been analyzed in a set of 288 controls and are currently being evaluated in 500 additional BRCAX cases. Results: By using the described workflow we have identified 16 candidate genes. They were selected based on the presence of rare missense variants that were not found in our control group, predicted as pathogenic using several algorithms (SIFT, PoplyPhen2, Condel and Mutation Taster). Currently, we are re-sequencing these genes in a set of BRCAX samples and complementarily performing loss-of-heterozigosity studies in tumor samples. Conclusions: Exome sequencing of 20 patients from 5 independent Spanish BRCAX families identified a list of putative new BRCAX genes that are currently in the process of validation. Identification of the genetic causes underlying breast and ovarian cancer in BRCAX families is paramount to perform genetic counseling and individual risk assessment in these patients.

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Mikulasova, Aneta, Brian A. Walker, Christopher P. Wardell, Eileen M. Boyle, Alexander Murison, Zuzana Kufova, Ludek Pour, Petr Kuglik, Roman Hajek et Gareth J. Morgan. « Somatic Mutation Spectrum in Monoclonal Gammopathy of Undetermined Significance Compared to Multiple Myeloma ». Blood 124, n^o 21 (6 décembre 2014) : 3346. http://dx.doi.org/10.1182/blood.v124.21.3346.3346.

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Abstract Introduction: Malignant transformation of normal to tumour cells is a multistep process followed by sequential aggregation of hits at different molecular levels. Genetic events including single nucleotide variants (SNVs), insertion-deletion changes (indels) as well as copy number variants (CNVs) affect the phenotype of the tumour population and consequently patient prognosis. Transformation from a symptomless state, monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) can be used as a unique model for cancer development studies. To date, there is very little data regarding the mechanisms leading to disease progression at molecular level. In our study, we performed exome sequencing together with SNP array analysis on 33 MGUS patients to describe the premalignant phenotype and compared these to advanced tumour cells at the DNA level. We hypothesised that increased genetic instability indicated MGUS patients with a high risk of progression to MM. Methods: 33 MGUS patients (M:F 1.5:3; median age 61, range: 35-86) were included in this study. Plasma cells were isolated from bone marrow by FACSAria (BD Biosciences) system using CD138, CD19 and CD56 markers to obtain a pure abnormal plasma cell population with a purity >90%. Tumour DNA was isolated using Gentra Puregene Kit and amplified using REPLI-g Midi Kit (both Qiagen); control DNA was gained from peripheral white blood cells by MagNA Pure System (Roche Diagnostics). For exome sequencing, NEBNext kit (NEB) and SureSelect Human All Exon V5 (Agilent Technologies) were used and samples were sequenced by HiSeq2000 (Illumina) using 76-bp paired end reads. Unbalanced CNVs were tested by SurePrint G3 CGH+SNP, 4x180K (Agilent Technologies). Results were compared to 463 MM patients. Results: In our analysis, we found acquired SNVs in 100% (33/33) MGUS patients with a median of 89 (range 9-315) SNVs per patient. Non-synonymous SNVs (NS-SNVs) were present in 97% (32/33) cases with a median 19 (range 0–70) NS-SNVs per patient. Overall, 42 genes were recurrently mutated in at least 2 patients and 6 genes were mutated in at least 3 cases including MUC16, IGK, TTN, KLHL6, AKAP9 and NPIPL2. We identified 7 genes which were significantly mutated in MM in our previous study including KRAS (n=2), HIST1H1E (n=2) and NRAS, DIS3, EGR1, LTB, PRKD2 (all n=1). IGH translocations were identified in 27% (9/33) of patients: t(11;14) in 12% (4/33), t(4;14) in 9% (3/33), t(14;16) in 3% (1/33) and t(14;20) in 3% (1/33). We did not find any translocations involving MYC (8q24.21) or the light chain loci IGK (2p12) and IGL (22q11.2). Using SNP arrays, unbalanced CNVs were presented in 67% (22/33) of MGUS patients and detected CNVs showed similarity to MM across the cohort. As previously described in MM, only one type of IGH translocation was found per patient and all 9 cases with IGH translocation did not have additional hyperdiploidy. Furthermore, we identified a patient with two CCND1 (p.K50T, p.E51D) mutations and a t(11;14), a case with a DIS3 (p.D488N) mutation and a 13q loss. Moreover, we noticed a co-segregation of cases t(4;14) and t(14;16) who all had a 13q loss (100%, 4/4). In contrast none of the patients (0/5) with a t(11;14) or a t(14;20) had a 13q loss. Of note 29% (7/24) patients without any IGH translocation had a 13q loss. Sixty seven percent (2/3) of patients with a t(4;14) and the one case with a t(14;16) also had a 1q gain. In comparison, none of patients with a t(11;14) (0%; 0/4) had a 1q gain. Unlike what has previously been described in MM, neither of the 2 MGUS patients with a KRAS (p.Q61L and p.A146T) mutations had a t(11;14). We also identified a patient with both a KRAS (p.Q61L) and an NRAS (p.G13R) mutation which are although not mutually exclusive, negatively correlated in MM. Importantly, we did not find any mutations in TP53, ATM, ATR and ZFHX4 genes involved in DNA repair pathway alterations which were identified as unfavourable factors in survival of MM patients. Summary: We have performed the first comprehensive analysis of 33 MGUS patients using exome sequencing together with SNP arrays and described the main genetic events that are already present in this premalignant state. We found similarities to MM in terms of SNVs, CNVs and their correlations. We identified 6 MGUS cases with NS-SNVs in potential key genes that could indicate a potential high risk to progression. Support: IGA MH CZ NT13492, OPVK CZ.1.07/2.3.00/20.0183. Disclosures Walker: Onyx Pharmaceuticals: Consultancy, Honoraria.

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Mahjoubi, Frouzandeh, Samira Shabani, Sogand Khakbazpour et Aylar Khaligh Akhlaghi. « Novel EPG5 Mutation Associated with Vici Syndrome Gene ». Case Reports in Genetics 2022 (5 juillet 2022) : 1–3. http://dx.doi.org/10.1155/2022/5452944.

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Introduction. Vici syndrome (also known as immunodeficiency with cleft lip/palate, cataract, and hypopigmentation and absent corpus callosum) is considered as a progressive neurodevelopmental multisystem disorder. Till date, only 80 cases, including our patient, with this syndrome have been reported .This syndrome is characterized by agenesis of the corpus callosum, hypopigmentation of the eyes and hair, cataract, cardiomyopathy, combined immunodeficiency, hearing loss, seizures, and additional multisystem involvements which have been reported as case reports in the past. Clinical Manifestation. A 5-year-old girl, who is a product of consanguineous marriage, was referred to our center with developmental delay, optic atrophy, blindness, spasticity, seizure, movement disability, and spasticity. Her magnetic resonance imaging (MRI) test showed agenesis of the corpus callosum and her metabolic test reported normal. Materials and Methods. In our laboratory, blood sample was obtained from the patient. DNA was extracted from lymphocytes, and whole exome sequencing (WES) using next generation Illumina sequencing was performed. Result. A novel (private), homozygous, nonsynonymous mutation c.A3206G (p.Y1069C Het) in EPG5 gene was detected; in continuum, testing for this specific variant in her parents was carried out. DNA sequencing of the PCR-amplified product of the EPG5 exon 17 showed that her parents were heterozygote for this variant. These mutations have not been reported before and therefore classified as variation of unknown significance (VUS). Mutation in this gene is shown to cause autosomal recessive Vici syndrome. Conclusion. Since clinical features of Vici syndrome has overlap, its diagnosis is differential and developmental delay occurs in 98% of reported cases. Vici syndrome can be considered as one of the main causes of developmental delay, and this syndrome can be introduced as a novel group of inherited neurometabolic conditions and congenital disorders.

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Sacco, Antonio, Yawara Kawano, Michele Moschetta, Jihye Park, Oksana Zavidij, Michaela Reagan, Yuji Mishima et al. « Dual Conditional Loss of BLIMP-1 and p53 in B-Cells Drives B-Cell Lymphomagenesis ». Blood 128, n^o 22 (2 décembre 2016) : 4169. http://dx.doi.org/10.1182/blood.v128.22.4169.4169.

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Abstract Background. p53 is a well defined tumor suppressor involved in the modulation of cell proliferation, cell cycle progression and programmed cell death. BLIMP-1 plays a crucial role in modulating B-cell differentiation towards Ig-secreting plasma cells, and it acts as a tumor suppressor, as documented in both diffuse large B-cell lymphoma and Burkitt lymphoma. Whether B-cell specific loss of both p53 and BLIMP-1 may favor a B-cell lymphoma phenotype remains unanswered. We therefore aimed to generate in vivo dual p53/BLIMP-1-floxed conditional inactivation in B-cells, and to define the functional relevance of both p53 and BLIMP-1 n B-cell lymphomagenesis in vivo Methods.Cre recombinase under the control of CD19 promoter (C57BL/6 CD19Cre/Cre) mice were crossed with either C57BL/6 BLIMPflox/flox or C57BL/6 p53flox/flox mice to achieve deletion of BLIMP or p53, respectively, in B cells. Secondly, CD19Cre/Cre BLIMPflox/flox mice were crossed with CD19Cre/Cre p53flox/flox to achieve concomitant deletion of both BLIMP and p53 in B cells (CD19Cre/Cre BLIMPflox/flox p53flox/flox), referred as CD19/Bl-/p53- mice. Transgenic experimental mice (CD19/Bl-/p53-) where characterized for B cell infiltration using immunohistochemistry, flow cytometry; clonotypic immunoglobulin heavy-chain rearrangement was assessed by Southern Blotting. Whole exome sequencing was performed using DNA isolated from B220+ selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice and from matched tail-derived tissues, used as germline (Illumina HiSeq 2500 platform; Agilent SureSelectXT). MTT assay was used to BTK-inhibitor-dependent cytotoxicity using CD19/Bl-/p53-derived B220 cells. Results.We generated dual p53/BLIMP-1-floxed conditional inactivation in B-cells, using mice expressing Cre recombinase under the control of CD19 promoter. 100% of the CD19/Bl-/p53- mice presented with diffuse lymphadenomegalies, and splenomegaly, hepatomegaly (90.3% and 77.4%, respectively). Other clinical manifestations included presence of ascites and hind lymb paralysis (12.9% and 19.3%, respectively). The CD19/Bl-/p53- showed worse survival compared to Bl-/p53- mice non-expressing the CD19/Cre recombinase, CD19/p53-, or CD19/Bl- (363, 469.5, 460.5, and 770 days, respectively). H.E. staining of CD19/Bl-/p53--derived lymph nodes, defined a nodal architecture with a monomorphic population of large sized atypical lymphoid cells with finely clumped and dispersed chromatin, and multiple basophilic medium sized, paracentrally situated nucleoli. A "starry sky" pattern was also observed. Overall, these features are compatible with a high-grade lymphomas. IHC analysis confirmed a marked positivity for B220 staining (TdT, Bcl6, CD138 and CD4, CD8 negative). Tumors were confirmed to be B220+/IgM+, with either Igk- or Ig-lambda-restriction as demonstrated by flow cytometry; and either mono- or bi-clonal, as demonstrated by Southern blotting, thus further confirming the clonal transformation induced by dual BLIMP/p53 deletion in B cells. Whole exome sequencing was performed from B220+ selected cells obtained from pathological lymph nodes of CD19/Bl-/p53- mice and identified 143 SNVs. Among them, non-synonymous somatic mutations were mapped on genes involved in the regulation of focal adhesion, PDGF signaling, p53-downstream pathway, and lipoprotein metabolism. B220+ cells selected from CD19/Bl-/p53--derived lymph nodes were implanted subcutaneously into recipient SCID/Bg mice (n: 10), and presented with 100% engraftment, with a monomorphic lymphoid infiltration of B220+ and IgM+ cells. B220 positive cells were selected from the s.q. tumor and intravenous injected into recipient SCID/Bg (n: 10) and BL/6 mice (n: 10). Engraftment was demonstrated in all the mice, where hepatomegaly, splenomegaly and hind lymb paralysis were observed. Infiltration of B220+ cells was documented within bone marrow, liver and spleen. We next investigated the anti-tumor activity of BTK-inhibitor, and found that B220+ cells selected from lymph nodes harvested from CD19/Bl-/p53-mice were sensitive to ibrutinib treatment. Conclusion. These studies demonstrate that the specific dual inactivation of p53 and BLIMP in B-cells promotes oncogenic transformation, resulting in aggressive B-cell lymphoma development. Disclosures Ghobrial: Celgene: Other: Advisory Board; BMS: Other: Advisory Board; Amgen: Other: Advisory Board; Takeda: Other: Advisory Board; Janssen: Other: Advisory Board. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria.

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Smits, Willem K., Carlo Vermeulen, Rico Hagelaar, Shunsuke Kimura, Eric Vroegindeweij, Jessica G. C. A. M. Buijs-Gladdines, Ellen Van De Geer et al. « Elevated Enhancer-Oncogene Contacts and Higher Oncogene Expression Levels By Recurrent CTCF inactivating Mutations in T Cell Acute Lymphoblastic Leukemia ». Blood 138, Supplement 1 (5 novembre 2021) : 501. http://dx.doi.org/10.1182/blood-2021-152221.

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Abstract Introduction. The CCCTC-binding factor (CTCF) regulates the 3D chromatin architecture by facilitating chromosomal loops and forming the boundaries of structural domains. In addition, CTCF is an important transcription factor and regulator of antigen receptor and T cell receptor recombination events. CTCF inactivating events have been found in various human cancers. Loss-of-heterozygosity (LOH) or inactivating missense mutations in specific zinc- fingers have been identified in many human cancers including sporadic breast cancer, prostate cancer, Wilms-tumors and acute lymphoblastic leukemia (ALL). Heterozygous deletions or point mutations have been identified in over half of the patients with breast cancer or uterine endometrial cancers, deregulating global gene expression by altering methylated genomic states and poor survival. Here, we investigated the functional significance and molecular-cytogenetic associations of CTCF aberrations in T-cell acute lymphoblastic leukemia patients. Methods. Biopsies from a cohort of 181 pediatric T-ALL patients who enrolled on DCOG or COALL protocols and/or their derivative patient-derived xenograft models were screened for alterations in global DNA copy number, methylation status, topologically associating domain organization and CTCF and cohesion binding patterns and changes in local TLX3 and BCL11B promoter enhancer loops using array-comparative genomic hybridization, single molecule Molecular Inversion Probe sequencing, targeted locus amplification, gene expression and DNA methylation microarrays, Hi-C sequencing, Chromatin Immunoprecipitation and/or real-time quantitative PCR. Ctcf f/fl mice 1 were crossed on a the Lck-cre transgenic background 2 to study the impact of Ctcf loss during early T-cell development. Results. We here describe that inactivation of CTCF can drive subtle and local genomic effects that elevate oncogene expression levels from driver chromosomal rearrangements. We find that for T cell acute lymphoblastic leukemia (T-ALL), heterozygous CTCF deletions or inactivating mutations are present in nearly 50 percent of t(5;14)(q35;q32.2) rearranged patients that positions the TLX3 oncogene in the vicinity of the BCL11B enhancer. Functional CTCF loss results in diminished expression of the αβ-lineage commitment factor BCL11B from the non-rearranged allele and γδ-lineage development. Unexpectedly, it also drives higher levels of the TLX3 oncogene from the translocated allele. We demonstrate that heterozygous CTCF aberrations specifically occur in TLX3-rearranged patients with distal breakpoints that preserve CTCF bindings sites in the translocation breakpoint areas in between the BCL11B enhancer and the TLX3 oncogene. We show that these intervening CTCF sites insulate TLX3 from the enhancer by forming competitive loops with TLX3. Upon loss of CTCF, or the deletion of the intervening CTCF sites, these competitive loops are weakened and loops with the BCL11B enhancer are stimulated, boosting TLX3 oncogene expression levels and leukemia burden in these T-ALL patients. Conclusions. CTCF aberrations are especially associated with t(5;14)(q35;q32.2) rearranged T-ALL patients who maintain TLX3-proximal CTCF sites reflects a necessity to neutralize these sites in order to topologically enable the distal BCL11B enhancer to interact with the TLX3 oncogene and to boost its expression. Collectively, this provides direct demonstration of a mechanism in which loss of CTCF result in removal of enhancer insulation that facilitates elevated levels of an oncogene in leukemia. References. 1. Heath H, Ribeiro de Almeida C, Sleutels F, et al. CTCF regulates cell cycle progression of alphabeta T cells in the thymus. EMBO J. 2008;27(21):2839-2850. 2. Lee PP, Fitzpatrick DR, Beard C, et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity. 2001;15(5):763-774. Disclosures Splinter: Cergentis BV: Current Employment. Van Eyndhoven: Agilent Technologies Netherland: Current Employment. Van Min: Cergentis BV: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.

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Zemanova, Zuzana, Kyra Michalova, Karla Svobodova, Jana Brezinova, Halka Lhotska, Libuse Lizcova, Iveta Sarova et al. « Chromothripsis in High-Risk Myelodysplastic Syndromes : Incidence, Genetic Features, Clinical Implications, and Impact on Survival of Patients Treated with Azacytidine (Data from Czech MDS Group) ». Blood 132, Supplement 1 (29 novembre 2018) : 1815. http://dx.doi.org/10.1182/blood-2018-99-114151.

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Abstract Introduction: Chromothripsis is a recently identified genomic instability phenomenon that plays a role in the genesis and progression of cancer. It is a one-step catastrophic genomic event involving multiple chromosomal breakages and random DNA rejoining. This genetic abnormality can affect an entire chromosome, a chromosomal arm, or a single chromosomal region. Chromothripsis is associated with highly complex karyotypes and a very poor prognosis, and has been detected in a wide range of tumor entities, including hematological malignancies. However, this complex genomic abnormality has not been comprehensively studied in patients with myelodysplastic syndromes (MDS). The aim of the study was to assess the incidence, associated genetic features, and clinical significance of chromothripsis in a large homogeneous cohort of patients newly diagnosed with high-risk MDS and complex karyotypes. Methods: A detailed genome-wide analysis of fixed bone-marrow cells from adults with complex karyotypes (≥ 3 aberrations), identified with conventional G-banding at the diagnosis of MDS, was performed. The complex rearrangements were studied with integrative genetic methodologies: fluorescence in situ hybridization (FISH) with Vysis DNA probes (Abbott, Des Plaines, IL), multicolor FISH (mFISH) and/or multicolor banding (mBAND) methods with the 24XCyte and the XCyte color kits (MetaSystems, Altlussheim, Germany), and array-based comparative genomic hybridization with CytoChip Cancer SNP 180K (Illumina, San Diego, CA) or the SurePrint G3 Cancer CGH+SNP 4x180K Microarray (Agilent, Santa Clara, CA). A mutational analysis of the TP53 gene was also performed in selected cases using amplicon-based deep sequencing on a 454 GS Junior System (Roche, Basel, Switzerland) or the TruSight Myeloid Sequencing Panel on MiSeq sequencing instruments (Illumina). Results: In total, 265 patients with complex karyotypes and newly diagnosed high-risk MDS were included (131 females, 134 males; median age, 70 years). The hallmarks of chromothripsis were detected in 67.7% of cases, in both the main clones and one or more subclones. At the cytogenetic level, chromothripsis was apparent as multiple deletions, insertions, ring chromosomes, amplification of individual genes or chromosomal regions, and/or the formation of chaotically reassembled chromosomes. Chromothripsis affected almost all the chromosomes, except the Y chromosome. The most frequently involved were chromosomes 5 (33.3% of events), 7 (28.1% of events), 17 (18.9% of events), 11 and 12 (15.6% of events each). In samples with signs of chromothripsis, the higher frequency of aberrations on chromosome 17p was observed (loss of heterozygosity [LOH], copy-number neutral LOH [cnLOH] and/or homozygous mutation of TP53) (p = 0.05). Patients with chromothripsis had significantly worse overall survival (OS; median, 3 months). We also investigated the effect of chromothripsis on the survival of 183 patients treated with azacytidine. In this cohort, the median OS of chromothripsis-positive patients was 10.1 months (mean, 12.2 months), whereas the median OS of chromothripsis-negative patients was 17.3 months (mean, 31.0 months). Conclusions: Our results demonstrate that chromothripsis is a frequent genomic abnormality in patients with high-risk MDS, which influences the patient's prognosis and disease biology. Chromothripsis was associated with a higher frequency of LOH/cnLOH 17p, rapid disease progression, and short survival. The adverse outcomes may be attributable to the effects on the functions of many important genes. The OS of patients with high-risk MDS with chromotripsis may be slightly improved by azacytidine treatment, but the prognosis of these patients remains very poor. Therefore, a better understanding of the mechanistic basis of chromothripsis is extremely important and could lead to the development of new treatment strategies based on drugs that target the genes present in amplified or deleted regions and/or the DNA damage response pathways. This study was supported by research projects RVO-VFN64165, GACR P302/12/G157, AZV 16-27790A , Progres Q26 and Q28/LF1, GACR 18-01687S, MHCR 00023736 and UNCE/MED/016 . Disclosures No relevant conflicts of interest to declare.

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Hamada, Motoharu, Hideki Muramatsu, Yusuke Okuno, Ayako Yamamori, Taro Yoshida, Masayuki Imaya, Manabu Wakamatsu et al. « Diagnostic Whole Exome Sequencing for 166 Patients with Inherited Bone Marrow Failure Syndrome ». Blood 136, Supplement 1 (5 novembre 2020) : 9. http://dx.doi.org/10.1182/blood-2020-143241.

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BACKGROUND: Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of genetic disorders characterized by bone marrow failure, physical anomalies, and various kinds of organ complications. In addition to classical IBMFSs, such as Fanconi anemia, Diamond-Blackfan anemia, Dyskeratosis congenita, Shwachman-Diamond syndrome, and familial platelet disorders, many types of unclassified IBMFSs are reported. Over 100 genes are considered causative genes; however, the precise genetic diagnosis of IBMFSs remains challenging. We developed a capture-based target sequencing method for IBMFSs that covers more than 180 associated genes. Our system achieved genetic diagnosis for 225 (35%) of 738 patients between 2013 and 2018. However, the causative gene remained unknown for 513 (65%) patients, and further genetic analysis of these "target-negative" cases was necessary to achieve a precise diagnosis. METHODS: We performed whole exome sequencing (WES) for patients who were "target-negative" but strongly suspected of having IBMFS based on the following clinical characteristics: physical or organ anomalies (skin, nail, hair, skeletal, growth, cardiac, lung, liver, or genitourinary), family history of hematological disorder, young age (≤2 years), short telomere length (<-2.0 SD), and hyper sensitivity to the chromosome breakage test. A sequencing library was prepared using the SureSelect Human All Exon 50Mb kit (Agilent Technologies, Santa Clara, CA, USA) and it was sequenced using the HiSeq2000 platform (Illumina, San Diego, CA, USA), according to manufacturers' instructions. The candidate germline variants were detected through our Genomon-exome analysis pipeline. With mean coverage of 100×, ≥ 85% of all protein coding bases were covered at 20× or more. RESULTS: Among the 513 "target-negative" cases, 166 patients were evaluated, of whom 17 patients' parents were also analyzed in a trio-based analysis. New pathogenic variants were identified in 18 of the 166 (11%) patients according to the American College of Medical Genetics (ACMG) guidelines, of which 5 variants were revealed to be de novo. Diagnostic variants were identified in FANCF, SRP54, RPL19, RPL5, RTEL1, RUNX1, MECOM, CDC42, GNE, SLNF14 (all n = 1). In addition to IBMFS-associated genes, causative genes for congenital hemolytic anemia (G6PD, PKLR), inborn error of metabolism (SLC46A1), and primary immune deficiency (NFKB2, LRBA) are also identified (all n = 1). Moreover, loss-of-function mutation of ADH5 gene are identified in three patients that seems to be associated to novel IBMFSs. On the other hand, no pathogenic variant in GATA2, ERCC6L2, LIG4, and SAMD9/SAMD9L genes that are reported as unclassified IBMFSs in Europe and United States are identified in our cohort. CONCLUSION: Our findings support the utility of WES (especially trio-based analysis) as a diagnostic tool for IBMFSs. Furthermore, genetic background of IBMFSs in East Asia seems to be different from that of Europe and United States. Disclosures No relevant conflicts of interest to declare.

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Garg, Tarun K., Ricky D. Edmondson, Shweta S. Chavan, Katie Stone, Justin M. Stivers, Jessica I. Warden, Veronica Macleod et al. « Differential ICAM3 Gene Expression Correlates with Susceptibility to Natural Killer Cell-Mediated Lysis in Multiple Myeloma ». Blood 126, n^o 23 (3 décembre 2015) : 2990. http://dx.doi.org/10.1182/blood.v126.23.2990.2990.

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Abstract Introduction We previously reported on the generation of highly activated/expanded natural killer cells (ENKs) after coculture with K562 cells modified to express membrane bound IL15 and 41BB-ligand. These cells have potent antimyeloma properties in vitro, in a NGS mouse model, and are safe when given to advanced multiple myeloma (MM) patients. (Szmania et al, J Immunother 2015) A potential obstacle to the effectiveness of ENK-based immunotherapy of MM is the evasion of immune recognition. We have generated 4 MM cell lines (OPM2, JJN3, ANBL6, and INA-6) which are resistant to ENK-mediated lysis to study mechanisms of resistance. These lines were derived from parental lines by repeated challenge with ENKs and maintained resistance long term when cultured without further exposure to ENKs.(Garg et al, Blood 2012, 120:4020) We have shown by stable isotope labeling with amino acids in cell culture-mass spectrometry, gene expression profiling (GEP), and flow cytometry that ICAM3 is downregulated in the ENK-resistant version of OPM2 (OPM2-R) compared to the parental OPM2. (OPM2-P; Garg et al, Blood 2013, 122:3105) We investigated OPM2-P and OPM2-R by whole exome sequencing (WES) and RNA sequencing (RNAseq) with a focus on ICAM3, evaluated ICAM3 cell surface expression on patient myeloma cells, and studied the importance of ICAM3 expression on ENK functionality. Methods DNA and RNA were extracted from OPM2-P and OPM2-R cells using the Qiagen AllPrep kit. WES libraries were prepared with the Agilent qXT and Agilent SureSelect Clinical Research Exome kits with additional baits covering the Ig and MYC loci. RNAseq libraries were prepared using the Illumina TruSeq stranded mRNA kit. Samples were sequenced 100bp PE on an Illumina HiSeq2500. Samples for WES were sequenced to a mean coverage of >120x and RNAseq to a target of >100M reads. WES data were aligned to the Ensembl GRCh37/hg19 human reference using BWA mem. Somatic variants were called MuTect. RNAseq data were analyzed using Tuxedo Suite. Data were aligned to the Ensembl GRCh37/hg19 human reference using TopHat with Bowtie2. Transcriptome reconstruction, quantification and differential analysis was performed using CuffLinks. ENK-mediated lysis of myeloma cells was measured by 4 hour chromium release assay in the presence of isotype or ICAM3 blocking antibody. Bone marrow aspirates were obtained from MM patients after informed consent in accordance with the Declaration of Helsinki. Primary myeloma cells were selected with CD138-coated immunomagnetic beads and ICAM3 expression was assessed by flow cytometry gated on viable CD138 positive cells. Results There was no mutation in ICAM3 in OPM2-R by WES, but RNAseq found a significant reduction in ICAM3 RNA in OPM2-R compared to OPM2-P (p <0.008). Loss of ICAM3 expression on OPM2-R correlated with a reduction in sensitivity to ENK-mediated lysis compared to OPM2-P (mean 83%, range 77-88%, N=7 assays; E:T ratio 10:1). Blocking of ICAM3 on OPM2-P similarly reduced susceptibility to ENK-mediated cytotoxicity (mean 45%, range 30-56%, N=4 assays; E:T ratio 10:1). We next examined ICAM3 expression on primary myeloma cells by flow cytometry (N=49; GEP-defined high-risk n=43) and found that there is considerable biological inter-patient variation in ICAM3 expression (median MFI 922; range 97-5882, Figure 1A). Further, the majority of patients studied exhibited ICAM3-negative myeloma subpopulations (0.01%-19.4% of CD138 positive myeloma cells, Figure 1B). Functional studies will be presented to correlate the level of ICAM3 expression on primary myeloma cells with sensitivity to ENK-mediated lysis and resulting data shall be presented. Conclusion Our findings demonstrate that MM patients harbor ICAM3-negative myeloma populations in varying frequencies, and we hypothesize that these cells may be similarly resistant to ENK-mediated lysis. Functional assays exploring this question are in progress. By understanding the mechanisms of ENK resistance and immune escape in MM, we hope to elucidate a surrogate biomarker which will allow us to select subjects who are most likely to benefit from cellular immunotherapeutic strategies for enrollment in future ENK-based clinical trials. Additionally, the ICAM3/LFA-1 interaction is also important for adhesion of T cells to their targets; therefore, down-regulation of ICAM3 may also have functional implications in the efficacy of T cell-based therapies for MM. Disclosures Garg: University of Arkansas for Medical Sciences: Employment. Chavan:University of Arkansas for Medical Sciences: Employment. Stone:University of Arkansas for Medical Sciences: Employment. Stivers:University of Arkansas for Medical Sciences: Employment. Warden:University of Arkansas for Medical Sciences: Employment. Skinner:University of Arkansas for Medical Sciences: Employment. Lingo:University of Arkansas for Medical Sciences: Employment. Greenway:University of Arkansas for Medical Sciences: Employment. Khan:University of Arkansas for Medical Sciences: Employment. Johann:University of Arkansas for Medical Sciences: Employment. Heuck:Millenium: Other: Advisory Board; Celgene: Consultancy; Janssen: Other: Advisory Board; University of Arkansas for Medical Sciences: Employment; Foundation Medicine: Honoraria. Barlogie:University of Arkansas for Medical Sciences: Employment. Morgan:University of Arkansas for Medical Sciences: Employment; Weismann Institute: Honoraria; MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; CancerNet: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Epstein:University of Arkansas for Medical Sciences: Employment. van Rhee:University of Arkansa for Medical Sciences: Employment.

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Гусина, А. А., В. Ф. Иванова, К. А. Криницкая et Н. Б. Гусина. « SLC4A11-Associated Hereditary Corneal Endothelial Dystrophies : Literature Review and Case Report ». Офтальмология. Восточная Европа, n^o 4 (9 février 2021) : 555–67. http://dx.doi.org/10.34883/pi.2020.10.4.027.

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Введение. Наследственные дистрофии роговицы – гетерогенная группа генетически детерминированных двусторонних, симметричных, медленно прогрессирующих поражений роговицы невоспалительного характера. Ген SLC4A11 кодирует синтез интегрального мембранного белка, который участвует в транспорте воды, ионов, аммиака и функционирует как молекула клеточной адгезии. Мутации в гене SLC4A11 в гетерозиготном состоянии описаны у пациентов с эндотелиальной дистрофией роговицы Фукса 4-го типа (OMIM: 613268). Гомозиготное или компаундное гетерозиготное носительство мутаций в гене SLC4A11 является причиной врожденной наследственной эндотелиальной дистрофии роговицы (OMIM: 217700), а также эндотелиальной дистрофии роговицы с перцептивной тугоухостью (синдром Харбойяна, OMIM: 217400). В этой работе мы представляем редкое наблюдение синдрома Харбойяна, собственный опыт молекулярной диагностики и лечения этого заболевания.Материалы и методы. Пробанд – девочка 9 лет, направлена к генетику для исключения наследственных заболеваний обмена веществ в связи с диффузным двусторонним помутнением роговицы. Образцы ДНК, полученные от пробанда и ее матери, были исследованы методом высокопроизводительного секвенирования с использованием панели TruSight Inherited Disease, Illumina. Наличие мутаций в гене SLC4A11 у пациентки и ее матери подтвердили методом прямого секвенирования.Результаты и обсуждение. Девочка родилась в неродственном браке с нормальными показателями длины и массы тела. Мать впервые заметила помутнение роговицы у дочери в возрасте 8 месяцев. Заболевание медленно прогрессировало: ухудшалась острота зрения, появились светобоязнь и боль в глазах. В возрасте 9 лет в связи со значительным снижением остроты зрения, наличием роговичного синдрома, тотальным помутнением роговицы, дефицитом эндотелиальных клеток, дефектами десцеметовой оболочки, буллезными изменениями эпителия произведена субтотальная сквозная кератопластика на правом глазу. Послеоперационный период протекал без осложнений. Через 6 месяцев после операции роговичный трансплантат прозрачен, Visus OD = 0,1 н/кор.При проведении высокопроизводительного секвенирования у пациентки были выявлены 2 вероятно патогенные мутации в гене SLC4A11: делеция 2 нуклеотидов c.733_734delAT (p.Ile245LeufsTer28, NM_001174090.1) в 6-м экзоне гена и замена c.2321+1G>A в каноническом сайте сплайсинга в 17-м интроне. Мутация c.733_734delAT является новой, не описанной ранее в научной литературе, она не зарегистрирована в контрольных выборках GNOMAD и ExAC. Вариант c.2321+1G>A отмечен в контрольной выборке ExAC FIN с частотой 0,016%, однако у пациентов с наследственными дистрофиями роговицы ранее описан не был. У матери пробанда мутация c.2321+1G>A обнаружена в гетерозиготном состоянии.Заключение. По совокупности сведений, с учетом клинической картины заболевания у пробанда, мы классифицировали данный случай как врожденную эндотелиальную дистрофию роговицы с перцептивной тугоухостью, обусловленную компаундным гетерозиготным носительством мутации со сдвигом рамки считывания и мутации сплайсинга в гене SLC4A11. Introduction. Hereditary corneal dystrophies are a heterogeneous group of genetically determined bilateral, symmetrical, slowly progressive, non-inflammatory corneal lesions. Heterozygous mutations in the SLC4A11 gene have been described in patients with type 4 Fuchs endothelial corneal dystrophy (OMIM: 613268). Homozygous or compound heterozygous mutations in the SLC4A11 gene have also been shown to cause congenital hereditary endothelial corneal dystrophy (OMIM: 217700) and endothelial corneal dystrophy with perceptual hearing loss (Harboyan syndrome, OMIM: 217400). Here we present a rare observation of Harboyan’s syndrome and our own experience in molecular diagnostics and treatment of this disease.Materials and methods. Proband – a 9-year-old girl suffered from diffuse bilateral corneal opacity was brought to the geneticist to exclude hereditary metabolic diseases. Next generation sequencing using a TruSight Inherited Disease, Illumina panel was performed for the proband. The presence of mutations in the SLC4A11 gene in the patient and her mother was confirmed by direct sequencing. Results and discussion. The girl was born in non-consanguineous union with normal length and weight. Mother noticed corneal opacity in her daughter at the age of 8 months first. The disease progressed slowly: visual acuity worsened, photophobia and pain in the eyes appeared. At the age of 9 years, due to a significant decrease in visual acuity, presence of corneal syndrome, total corneal opacity, endothelial cell deficiency, Descemet membrane defects, and bullous changes in the epithelium, subtotal penetrating keratoplasty was performed on the right eye. The postoperative period was uneventful. The corneal graft is transparent 6 months after the operation, Visus OD =0.1 n / cor.Next generation sequencing revealed two likely pathogenic variants in the SLC4A11 gene: deletion of 2 nucleotides c.733_734delAT (p.Ile245LeufsTer28, NM_001174090.1) in exon 6 and splice site mutation c.2321 + 1G> A in 17 intron. The c.733_734delAT mutation is new, not previously described in the scientific literature; it was not registered in the GNOMAD and ExAC control samples. Variant c.2321 + 1G> A was noted in the control sample ExAC FIN with a frequency of 0.016%; however, it was not previously described in patients with hereditary corneal dystrophies. Heterozygous mutation c.2321 + 1G> A was found in proban’s mother.Conclusion. We classified this case as congenital endothelial corneal dystrophy with perceptual hearing loss (Harboyan syndrome) caused by the compound heterozygous mutations compound heterozygous mutations affecting the SLC4A11 gene.

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Neumann, Martin, Marco Seehawer, Cornelia Schlee, Sebastian Vosberg, Sandra Heesch, Eva von der Heide, Alexander Graf et al. « FAT1 Expression and Mutation Status In Adult Acute Lymphoblastic Leukemia ». Blood 122, n^o 21 (15 novembre 2013) : 2564. http://dx.doi.org/10.1182/blood.v122.21.2564.2564.

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Abstract Introduction FAT1 belongs to the FAT protocadherin family, a drosophila homologous gene involved in development processes. Recently, FAT1 gained large interest as it is mutated in various cancers. Besides the known function of cell-cell interaction and polarity, FAT1 loss of function mutations have been linked to dysregulation of the WNT pathway in solid tumors. In acute lymphoblastic leukemia (ALL), aberrantly high expression of FAT1 was claimed to be associated with inferior outcome in pediatric B-lineage ALL. Herein, we investigated the yet unknown frequency and relevance of FAT1 expression and mutation in a large, homogenously treated cohort of adult ALL patients. Patients and Methods We investigated FAT1 expression in diagnostic bone marrow (BM) samples of 112 T-ALL, 122 B-lineage ALL, and additional 63 early T-cell precursor (ETP) ALL patients by real time (RT)-PCR. Patients were enrolled in trials of the German Multicenter Study Group for Adult ALL (GMALL) and outcome was investigated for patients into GMALL trials 06/99 and 07/03. Using the T-cell line BE13 as reference, we defined patients with FAT1 expression higher than BE13 as FAT1pos (0.01-38.5) and patients with a lower expression as FAT1neg (<0.01). We additionally examined peripheral blood (PB), BM, CD34+-, CD3+-cells from healthy donors. FAT1mutation status was investigated in 68 T-ALL patients. For mutation analyses customized biotinylated RNA oligo pools (SureSelect, Agilent) were used to hybridize the targeted regions, followed by 76-bp paired-end sequencing on an Illumina Genome Analyzer IIx platform. Results Normal hematopoietic cells including unselected BM cells, CD34+-progenitors, or CD3+ T-cells from healthy donors lacked FAT1 expression (<0.01) with the one exception of a CD34+-sample (0.03). In contrast, ALL samples aberrantly expressed FAT1: 32% of B-lineage ALL (0.01-38.5) and 54% of T-ALL cases (0.01-31.2) showed elevated FAT1 expression, with a lower frequency in the immature ETP-ALL subgroup (17%; 0.01-15.2). FAT1 expression was associated with a more mature immunophenotype (T-ALL: thymic 74%, mature 45%, early 4%, p<0.001; B-lineage ALL: pre B-ALL 57%, common ALL 26%, pro B-ALL 9%, p=0.04). No significant differences between FAT1pos and FAT1neg patients were observed regarding age and sex. FAT1pos T-ALL patients more frequently showed a white blood cell count (WBC) >30.000/µL at diagnosis compared to FAT1neg T-ALL patients (78% vs. 42%, p<0.01). Whereas no negative prognostic impact was observed for FAT1 expression in B-lineage ALL or T-ALL with respect to overall survival or remission duration, lack of FAT1 expression was associated with primary resistance to induction therapy in T-ALL (T-ALL: FAT1neg 12%, FAT1pos 0%, p=0.04). In addition, we found an unexpected high rate of FAT1 mutations (exclusively missense mutations) with 8 of 68 T-ALL patients (12%). The mutation spectrum, mainly located in the cadherin domains, was similar to the distribution of FAT1 mutations in solid tumors. No difference were observed between FAT1 mutated (FAT1mut) and FAT1 wild-type patients (FAT1wt) with regard to sex, age, WBC, and presentation of antigens associated with an early differentiation stage. FAT1 mutations were more frequent in early T-ALL (3/12, 25%) and in thymic T-ALL (5/41, 12%) than in T-ALL patients with a mature immunophenotype (0/15, 0%). FAT1 expression was more common in FAT1wt T-ALL compared to FAT1mut T-ALL patients (50% vs. 25%). Conclusion This first comprehensive analysis on FAT1 in a large cohort of adult patients with ALL shows a high frequency of FAT1 expression. Higher FAT1 expression occurred in ALL patients with more mature immunophenotype linking FAT1 to cell-cell adhesion and polarity, thymic homing and interaction with the BM niche. This yet unreported high mutation rate of 12 % in adult T-ALL makes FAT1 to one of the most frequently mutated genes in T-ALL. The link of inactivating FAT1 mutations to aberrant activation of the WNT pathway, as reported in solid tumors, might allow the development of refined treatment options. In summary, these data make FAT1 a promising candidate for disease monitoring, risk stratification and development of targeted therapies. Disclosures: Krebs: Illumina: Honoraria. Greif:Illumina: Honoraria.

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Vosberg, Sebastian, Tobias Herold, Klaus H. Metzeler, Stephanie Schneider, Bianka Ksienzyk, Alexander Graf, Stefan Krebs et al. « Copy Number Alteration (CNA) Analysis in Targeted Sequencing Data from Acute Myeloid Leukemia (AML) Patients with Chromosome 9q Deletion ». Blood 124, n^o 21 (6 décembre 2014) : 1058. http://dx.doi.org/10.1182/blood.v124.21.1058.1058.

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Abstract Whole exome sequencing (WES) or customized gene panel sequencing (GPS) in acute myeloid leukemia (AML) is commonly used to detect point mutations and small insertions/deletions that might contribute to leukemogenesis. Beyond sequence variant detection, targeted sequencing also allows to detect gain or loss of genomic material in tumor cells (i.e. copy number alteration; CNA) based on the comparison of sequence coverage in target regions between samples. This approach allows not only for the detection of whole chromosome aneuploidies but also submicroscopic deletions or amplifications affecting small regions of the genome. We selected cytogenetically well characterized AML patients with partial deletions of the long arm of chromosome 9 (AML del(9q), n=5) and performed WES at diagnosis and at complete remission (SureSelect, Agilent; Illumina paired-end sequencing). At least 75% of the target region was sequenced with coverage ≥ 10x. As state of the art method for CNA detection, we also performed SNP array profiling (Affymetrix) of the 5 diagnostic AML samples. In addition, we performed GPS of 140 genes (total target 492 kb; mean coverage 356x, range 112-995x; targets on chromosome 9 with mean coverage 275x, range 71-705x) in the diagnostic samples from 26 cases of AML del(9q) (including the 5 exome cases) and 21 AML patients without any detectable cytogenetic aberration on chromosome 9 (control cohort). Our custom gene panel (Haloplex, Agilent) included known mutational targets in AML and candidate genes located on 9q. We used a linear regression model to normalize the mean read count of exon regions for target enrichment efficiency and to model the test sample coverage as a linear function of the control sample coverage (Rigaill et al., 2012, Bioinformatics). This approach is able to deal with regions of zero coverage, monoallelic deletions and tolerates outliers. An exact segmentation algorithm was applied to each chromosome individually in order to separate regions of equivalent exon coverage from regions of different exon coverage between test and control samples. Thereby, regions of genomic alterations can be defined as well as ranges for the flanking breakpoints. We defined a maximum of 5 regions per chromosome and a minimum size of 2 exons per region. For WES analysis, diagnostic AML del(9q) data sets were used as test samples and matching remission data sets were used as control. The minimum mean exon coverage was set to 10x. For custom GPS analysis, the minimum coverage was set to 50x and each AML del(9q) patient was compared to each control patient. Only chromosome 9 was included in the analysis, as patients of the control cohort harbor additional alterations on other chromosomes. CNAs were defined as regions that differ from the majority of control samples. Overlapping CNAs of AML del(9q) patients were subsequently identified as common altered regions. CNA profiling based on WES data sets of AML del(9q) patients showed somatically acquired stretches of significantly reduced read counts for genes located on 9q in 2 of the 5 patients (Figure 1A), consistent with the corresponding SNP array results. CNA profiling based on GPS from 26 AML del(9q) patients and 21 control patients defined a common deleted region ranging from at least 79.2 Mb to 87.6 Mb (Figure 1B). The deletion was detected in 18 out of 26 (70%) of the AML del(9q) patients. Neither the comparison of the test samples to each other nor the comparison of the control samples to each other resulted in CNA calling. It is very likely that the varying clone size harboring the 9q deletion in the diagnostic samples is limiting for CNA detection. This is also supported by the observation that patients without detectable 9q deletion in our CNA analyses tended to have fewer metaphases with 9q deletion (median 27%, range 8-77%) compared to patient samples with detectable 9q deletion in the CNA analyses (median 95%, range 24-100%; p = 0.001), as reported by routine cytogenetics. Our study confirms that, despite the experimental variability of target enrichment, sequencing data can be efficiently used not only to identify somatic mutations with single nucleotide resolution, but also to detect recurring and/or somatic CNAs in AML. Similar to CNA detection by SNP array analysis, the clonal architecture of the tumor is limiting for sensitivity. However, this limitation might be overcome by increasing the read depth. Figure 1: Figure 1:. Detection of del(9q) in WES (A) and GPS data sets (B) Disclosures No relevant conflicts of interest to declare.

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Rose-Zerilli, Matthew JJ, Gibson Jane, Jun Wang, William J. Tapper, Helen Parker, Anton Parker, Zadie Davis et al. « Tracking Subclonal Mutations in IGHV-Mutated CLL with Progressive Disease ». Blood 124, n^o 21 (6 décembre 2014) : 1962. http://dx.doi.org/10.1182/blood.v124.21.1962.1962.

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Abstract Most CLL is diagnosed with a low tumor burden with no indication for therapy. Biomarkers, such as unmutated IGHV genes, TP53 loss/mutation and raised β2M predict short time to first treatment and overall survival; however there remain patients with good risk biomarkers who nevertheless develop progressive disease. Advances in genomics and immunogenetics have lead to the discovery of new biomarkers and their integration with cytogenetic data refines outcome prediction. However these novel markers are predominantly found in IGHV unmutated cases (U-CLL). To identify novel genetic mechanisms that may contribute to progression, we have studied 13 patients (pts) presenting with cMBL (n=3) or Stage Binet A/Rai 0 disease and good risk markers: IGHV-mutated, excluding poor risk stereotypes (n=13), no 17p or 11q deletion by FISH, (n=13), sole del13q14 (n=8), low CD38 expression (n=13) who all developed lymphocytosis (n=13), between two untreated timepoints (TP1 & TP2), 10 of whom subsequently required treatment. Copy number analysis (SNP6), whole exome sequencing (WES; Agilent SureSelect & Illumina sequencing) of tumour-germline pairs and targeted deep sequencing (TDS; Haloplex, Agilent) of the the WES-identified variants and the 22 most frequently mutated genes in CLL, to a mean depth of 3681 fold, were performed at TP1 and TP2. TP1 was close to diagnosis (median of 1 yr, range 0.11-7.33) with a median time to TP2 of 4.5 yrs (0.2-8.9). In addition, TDS was undertaken at later time points in one patient described in point 4), who relapsed and ultimately transformed. Our analysis shows the following potential mechanisms: 1. Our germline WES data revealed 5 heterozygous missense/frameshift variants in 5 genes in 5 pts, also known to be targeted by somatic mutation in CLL (eg: FBXW7, POT1, SAMHD1. Fig1). 2. We then established the somatically-acquired mutation profile of each patient. We validated 72% (224/312) of the mutations discovered by WES using TDS and identified clinically relevant mutations earlier on in disease, supporting the hypothesis that sub-clonal mutations in genes in addition to TP53 may drive a progressive clinical course. At diagnosis (TP1) by WES/TDS, 5/13 pts had mutations in CLL driver genes (ATM, NOTCH1, SF3B1, TP53) and 2/13 pts had mutations in genes of undetermined clinical significance (CHD2, NFKBIE, ZMYM3). One patient was MYD88 mutated at TP1 and remains untreated after follow up of 12 yrs. In total, the following 9 genes (ATM, CHD2, DDX3X, MYD88, NOTCH1, NFKBIE, SF3B1, TP53 & ZMYM3) were mutated in 62% (8/13) pts at TP1. 3. Of the remaining 5/13 pts lacking a detectable mutation in any of the established CLL genes, we observed on average 7 mutations/patient in genes involved in cancer and each patient harboured one or more mutated genes with a role in haematological malignancy (eg. ITGA6, KLHL6, LTF, TNFAIP3). 4. One patient exhibited a remarkable temporal shift in copy number changes and mutations. At TP2, SNP6 analysis could not detect the del13q observed at TP1, and a clonal trisomy 12 had emerged, along with several mutations associated with progressive disease (BIRC3, IRF4, NOTCH1), that predominate in U-CLL. As a consequence we re-analysed the IGHV mutational status at TP2, and showed that rather than the IGHV3-48 with 92% germline identity identified at diagnosis, our patient exhibited an additional and dominant IGHV5-10-1*01 (100% identity) clone at TP2, 8 yrs after TP1. Additional analysis of intermediate samples detected the unmutated clone as far back as 4 yrs post diagnosis, and TDS analysis showed the NOTCH1 mutation was a minor subclone at diagnosis (0.06% VAF). Ultimately, this patient developed Richters syndrome with expansion of the NOTCH1 mutation (27% VAF). Retrospective sequential immunogenetic analysis of the other 12 cases yielded no other example of this phenomenon. In summary, IGHV-mutated cMBL/early stage CLL with a progressive outcome can be associated with, the presence of germline or subclonal gene mutations of known or putative importance in CLL, or the emergence of a IGHV-unmutated clone. Our data supports deep sequencing in the clinical setting for earlier detection of pathogenetic mutations and emerging immunogenetically distinct subclones in patients with early stage 'good risk' disease. Figure 1: Heatmap representation of the cohorts clinical features and DNA mutation. Patient 287 haboured the IGHV-unmutated clone at TP2-5. Figure 1:. Heatmap representation of the cohorts clinical features and DNA mutation. Patient 287 haboured the IGHV-unmutated clone at TP2-5. Disclosures No relevant conflicts of interest to declare.

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Lawrie, Alastair, Timothee Cezard, Dominic J. Culligan et Mark A. Vickers. « Exome Sequencing and Linkage Analysis Implicates Two Candidate Genes On Chromosome 3p in Familial Hodgkin Lymphoma ». Blood 120, n^o 21 (16 novembre 2012) : 53. http://dx.doi.org/10.1182/blood.v120.21.53.53.

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Abstract Abstract 53 Background It is well recognised that a genetic component exists in classical Hodgkin lymphoma. Higher rates are seen in first-degree relatives of affected individuals, with high concordance observed in monozygotic compared to dizygotic twins. Numerous associations with HLA alleles have been demonstrated in both Epstein-Barr virus-positive and negative forms of the disease and several non-HLA genes have now been implicated. However, these genetic factors are insufficient to account for all the observed inherited risk. Methods We report a family from North-East Scotland containing five related individuals with classical Hodgkin lymphoma. DNA was extracted from either whole peripheral blood or saliva. Affected individuals were genotyped using the Affymetrix 500Kb SNP array. Genome-wide linkage analysis was performed using easyLINKAGE plus. Targeted exome capture was performed on four affected individuals using the Agilent SureSelect 50Mb kit. Exome sequencing was performed on the Illumina HiSeq2000 to produce 101bp paired-end reads. Following quality control, raw fastq reads were aligned to the human genome (GRCh37) using bwa. SNP/indel calls were performed using Samtools. Variants identified were filtered by quality to include only those with a read coverage of ≥ 20 and phred score of ≥ 30. Given the high penetrance in this family, we hypothesised that a causative genetic variant would not have been described before so eliminated all SNPs present in dbSNP (version 132) or the 1000 genomes project. Finally, only SNPs affecting protein structure were considered further. SIFT and Polyphen-2 were used to predict impact of genetic variation upon protein structure/function. Results Karyotypic analysis was performed on one individual at diagnosis. This was normal, suggesting that a consitutional chromosomal disorder is not responsible for classical Hodgkin lymphoma in this family. The two regions exhibiting strongest linkage with disease were observed on chromosome 3p (LOD scores 1.5 & 1.4). Exome sequencing revealed seven novel, non-synonymous, heterozygous SNPs present in all four analysed individuals. Three were considered biologically plausible (FAM107A:A89S, ALS2CL:L440V, IGSF3:E414G) on the basis of known function and pattern of expression. Two of these three genes (FAM107A and ALS2CL) are on chromosome 3p, in regions similar to those identified in the linkage analysis, and have been implicated as candidate tumor suppressor genes in other malignancies. All three variants were located in evolutionary conserved regions. However, only the mutations in FAM107A and IGSF3 were predicted to disrupt protein function. Discussion Two genes on chromosome 3p represent candidate loci for causing familial classical Hodgkin lymphoma. FAM107A protein is downregulated in several malignant cell lines and primary tumor cells. Overexpression can result in suppression of tumor growth. A study of head and neck carcinoma identified frequent missense mutations and/or loss of heterozygosity at ALS2CL, suggesting that this gene can contribute to tumorigenesis. We are evaluating these genes further by assessing gene expression and protein levels in both Hodgkin cell lines and primary Reed-Sternberg cells. Finally, we intend to evaluate the presence of these candidate genetic variants in other, unaffected members of this family and sporadic cases. Disclosures: No relevant conflicts of interest to declare.

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Nussenzveig, Roberto H., Mohamed E. Salama, Sherrie L. Perkins, Josef Prchal et Archana M. Agarwal. « The Clinical Utility Of Next-Generation Sequencing In The Diagnosis Of Polycythemia ». Blood 122, n^o 21 (15 novembre 2013) : 2185. http://dx.doi.org/10.1182/blood.v122.21.2185.2185.

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Abstract Polycythemia or erythrocytosis is a disorder characterized by expansion of the RBC mass, and can be primary or secondary. Primary polycythemia is caused by an acquired or inherited mutation, it includes polycythemia vera and familial/congenital variants. e.g. due to gain of function mutations in the erythropoietin receptor (EPOR), or mutations in the hypoxia sensing pathway. Secondary polycythemia is caused by a circulating factor stimulating erythropoiesis, usually erythropoietin (EPO). It is most often due to an EPO response to hypoxia, but can also result from an EPO-secreting tumor. Either primary or secondary polycythemias can be inherited, i.e. due to germline mutations. The hypoxic response, mediated by hypoxia inducible transcription factors (HIFs), is central to the control and development of many essential biological functions, including erythropoiesis. Mutations in this pathway, causing polycythemia, have been identified in negative regulators of HIFs, such as the von Hippel-Lindau (VHL) gene, the HIF-prolyl-hydroxylase 2 (PHD2) gene, and gain-of-function mutations of the HIF-2-alpha (HIF2A) gene. Routine diagnostic testing can be challenging with specialized testing often only available in specialized research laboratories. Comprehensive, coding region analysis of all candidate genes by selective amplification of DNA regions of interest by PCR followed by the sequencing and analysis of amplified DNA fragments involved can be daunting due to molecular heterogeneity of causative genes as well as the size of the genes involved. Targeted molecular analysis is now being developed for both acquired (somatic) mutations or inherited (germline mutations) causing acquired and inherited diseases. We developed a novel, high-throughput, sensitive sequencing assay for diagnosis of congenital causes of polycythemias and polycythemia vera. Our diagnostic panel includes 9 genes and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Custom targeted gene capture and library construction for next-generation sequencing (NGS) was performed using HaloPlex as described by the manufacturer (Agilent Technologies, Santa Clara, CA). One hundred base-pair paired-end sequencing was done on a HiSeq 2000 system (Illumina, San Diego, CA). Bioinformatic analysis was based on an “in house” pipeline using standard open-source software. A total of 10 patients with clinically suspected polycythemia, and 30 normal controls were tested in our assay. Whole blood genomic DNA was isolated from the patients and targeted gene capture performed. Mutations in the target genes were identified in 3/10 patients, two of these being novel. All identified mutations were confirmed by Sanger sequencing. In one of these patients, a child with increased RBC mass, with EPO hypersensitive BFU-E colonies, a novel pathogenic, nonsense mutation was found in exon 8 of the EPOR gene (Q434X) resulting protein truncation and absence of the C-terminal negative regulatory domain of the receptor. In a different patient with suspected primary congenital/familial polycythemia due to EPO hypersensitive BFU-E colonies, we identified a novel pathogenic mutation in exon 3 of the MPL gene. This novel mutation, a single base deletion causes a frame-shift in codon 126 (F126L) and early termination at codon 130. To the best of our knowledge, this is the first report of a loss of function mutation in the MPL gene in a patient with polycythemia. Analysis of greater cohort of polycythemic patients is now in progress. Disclosures: No relevant conflicts of interest to declare.

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Weber, Simone, Manja Meggendorfer, Niroshan Nadarajah, Karolína Perglerová, Susanne Schnittger, Claudia Haferlach, Wolfgang Kern et Torsten Haferlach. « Molecular Characterization of Philadelphia Chromosome Positive Acute Myeloid Leukemia - New Provisional Entity ? » Blood 126, n^o 23 (3 décembre 2015) : 3846. http://dx.doi.org/10.1182/blood.v126.23.3846.3846.

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Abstract Introduction Philadelphia chromosome positive (Ph+) acute myeloid leukemia (Ph+AML) is discussed to be a new provisional entity for the upcoming WHO classification. Whether Ph+AML represents a distinct entity or rather embodies chronic myeloid leukemia in myeloid blast crisis (CML-BC) without preceding clinical manifestation is under debate mostly due to lack of robust criteria to reliably differentiate these two diseases. Further, while Ph+AML is clearly distinguishable from Ph+ acute lymphoblastic leukemia (Ph+ALL) based on immunophenotyping, recent studies demonstrated that Ph+AML retain typical characteristics of lymphoid disease. Aim Ph+AML, CML-BC and Ph+ALL were analyzed by a panel of 24 genes and array CGH to get more insights into these Ph+ leukemias and to potentially define delimiting genetic features. Patients and Methods We examined 24 pts with Ph+AML (11 females/13 males, median age: 58 (24-83)), 11 CML-BC (7 females/4 males, median age: 60 (32-79)) and 11 Ph+ALL (7 females/4 males, median age: 67 (44-77)). AML and ALL were diagnosed according to WHO classification by morphology, MPO and flow cytometry. CML-BC all were diagnosed as CML before and treated accordingly. All cases revealed the BCR-ABL1 fusion gene. Next generation sequencing was performed for ASXL1, BCOR, CBL, CSF3R, DNMT3A, ETV6, FLT3 tyrosine kinase domain(FLT3 -TKD), IDH1/2, JAK1/2/3, KRAS, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1 and ZRSR2 using the MiSeq Instrument (Illumina, San Diego, CA). Partial tandem duplications in MLL (MLL- PTD), internal tandem duplications in FLT3 (FLT3- ITD)and deletions in IKZF1 were analyzed by quantitative real-time PCR or genescan analysis. 45 cases were investigated by array CGH (Agilent, Waldbronn, Germany). Results With respect to cytogenetic abnormalities besides Philadelphia chromosome, several unbalanced abnormalities were found in Ph+ALL (mean: 9, range: 2-31), while less aberrations were found in Ph+AML and CML-BC (mean: 4, range: 0-28 and mean: 4, range: 0-16, respectively; p=0.03). Of these the most prominent aberrations which were present in all three groups included loss of 7p encompassing IKZF1 (Ph+AML: 7/23, 30%; Ph+ALL: 8/11, 73%; CML-BC: 2/10, 20%), loss of 9p encoding CDKN2A/B (Ph+AML: 2/23, 9%; Ph+ALL: 6/11, 55%; CML-BC: 2/10, 20%) as well as gain of 8q (Ph+AML: 6/23, 26%; Ph+ALL: 3/11, 27%; CML-BC:4/11, 36%). Loss of 5q (5/23, 22%), gain of 13q (4/23, 17%) and loss of 21q (3/23, 9%) was exclusively present in Ph+AML. While in Ph+ALL loss of 10q (3/11, 27%), 2p (3/11, 18%), 11q (3/11, 18%) and gain of 4q (3/11, 18%) was exclusively found. Regarding recurrent balanced aberrations no rearrangements were found in Ph+AML and Ph+ALL, while 3/11 (27%) CML-BC pts harbored balanced 3q26-rearrangements. With respect to molecular genetics, alterations were found in 15/24 (63%) Ph+AML, 8/11 (73%) CML-BC and 8/11 (73%) Ph+ALL pts. Commonly shared molecular aberrations were deletions in IKZF1 (Ph+AML: 3/24, 13%; Ph+ALL: 8/11, 73%; CML-BC: 2/10, 20%) as well as mutations (mut) in RUNX1 (Ph+AML: 5/19, 26%; Ph+ALL: 1/7, 14%; CML-BC: 5/10, 50%). Further, mut found in Ph+AML and CML-BC affected ASXL1 (Ph+AML: 2/22, 9%; CML-BC: 2/11, 18%) and IDH1 (Ph+AML: 2/22, 9%; CML-BC: 1/11, 9%). Additionally, Ph+AML harbored alterations in TP53 (3/21, 14%), TET2 (2/21, 10%) and DNMT3A (1/20, 5%). For CML-BC, additional mut were found in WT1 (2/9, 22%), ETV6 (1/9, 11%) and KRAS (1/9, 11%). Regarding FLT3 -ITD, NPM1 and the remaining genes no alterations were found. Overall, Ph+ALL differed from the combined cohort of Ph+AML and CML-BC in that mut in ASXL1, DNMT3A, ETV6, IDH1, KRAS, TET2 and WT1 as well as MLL -PTD occurred not in the former but only in the latter (12/31 cases with at least one gene mutated, p=0.07). Intriguingly, the mean±SD number of mut in these genes did not significantly differ between Ph+AML and CML-BC cases (0.36±0.58 vs. 0.67±0.71, p=0.23). Conclusion Comparing cytogenetic alterations Ph+AML could be clearly distinguished from CML-BC or Ph+ALL by harboring loss of 5q and gain of 13q, which are typically found in myeloid diseases. Beside, Ph+AML and CML-BC showed a high frequency of molecular mutations which were hardly found in Ph+ALL. This data supports the concept discussed by the WHO that Ph+AML is a specific entity and can be distinguished from CML-BC and Ph+ALL. However, further studies are warranted to define the most appropriate parameters to distinguish Ph+AML from CML-BC. Disclosures Weber: MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Furness, Caroline L., Marcela B. Mansur, Victoria J. Weston, Sarah Jenkinson, Frederik W. van Delft, Lyndal Kearney, Ian Titley et al. « The Sub-Clonal Complexity of STIL-TAL1 T-ALL ». Blood 124, n^o 21 (6 décembre 2014) : 3788. http://dx.doi.org/10.1182/blood.v124.21.3788.3788.

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Abstract Introduction The STIL-TAL1 fusion is found in 16% cases of paediatric and adolescent T-ALL, making it one of the most common T-ALL subgroups. Our study considers this leukaemia subtype in the context of a complex ecosystem that is diverse, evolving and subject to selective pressures. We used single cell methods to understand the order of co-operating mutational events and the clonal evolution of mutations in genes that are re-iteratively targeted, such as PTEN. Methods Diagnostic DNA from five STIL-TAL1 positive T-ALL cases was exome sequenced using Agilent SureSelect Human all Exon kit plus Illumina paired end sequencing. Driver copy number alterations and NOTCH1/PTEN exon 7 mutation status had been identified in a previous study and candidate driver mutations for inclusion in single cell experiments were validated by sequencing or Q-PCR using custom assays. Where more than one mutation was present within the same exon of a candidate driver gene, cloning experiments were carried out to verify the independent mutation sequences. Material from xenograft transplants was available in three of the five cases to assess their clonal heterogeneity in the leukaemia initiating cell compartment. Single cell multiplex Q-PCR was used to examine the single cell genetics of the pre-defined mutation events. Briefly, single cells were sorted and lysed prior to multiplex specific (DNA) target amplification and Q-PCR using the 96.96 dynamic microfluidic array and the BioMark HD (Fluidigm, UK). Copy number assays for the 1p33 deletion and custom assays for the patient specific STIL-TAL1 fusion breakpoints were used to confirm that the 1p33 deletion leading to this gene fusion was a clonal event. Results The only aberrant events common to all five samples were CKDN2A copy number loss and the 1p33 deletion that results in the STIL-TAL1 fusion. Exome sequencing revealed further mutations in known T-ALL drivers including NOTCH1, PTEN and PHF6 as well as candidate driver mutations in FREM2, PIK3CD, RPL14, BMPR1A and CDH18. Both NOTCH1 and PTEN demonstrated re-iterative inactivation and this was investigated in detail for PTEN. Case 1 had multiple PTEN exon 7 mutations and sub-clonal copy number loss. Case 2 had parallel frameshift mutations in PTEN exons 5 and 7. Case 3 contained an exon 8 mutation and multiple PTEN exon 7 mutations. In this case the three most frequent PTEN exon 7 indels were validated and tracked in a single cell multiplex Q-PCR experiment. This revealed a branching sub-clonal genetic architecture (see figure 1) in which all malignant cells at the proposed apex of the branching architecture harboured the STIL-TAL1 fusion and CDKN2A deletion with copy number losses of 4p, 6q and FREM2 and PTEN mutations occurring as sub-clonal events. PTEN indels 2 and 3 were found co-localised in the same sub-clone. Preliminary analysis of the paired mouse xenograft bone marrow did not detect PTEN exon 7 indels 1 – 3 in 84 single cells. However, bulk Sanger Sequencing analysis did identify the PTEN exon 8 mutation in the mouse. Ongoing work is in progress to determine whether single cells of the xenograft carry alternative PTEN exon 7 mutations detected in the diagnostic sample exome data and to characterise in which diagnostic sub-clone the PTEN exon 8 mutation resides. Conclusions This study demonstrates how exome sequencing and single cell multiplex Q-PCR can be used as complementary tools to understand the sub-clonal complexity of STIL-TAL1 T-ALL. PTEN inactivation is sub-clonal by single cell analysis, demonstrating the parallel evolution of multiple independent PTEN inactivated sub-clones, highlighting PTEN inactivation as a key event in this T-ALL subgroup. In a wider cohort of 20 patients collected by our group at least 50% had PTEN inactivation as assessed by sequencing of exon 7 and copy number data alone. Results indicate a strong evolutionary pressure selecting for mutational events that result in inactivation of the PTEN-PI3Kinase pathway. These events occur via multiple mechanisms, including copy number loss and truncating mutations, which are not limited to the known T-ALL hotspot in exon 7. Current work is focussing on using a similar approach to examine the clonal evolution of NOTCH1 mutations in STIL-TAL1 T-ALL samples in diagnostic and xenograft samples of cases 4 and 5. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Culen, Martin, Zdenka Kosarova, Ivana Jeziskova, Adam Folta, Nikola Tom, Ondrej Venglár, Dana Dvorakova, Jiri Mayer et Zdenek Racil. « Persistence of Mutations during Remission in 114 AML Patients ». Blood 132, Supplement 1 (29 novembre 2018) : 2796. http://dx.doi.org/10.1182/blood-2018-99-110586.

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Abstract Introduction: The current prognostic stratification of acute myeloid leukemia (AML) patients recognizes several recurrently mutated genes in AML as prognostic markers at the time of diagnosis. However, previous studies indicate that additional prognostic information can be further obtained by detection of pre-leukemic mutations that persist in clinical remission and represent clonal hematopoiesis. Aim: To analyze persistence of pre-leukemic mutations in AML patients at the time of disease remission and assess their prognostic relevance. Methods: Paired diagnosis and remission samples were analyzed for 114 AML patients; all carried at least 1 pre-treatment mutation at the time of diagnosis. Remission samples were collected 1 - 7 months from the start of conventional induction therapy (median 3 months, none of the patients received transplantation therapy prior to the sampling). All patients achieved at least hematological remission or deeper molecular remission that was identified by standard minimal residual disease monitoring for NPM1 or FLT3-ITD mutations, KMT2A-rearragements, or RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes. Sequencing of 47 exons in 19 genes was performed by targeted amplicon sequencing with ClearSeq AML Haloplex panel (Agilent) on MiSeq and NextSeq instruments (Illumina); MPL gene was excluded for inadequate coverage. Sequencing data were processed using VarDict, VarScan and Pindel algorithms. Mutation detection threshold was set to 2% variant allele frequency (VAF). Detection of CEBPA mutations was confirmed by sanger sequencing. Results: Pre-leukemic mutations persisting in remission were detected in 38/114 patients (33%). In 31/38 (82%) patients, the persistence was accompanied by loss of other mutation/s. Persisting mutations were most frequently detected in genes: DNMT3A (mutation persistence in 24 of 41 patients with pre-treatment DNMT3A mutation, 58%), IDH2 (5/19 patients, 26%), TET2 (4/12 patients, 33%) and ASXL1 (5/11 patients, 45%). On the contrary, complete clearance of all mutations in remission below 2% VAF was observed for the following genes: NPM1, FLT3, NRAS, CEBPA and IDH1, except for a single persisting FLT3-TKD mutation. The median VAF of persisting mutations was 18% (range 2 - 94%). Moreover, in almost half of the patients (17/38, 45%), VAF of persisting mutation exceeded 25% - thus more than half of the hematopoiesis was clonal at remission. Persistence of any mutation in remission was associated with impaired overall survival (OS) and event-free survival (EFS), compared to patients with no persisting mutations (2-year OS: 50% vs 81%, p=0.001; 2-year EFS: 28% vs 57%, p=0.0007). Persistence of 2 or more mutations at remission did not show further effect on OS or EFS compared to patients with exactly one persisting mutation. DNMT3A was the single persisting gene in remission in 20/38 (53%) patients, and was associated with shorter EFS but not OS compared to patients without any persisting mutations (2-year EFS: 26% vs 57%, p=0.03). Persistence of mutations in other genes than DNMT3A was detected in 14/38 (37%) patients and was associated with shorter OS but not EFS compared to patients without any persisting mutations (2-year OS: 37% vs 81%, p=0.001). Conclusions: One third of AML patients in our cohort carried pre-leukemic mutations in remission. These persisting mutations were retained at high VAFs and were limited to specific genes, while at the same time mutations in other genes were cleared by AML therapy. This supports the evidence that persisting mutations represented surviving clonal hematopoiesis, not minimal residual disease. The persistence of mutations in remission showed prognostic impact, however analysis of a larger cohort of patients is required to assess how to combine results from the mutational screening at diagnosis and remission to provide the best prognostic stratification. Supported by Ministry of Health of the Czech Republic, grant nr. 15-25809A. All rights reserved. This report was written with the support of the Specific University Research (nr. MUNI/A/0968/2017) provided by MEYS. Disclosures Mayer: Novartis: Research Funding; Eisai: Research Funding; Johnson & Johnson: Research Funding.

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Mansouri, Larry, Lesley-Ann Sutton, Viktor Ljungstrom, Sina Bondza, Linda Arngarden, Sujata Bhoi, Jimmy Larsson et al. « Recurrent Mutations within the Nfkbie gene : A Novel Mechanism for NF-κB Deregulation in Aggressive Chronic Lymphocytic Leukemia ». Blood 124, n^o 21 (6 décembre 2014) : 297. http://dx.doi.org/10.1182/blood.v124.21.297.297.

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Abstract Dysregulated NF-κB signaling appears to be particularly important in B-cell malignancies, with recurrent mutations identified within both the canonical and non-canonical NF-κB pathways, as well as in components of the B-cell receptor (BcR) and Toll-like receptor (TLR) signaling pathways. In chronic lymphocytic leukemia (CLL), although recurrent mutations have been identified in MYD88 (TLR signaling) and BIRC3 (non-canonical NF-κB pathway), their frequency is low (<3%) and hence the extent to which genetic aberrations may contribute to constitutional NF-κB activation remains largely unknown. To gain further insight into this issue, we designed a HaloPlex gene panel (Agilent Technologies) and performed targeted next-generation sequencing (NGS) (HiSeq 2000/Illumina) of 18 NF-κB genes in a discovery cohort of 124 CLL patients, intentionally biased towards poor-prognostic patients with either unmutated IGHV genes or high-risk genomic aberrations. Using a conservative cutoff of >10% for the mutant allele, we identified mutations (n=35) within 30/124 (24%) patients in 14/18 NF-κB genes analyzed. IκB genes, which encode for cytoplasmic inhibitor proteins, accounted for 20/35 (57%) mutations, with IκBε (encoded by NFKBIE) mutated in 8 patients; notably, 3/8 cases carried an identical 4bp deletion within exon 1 of NFKBIE. Prompted by these findings, we proceeded to validate our findings in an independent CLL cohort (n=168) using the same methodology as above and primarily focusing on cases with poor-prognostic features. We identified 30 mutations within 28 CLL patients in 11/18 NF-κB genes analyzed. Strikingly, 13/30 mutations were found within IκBε, with 10/13 patients carrying the same 4bp NFKBIE deletion. Notably, investigations into whether additional cases (within both the discovery and validation cohort) may harbor mutations of low clonal abundance (<10% mutant allele), led to the detection of the NFKBIE deletion in another 18 cases. Owing to the prevalence of this 4bp deletion within the NFKBIE gene, we developed a GeneScan assay and screened an additional 312 CLL cases. Collectively, 40/604 (6.6%) CLL patients were found to carry this frame-shift deletion within the NFKBIE gene, which is in line with a recent publication reporting that 10% of Binet stage B/C patients carried this mutation (Damm et al. Cancer Discovery 2014). Remarkably, the majority of these NFKBIE mutations (16/40) were found in a subgroup of patients that expressed highly similar or stereotyped BcRs and are known to have a particularly poor outcome, denoted as subset #1. This finding thus alludes to a subset-biased acquisition and/or selection of genomic aberrations, similar to what has been reported for subset #2 and SF3B1, perhaps as a result of particular modes of BcR/antigen interaction. We utilized proximity-ligation assays to test the functional impact of the NFKBIE deletion by investigating protein-protein interactions. This analysis revealed reduced interaction between the inhibitor IκBε and the transcription factor p65 in NFKBIE-deleted CLL cells; IκBε-knock-down shRNA experiments confirmed dysregulated apoptosis/NF-κB signaling. Finally, to assess whether the NFKBIE deletion could also be present in other B-cell malignancies, we screened 372 mature B-cell lymphoma cases using NGS or the GeneScan assay and found the deletion in 7/136 (5.1%) mantle cell lymphomas, 3/66 (4.5%) diffuse large B-cell lymphomas and 3/170 (1.8%) splenic marginal zone lymphomas. Taken together, our analysis revealed that inactivating mutations within the NFKBIE gene lead to NF-κB activation in CLL and potentially several other B-cell-derived malignancies. Considering the central role of BcR stimulation in the natural history of CLL, the functional loss of IκBε may significantly contribute to sustained CLL cell survival and shape the disease evolution. This novel data strongly indicates that components of the NF-κB signaling pathway may be prime targets for future targeted therapies not only in CLL but also other mature B-cell lymphomas. Disclosures No relevant conflicts of interest to declare.

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Nishijima, Dai, Mitsuko Akaihata, Yuka Iijima-Yamashita, Tomomi Yamada, Yuichi Shiraishi, Hiroko Tanaka, Toshinori Hori, Satoru Miyano, Keizo Horibe et Masashi Sanada. « Capture Sequencing Is a Useful Method for Comprehensive Clonality Analysis Based on Ig/TCR Gene Rearrangements in Acute Lymphoblastic Leukemia ». Blood 132, Supplement 1 (29 novembre 2018) : 1543. http://dx.doi.org/10.1182/blood-2018-99-115624.

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Abstract Introduction Immunoglobulin (Ig)/ T-cell receptor (TCR) gene rearrangements are the most widely used clonal marker to detect residual leukemic cells in patients with Acute Lymphoblastic Leukemia (ALL). Ig/TCR gene rearrangements based molecular minimum residual disease (MRD) monitoring has become one of the most powerful prognostic indicators for patients with ALL. Although the standard method of real-time quantitative PCR (RQ-PCR) provides very good sensitivity in MRD measurement, the workflow is very complicated and time-consuming, requiring expert technique and much work for operation, which limits the number of patients to be examined for MRD monitoring and the number of testable markers per patient. Material and methods To reveal clonal architecture and detect appropriate MRD marker, we designed capture probes covering the coding and recognition signal sequences of V, D, J genes of the Ig/TCR loci. We performed high-throughput target-capture sequencing in 208 pediatric cases with BCP-ALL and 35 pediatric cases with T-cell ALL, including 20 relapsed cases and 14 MRD marker negative cases. Extracted DNA samples were enriched with about 420 capture probes (Agilent Technology) and sequenced by HiSeq2500 platform (Illumina) in order to obtain enough sequence coverage (> 500 mean depth). Sequenced data were analyzed with Ig/TCR recombination analysis tool Vidjil (Giraud et al, 2014) and V(D)J recombination clones were listed according to a number of detected read for each clone. Results Total 2379 clonal Ig/TCR gene rearrangements (median 9 per patient, range 0-82) were detected by capture sequencing among 236 (97%) cases. A clonal IGH sequence with V(D)J recombination was identified in 91% of BCP-ALL cases, followed by TRG (68 %), IGK (67 %), TRA+D (66%), TRD (59 %), TRB (49%), and IGL (15 %), respectively. On the other hand, clonal TRG V(D)J recombination was detected in 74% of T-ALL cases, followed by TRB (69%), TRD (57%), IGH (26%), and TRA+D (6%), respectively. About half of BCP-ALL cases were identified two independent IGH rearrangements. These frequencies agree with previous reports obtained by PCR based experiments. In the cases in this study with well-characterized clonal Ig/TCR gene rearrangements by PCR and Sanger sequencing, our capture sequencing was able to detect all rearrangements used in MRD measurements. Although 8 BCP-ALL cases in this study were marker-negative in standard PCR-MRD diagnostics, clonal Ig/TCR gene rearrangements were identified for 5 out of 8 cases by capture sequencing. Some of the hidden clonal rearrangements showed specific and good quantitative amplification by RQ-PCR and can be used as sensitive PCR-MRD targets. On the other hand, all the MRD marker negative 6 T-ALL cases were not detected clonal Ig/TCR gene rearrangements. Finally, we compared the clonal architecture based on Ig/TCR gene rearrangements between diagnosis and relapse in relapsed B-ALL patients. Changes in the clonal architecture were associated with remission duration. In very early relapse cases, detected Ig/TCR rearrangements and their proportion at relapse are very similar to those at diagnosis. In early to late relapse cases, some major Ig/TCR gene rearrangements were lost at relapse and other minor rearrangements expanded at relapse. Most of the identified Ig/TCR gene rearrangements were different between at diagnosis and at relapse in a case relapsed after more than 10 years. Loss of rearrangements were commonly seen in TRA, TRB, and IgL, while most of the IgK and TRD rearrangements were steady during disease course. Conclusion Introducing target capture sequencing enables to high throughput sample preparation and automated data analysis. Capture sequencing is a useful method for comprehensive detection of Ig/TCR gene rearrangements and contributes to better understanding clonal architecture and detecting appropriate MRD markers in ALL patients. Disclosures No relevant conflicts of interest to declare.

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DONG, Gehong, Qiang Gong, Jinhui Wang, Xiwei Wu, Yuping Li, Leticia Quintanilla-Martinez Fend, Jingwen Wang, Hong-gang Liu, Timothy W. McKeithan et Wing Chung Chan. « Driver Mutations Affecting Natural Killer/T Cell Lymphoma ». Blood 128, n^o 22 (2 décembre 2016) : 4109. http://dx.doi.org/10.1182/blood.v128.22.4109.4109.

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Abstract Natural killer/T cell lymphoma (NKTCL) is an aggressive subtype of non-Hodgkin lymphoma that is rare overall but has a higher prevalence in Chinese and Hispanic populations. Recent sequencing efforts have improved the understanding of the disease and revealed a number of genes that may drive the pathogenesis of NKTCL. These efforts were largely limited by the number of cases studied. Based on previously reported whole exome sequencing data in NKTCL and other lymphoid malignancies and on the frequency and potential biological significance of the mutant, we designed a 334-gene panel and sequenced 105 NKTCLs. We characterized single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations (CNAs) that may have pathogenic roles. Tumor DNA was isolated from 74 Chinese and 31 Hispanic NKTCL patients. For 7 of these, matched normal DNA was extracted from peripheral blood. The 334 genes in the custom panel were captured using the Agilent SureSelect platform, and the exons of these genes were sequenced on an Illumina HiSeq 2500 instrument. For cases without matched normal samples, we discriminated between somatic and germline variants with an in-house machine learning algorithm which is based on information of public variant databases, variant annotations, and sequencing statistics, and followed by manual inspection. CNAs were determined based on the untargeted sequences using an R package, CopywriteR. We identified a total of 479 SNVs and 113 indels in 155 genes in 102 out of 105 patients. Mutations in STAT3 and JAK3 were identified in 30% and 7% of the cases, respectively. The hotspot mutations Y640F, S614R, and G618R in the SH2 domain of STAT3 and A573V of the JH2 domain of JAK3 were observed in most of these cases. 46% of the cases harbored one or more mutations in epigenetic regulators ARID1A, EP300, MLL2, MLL3, and TET2. Transcription co-repressors BCOR and NCOR2 were mutated in 25 of the cases. 13% of the cases contained mutations in HLA-A or CIITA, most of which were inactivating, suggesting possible defects in immune surveillance. DDX3X and TP53 mutations were detected in 19% and 10% of the cases, respectively. TP53 mutations were mutually exclusive with mutations in DDX3X and, except for one case, with STAT3 also. MGA mutations, which were present in 12% of the cases, were usually concurrent with STAT3 mutations. The mutational profiles of Chinese and Hispanic cases were similar, and only CD58 mutations were significantly more common in Hispanic patients (p=0.03). Comparison between NKTCL with localized and systemic presentation demonstrated that CCR7 was mutated more frequently in systemic cases (p=0.02). Prognostic analysis identified EP300 mutations as a potential marker for poorer survival (p=0.01). Frequent copy loss on chromosome 6q was observed and PRDM1, which is present on 6q, was mutated in 10% of the cases. In summary, we identified the profile of mutated genes in a large series of NKTCL. Most of the genes have been previously reported to be mutated in NKTCL, but the frequencies for some of the mutants were quite different. Interestingly, the mutational profiles of NKTCL in Chinese and Hispanic patients were very similar despite the geographic differences. Mutations leading to abnormal activation of the JAK-STAT3 pathway and abnormal chromatin modification were highly prevalent. BCOR, DDX3X and TP53 mutations were also frequent. Variants in CCR7 and EP300 were potential markers for systemic disease and poor prognosis, respectively. Figure. Mutation profiles of genes with mutation frequency larger than 5%. Genes that are not expressed in normal NK cells were excluded. Figure. Mutation profiles of genes with mutation frequency larger than 5%. Genes that are not expressed in normal NK cells were excluded. Figure. Overall survival curves of cases with and without mutations in EP300. Figure. Overall survival curves of cases with and without mutations in EP300. Disclosures No relevant conflicts of interest to declare.

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Gerrard, Gareth, Mikel Valgañón, Hui En Foong, Dalia Kasperaviciute, Michael Müller, Laurence Game, Deena Iskander et al. « Target Enrichment and High-Throughput Sequencing of 80 Ribosomal Protein Genes to Identify Mutations Associated with Diamond-Blackfan Anaemia. » Blood 120, n^o 21 (16 novembre 2012) : 2369. http://dx.doi.org/10.1182/blood.v120.21.2369.2369.

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Abstract Abstract 2369 Diamond-Blackfan anaemia (DBA) is a rare autosomal dominant disorder associated with inactivating mutations in ribosomal protein (RP) genes, causing defects in erythroid progenitor and precursor cell development. Many cases are due to de novo mutations and in family cases there is often clinical heterogeneity due to variable penetrance. Mutations in RPS19 account for 25% of all DBA cases and single nucleotide variations (SNV), indels and allele-loss deletions have been found in 11 other RP genes in a further ∼50% of patients. Around 25% of patients with DBA have no identifiable mutations. Given that (with the exception of 2 cases with GATA1 mutations) all mutations in DBA characterised so far affect RP genes, it is likely that mutations in one of the 80 RP genes will be eventually identified in a significant proportion of the patients. Current screening methods are primarily based on Sanger sequencing on a per-exon/per-gene basis, with the associated time, labour and cost restrictions. We therefore aimed to evaluate high-throughput sequencing technology, including a bespoke target enrichment platform, to screen all 80 known RP genes to facilitate rapid, cost-effective identification of DBA associated mutations. DNA was extracted from peripheral blood samples that had been referred to Imperial Molecular Pathology for DBA screening from 10 individuals, including 3 family pairs: affected mother and daughter; 2 affected siblings; and another sibling pair, one of whom was unaffected/low-penetrance (no defining clinical symptoms, except for high adenine deaminase). Only one patient had a known mutation (RPS19 c.280C>T) and was included as a control. Agilent SureSelect XP was used for the target enrichment, which employed a custom designed tiled-RNA bait hybridisation solution to capture the target genes, including non-masked intronic regions and 500bp of flanking sequence. The DNA was sheared using a Covaris e220, QC was performed via QIAxcel capillary electrophoresis and the hybridisation was carried out at 65°C for 48h. Individual libraries were quantified using qPCR against the supplied standard curve and pooled proportionally. The sequencing was performed on an Illumina MiSeq, using 150bp paired-end reads and multiplexed using the supplied ScriptSeq barcodes. The sequencing reads were aligned to the build 37 reference genome using BWA software, and the variant calls made using GATK. Annovar was used for functional annotations of the variants. Protein truncating mutations were found in RP genes in 7 of the 10 samples, including the positive control and 6 of the 8 clinically confirmed DBA patient samples., All mutations were in RP genes previous described as being involved in DBA, although 3 affected novel codons: RPL5 c.G244T (stop-gain SNV; novel; mother-daughter pair); RPL5 c.166_169delACAA (frameshift); RPS10 c.C337T (stop-gain SNV); RPL11 c.472–473delAA (frameshift; novel); RPS26 c.212–213insA (frameshift; novel). Validation was by Sanger sequencing and further confirmation testing will include unaffected family members. The remaining 2 DBA patients, a brother-sister pair, showed no definable mutations in the captured regions and neither did the unaffected/low-penetrance sibling of the RPS10 patient. In summary, a rapid and cost effective methodology for screening genetic lesions associated with the causation of DBA is warranted, especially given the magnitude of attaining global coverage by conventional techniques. Whole-gene enrichment followed by multiplexed runs on a bench-top class high-throughput sequencing platform is arguably the approach of choice; although as the cost of exome and even genome sequencing continues to fall, these may well become realistic options in the coming few years. This work is ongoing, with a second group of 10 samples already sequenced and undergoing analysis, and bioinformatic refinements, especially for the detection of larger deletions, may yet yield results for the two undetected samples. These preliminary results suggest that high throughput sequencing technology with a bespoke target enrichment platform for RP genes is a feasible, efficient and relatively rapid diagnostic tool for detection of causative mutations DBA. Disclosures: No relevant conflicts of interest to declare.

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Heuck, Christoph, Niels Weinhold, Erich Allen Peterson, Michael Bauer, Caleb K. Stein, Timothy Ashby, Shweta S. Chavan et al. « The Impact of Combination Chemotherapy and Tandem Stem Cell Transplant on Clonal Substructure and Mutational Pattern at Relapse of MM ». Blood 126, n^o 23 (3 décembre 2015) : 372. http://dx.doi.org/10.1182/blood.v126.23.372.372.

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Abstract Introduction: Next generation sequencing of over 800 newly diagnosed multiple myeloma (NDMM) cases has established the mutational landscape and key cancer driver pathways. The mutational basis of relapse has not been systematically studied. Two previous studies (Keats et al.; Bolli et al.) identified 4 patterns of clonal evolution. Neither study included uniformly treated patients and looked at the impact of therapy on clonal structure at relapse. Understanding the mutational patterns underlying relapse and how they relate to specific therapies is crucial in order to improve MM outcomes, especially for high-risk (HR) MM. In this study we compare the clonal structure at presentation (PRES) and at relapse (REL), after exposure to Total Therapy (TT). Materials and Methods: We studied 33 pairs of tumor samples collected at PRES and REL. 9 patients were treated on TT2, 13 on TT3, 10 on TT4 and 1 on TT5-like regimen. Eleven patients had HR disease at PRES. DNA was extracted from CD138+ selected cells from random bone marrow aspirates. Germline controls were obtained from leukapheresis products. Whole exome sequencing libraries were prepared using the Agilent qXT kit and the Agilent SureSelect Clinical Research Exome kit with additional baits covering the Ig and MYC loci. All samples were sequenced on an Illumina HiSeq2500 to a median depth of 120x. Sequencing data were aligned to the Ensembl GRCh37/hg19 human reference using BWA. Somatic variants were called using MuTect. Translocations were identified using MANTA. Copy number variations were inferred using TITAN. Gene expression profiles (GEP), generated using the Affymetrix U133plus2 microarray, were available for all tumor samples. Nonnegative matrix factorization (NMF) was used to define mutation signatures. Results: The median time to progression was 30 months with a median follow up of 9.5 years. 22 cases achieved a complete remission (CR) or near CR. There were 11 cases of HR at PRES. Of the 22 cases with low risk (LR) MM, 7 relapsed with HR disease. There were on average 478 SNVs per sample at PRES and 422 at REL. All but 2 cases had evidence of new mutations at REL. There were no consistent patterns or number of mutation associated with REL or GEP-defined risk. Patients of the MF molecular subgroup had more mutations compared to other molecular subgroups (657 vs. 379) and were enriched for mutations with an APOBEC signature. We did not detect any mutation signature consistent with chemotherapy-induced alterations, providing evidence that TT itself does not cause additional mutations. Primary recurrent IgH translocations called by MANTA were confirmed by GEP data. A number of new translocations were identified , several only at REL. In particular we demonstrate a case with a newly acquired MYC translocation at relapse, indicating that it contributed to progression. We identified 5 patterns of clonal evolution (Figure 1): A) genetically distinct relapse in 3 patients, B) linear evolution in 8 patients, C) clonal selection in 9 patients, D) branching evolution in 11 patients, and E) stable clone(s) in 2 patients. Patterns A (distinct) and B (linear) were associated with low risk and longer survival, whereas patterns D (branching) and E (stable) were associated with high risk and shorter time to relapse and overall survival (Table 1). Conclusion: This is the first study to systematically analyze the pattern of clonal evolution using NGS in patients treated with combination chemotherapy and tandem ASCT. We identified 5 patterns of evolution, which correlate with survival. We identified 3 cases with a loss of the original clone and emergence of a new clone, suggesting high effectiveness of Total Therapy for those patients. The persistence of major clones despite multi agent chemotherapy in most other cases supports a concept of a tumor-initiating cell population that persist in a protective niche, for which new therapies are needed. Table 1. Pattern of Evolution GEP70 Pres.(high risk: ≥0.66) Proliferation Index Pres. GEP70 Rel.(high risk: ≥0.66) Proliferation Index Rel Mean OS Mean TTR A: distinct (n=3) -0.690 -3.34 -0.015 2.04 8.18 5.00 B: linear (n=8) -0.171 -0.34 0.618 9.22 5.70 4.05 C: selection (n=9) 0.366 3.20 0.569 6.97 3.95 2.64 D: branching (n=11) 0.710 5.17 1.173 11.15 3.84 2.21 E: stable (n=2) 1.532 7.42 1.124 2.54 0.96 0.35 Pres.: Presentation; Rel.: Relapse; OS: Overall Survival; TTR: Time to Relapse Figure 1. Patterns of Relapse Figure 1. Patterns of Relapse Disclosures Heuck: Foundation Medicine: Honoraria; Millenium: Other: Advisory Board; Janssen: Other: Advisory Board; Celgene: Consultancy; University of Arkansas for Medical Sciences: Employment. Weinhold:Janssen Cilag: Other: Advisory Board; University of Arkansas for Medical Sciences: Employment. Peterson:University of Arkansas for Medical Sciences: Employment. Bauer:University of Arkansas for Medical Sciences: Employment. Stein:University of Arkansas for Medical Sciences: Employment. Ashby:University of Arkansas for Medical Sciences: Employment. Chavan:University of Arkansas for Medical Sciences: Employment. Stephens:University of Arkansas for Medical Sciences: Employment. Johann:University of Arkansas for Medical Sciences: Employment. van Rhee:University of Arkansa for Medical Sciences: Employment. Waheed:University of Arkansas for Medical Sciences: Employment. Johnson:University of Arkansas for Medical Sciences: Employment. Zangari:University of Arkansas for Medical Sciences: Employment; Millennium: Research Funding; Onyx: Research Funding; Novartis: Research Funding. Matin:University of Arkansas for Medical Sciences: Employment. Petty:University of Arkansas for Medical Sciences: Employment. Yaccoby:University of Arkansas for Medical Sciences: Employment. Davies:University of Arkansas for Medical Sciences: Employment; Millenium: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Celgene: Consultancy. Epstein:University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Morgan:Weismann Institute: Honoraria; MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; University of Arkansas for Medical Sciences: Employment; CancerNet: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Malecka, Agnieszka, Gunhild Trøen, Anne Tierens, Ingunn Østlie, Jedrzej Malecki, Ulla Randen, Sigbjørn Berentsen, Geir E. Tjønnfjord et Jan M. A. Delabie. « High Frequency of Somatic Mutations of KMT2D and CARD11 Genes in Cold Agglutinin Disease ». Blood 128, n^o 22 (2 décembre 2016) : 2934. http://dx.doi.org/10.1182/blood.v128.22.2934.2934.

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Abstract Primary cold agglutinin disease (CAD) is a hemolytic anemia mediated by monoclonal anti-I autoantibodies. CAD is caused by an underlying low grade B-cell lymphoproliferative disease of the bone marrow with a typical histology that is different from lymphoplasmacytic lymphoma and, accordingly, does not display the MYD88 L265P mutation (Randen et al., Haematologica, 2014). Since CAD is a clonal lymphoproliferative disorder, we studied the mutational landscape to further characterize the disease and identify potential novel treatment approaches. We prospectively collected bone marrow samples of CAD patients, enrolled in a clinical trial (CAD5; www.clinicaltrials.gov, NCT02689986). Exome sequencing of six cases was performed and findings were confirmed in ten additional cases using targeted sequencing. For these analyses, clonal B cells and normal T cells, used as control, were purified from bone marrow samples using fluorescent activated cell sorting. All mutations were verified by Sanger sequencing. Whole-exome sequencing was performed at BGI Tech Solutions (Hongkong) using the Agilent SureSelect Human All Exon V4 Reagent Kit and Illumina HiSeq technology. The bioinformatics pipeline consistent of BWA alignment tool (aligned to hg19); Picard tools FixMateInformation and MarkDuplicates; the Genome Analysis Toolkit (GATK) IndelRealigner and BaseRecalibrator; somatic variant detection tools Strelka, MuTect and Pindel; the annotation tool SnpEff. Recurrent somatic mutations were found in KMT2D (11/16 cases, 69%) and CARD11 (5/16, 31%) (Table 1, Figure 1-2). 7/16 (44%) of KMT2D mutations were deemed high impact mutations by SnpEff and to result in inactive protein. 2/16 (12,5%) of KMT2D mutations are missense mutations and are predicted to impair SET domain function. 2/16 (12,5%) of KMT2D mutations are classified by SnpEff as low impact mutations of which functional tests are necessary to demonstrate potential consequences for protein function. Of interest, two additional patients showed rare germline KMT2D variants that have also been seen in Kabuki syndrome patients, although these patients do not have Kabuki syndrome. CARD11 was somatically mutated in 5/16 (31%) cases. Four of those patients had a concurrent KMT2D mutation. All CARD 11 mutations were classified as moderate impact mutations by SnpEff. Mutations were tightly clustered in a 20bp sequence of the coiled-coil domain sequence (Table 1, Figure 2). KMT2D is a histone lysine methyl transferase that represses B cell lymphoma development. Mono-allelic mutations of KMT2D seem to act in a dominant fashion and cause partial loss of protein expression with cell growth advantage. KMT2D is frequently mutated in follicular lymphoma, diffuse large B cell lymphoma and nodal marginal zone lymphoma. Mono-allelic constitutional mutations cause Kabuki syndrome, characterized by distinct facial characteristics and multiple organ malformations. Patients frequently develop immune-mediated thrombocytopenia as well as auto-immune hemolytic anemia. CARD11 coiled-coil domain mutations result in constitutive NF-kB activation and enhanced NF-kB activity upon antigen receptor stimulation. Mutations were previously detected in diffuse large B cell lymphomas of activated B cell origin. Mono-allelic CARD11 coiled coil mutations are not oncogenic per se in mice and humans, but result in B-cell proliferation and auto-antibody production. Since four CAD patients showed concurrent KMT2D and CARD11 mutations, it seems likely that the mutations act in concert with anti-I B-cell receptor stimulation to contribute to CAD-associated lymphoproliferative disease. In conclusion, we demonstrated a high frequency of KMT2D and CARD11 mutations in the bone marrow B cell lymphoproliferative disease of patients with CAD. These results confirm that CAD-associated B cell lymphoproliferative disease is a distinct disease different from other known B cell lymphoproliferative diseases of the bone marrow, most notably lymphoplasmacytic lymphoma. The identification of these recurrent mutations in CAD may allow the design of novel treatment modalities. Table 1 Mutations in KMT2D and CARD11 gene in CAD. Table 1. Mutations in KMT2D and CARD11 gene in CAD. Figure 1 Mutations within KMT2D gene and protein detected in CAD patients. Figure 1. Mutations within KMT2D gene and protein detected in CAD patients. Figure 2 Mutations within CARD11 protein detected in CAD patients. Figure 2. Mutations within CARD11 protein detected in CAD patients. Disclosures No relevant conflicts of interest to declare.

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McKerrell, Thomas D., Ignacio Varela, Nicolo Bolli, Postingl Hannes, Zemin Ning, German Tischler, Anthony Bench et al. « R.I.S.C.L : A Holistic Molecular Diagnostic Tool for Myeloid Malignancies ». Blood 124, n^o 21 (6 décembre 2014) : 2342. http://dx.doi.org/10.1182/blood.v124.21.2342.2342.

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Abstract The genomic landscapes of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD) and other related myeloid malignancies are now amongst the best characterized cancer genomes. These malignancies share most of their somatic driver mutations, many of which have therapeutic and prognostic significance (Patel et al, NEJM 2012). Patient prognostication and clinical decision-making can be greatly facilitated by testing for these mutations in parallel with established diagnostic assays. Here, we describe and validate RISCL (Rearrangements, Indels, Substitutions, Copy number and Loss-of-heterozygosity), a novel methodological and bioinformatic tool for the molecular diagnosis of myeloid malignancies. This tool employs targeted DNA capture to simultaneously: 1) identify coding mutations in 49 genes, 2) detect the four most important translocations in AML and 3) derive genome-wide copy number and zygosity data. Samples & methods 1. Samples Genomic DNA was extracted from bone marrow samples of 62 patients with AML (n=86 samples, including 24 remission samples) and 68 patients with MDS; and from blood granulocytes and mononuclear cells from 5 cord blood samples and 18 adults with normal hematopoiesis. 2. cRNA baits and sequencing The bait library (Agilent) contained 53,613 probes to capture: 1) all exons from 49 genes 2) intronic breakpoint sites for PML-RARA, CBFb-MYH11 and RUNX1-RUNX1T1 and MLL breakpoints 3) 9958 SNPs (minor allele frequency 0.40-0.45) for genome wide copy number and zygosity analysis. Barcoded sequencing was performed using Illumina HiSeq 2000 (100bp paired-end). 3. Bioinformatic analysis We used bespoke bioinformatics for detecting coding substitutions and indels (MIDAS; Conte et al, Leukemia 2013), chromosomal translocations (SMALT-FIT), copy number analysis (Avadis software) and detection of specific mutations such as MLL-PTD and FLT3-ITD (in-house scripts). 4. Verification of results To validate the sensitivity and specificity of our approach, we compared our findings to conventional diagnostic data and are also validating 30% of randomly selected variants. Results A mean of 94% of targeted bases were covered at least by 30x. In AML samples, the four most common coding mutations identified affected NPM1 (n=10), CEBPA (n=8), IDH1 (n=8) and NRAS (n=7). By comparison to conventional diagnostics, we detected 5/5 IDH1R132, 4/4 CEBPA, 1/1 IDH2R172K and 8/9 NPM1 mutations. In MDS samples, the top four mutations affected TP53 (n=15), TET2 (n=13), SRSF2 (n=9), ASXL1 (n=8) and mutations affecting spliceosome genes (n=18) that were mutually exclusive, as previously described (Yoshida et al, Nature 2011). RISCL detected 100% of known translocations (28/28) in AML patients, namely CBFb-MYH11 (n=8/8), PML-RARA (n=9/9), RUNX1-RUNX1T1 (n=4/4) and rearrangements of MLL (n=7/7). In every case of MLL rearrangement the gene partner was identified and in one case with t(X;11) we identified a novel gene partner to MLL, DIAPH2. Furthermore, we identified one patient with an MLL rearrangement not identified at diagnosis. Copy number analysis efficiently detected known large chromosomal deletions or monosomies in chromosome 5 (18/18) and 7 (10/12). Overall 47/54 large deletions were detected using Avadis software. Furthermore in one MDS patient we were able to detect a submicroscopic heterozygous deletion in chromosome 4 which included TET2. However this method was much less sensitive for detecting trisomies (13/27 trisomies detected overall). The reasons for this disparity between detection of deletions and amplifications using a standardized depth of coverage algorithm are unclear, but may include subclonal mutations, selection of karyotypically abnormal cells during metaphase preparation or limitations of our bioinformatic analysis, which we are currently investigating. Figure 1 shows an example of the results of our holistic analysis using RISCL from an informative case of AML. In summary we describe RISCL, a novel powerful holistic NGS tool for detailed characterization of myeloid malignancies that can be used for patient stratification and a personalized approach to malignancy in the molecular era. The same approach can be extended to other malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Romasko, Edward J., Sawona Biswas, Batsal Devkota, Jayaraman Vijayakumar, Sowmyra Jairam, Christopher S. Thom, Matthew C. Dulik et al. « Utility of Whole Exome Sequencing in Diagnosis of Pediatric Platelet Disorders : A Subanalysis of the Pediseq Study ». Blood 128, n^o 22 (2 décembre 2016) : 3726. http://dx.doi.org/10.1182/blood.v128.22.3726.3726.

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Abstract Background: Inherited Platelet Disorders (IPD) are individually rare disorders that have many different molecular causes. Diagnosis of IPD is often complicated by the need for complex testing that is not readily available at many centers and the lack of available testing to define the molecular cause of some disorders. While some platelet disorders are sufficiently defined by functional characterization, recent data suggests that some platelet disorders may predispose to significant other complications including cancer predisposition, myelofibrosis or hearing loss. Therefore, it may be important to establish a molecular diagnosis to better counsel families about necessary follow up and possible risks. The goal of this study was to determine the diagnostic yield of whole exome sequencing in a cohort of 22 pediatric patients with clinical presentation suggesting an underlying genetic cause (or positive family history of platelet disorder with no prior genetic diagnosis). Methods: Peripheral blood was collected from patients identified as likely to have an inherited platelet disorder after informed consent. Samples were also obtained from parents and siblings for co-segregation and variant calling. Genomic DNA was extracted manually using the Gentra Puregene Blood Kit. Exome capture was performed using the Agilent SureSelect v4 and 100 base paired end sequencing was done on an IlluminaHiSeq 2000 with 100X average coverage. Sequencing reads were generated in FASTQ format and mapped to human genome GRCh37 (hg19) and Novoalign v2.08 was used for optimal alignment. Disease-related variants were extracted from HGMD to identify variants that might be missed. Variant filtering and pathogenicity classification was performed using a customized pipeline and manual curation. We identified 53 genes of interest and on average across all exomes with an indication for a platelet disorder, bases were sequenced at a minimum depth of 15X to be considered covered within an exon. 80.4% of exons were 100% covered with this technology completely, while 10.4% of exons were partially covered (>40 to <100% bases) and 9.2% of exons were not covered (<40% of bases covered at a minimum of 15X depth). Results: 22 patients were enrolled over a 12-month period. Overall, 82% of patients had variants identified in platelet related genes on whole exome sequencing with 64% of patients returning at least one variant of uncertain significance (14) and 23% (5) patients returning definite positive results. One patient referred for further work up carried the initial diagnosis of ITP, but had macrothrombocytopenia since early childhood and bleeding out of proportion to the platelet count. Flow cytometry and functional studies performed on referral suggested possible Bernard Soulier Syndrome and sequencing confirmed homozygous pathogenic mutation in GP9. One patient with congenital thrombocytopenia and history of intracranial hemorrhage with a similarly affected sibling had confirmed pathogenic MYH9 mutation, allowing clinicians to offer prenatal diagnosis during a third pregnancy. One patient with a significant bleeding phenotype was a compound heterozygote for two novel RASGRP2 variants, but the functional significance of those variants is uncertain and further studies are underway to determine whether these variants are causative. 18% of patients (4) had negative sequencing results (no reportable variants in platelet related genes identified). Conclusions: Whole exome sequencing can be a powerful diagnostic tool in identifying the molecular cause of disease in a cohort of patients with suspected inherited platelet disorders. The majority of patients, however, will receive results of uncertain significance and centers that undertake this testing will require an infrastructure to allow for further functional evaluation, which will help in reclassification of these variants, and ensure that results are correctly interpreted. Clinicians who undertake ES to diagnose IPD need to understand limitations of the test as well as the full significance of results that may be returned. Disclosures Lambert: Novartis: Consultancy.

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Vrzalova, Zuzana, Katerina Stano Kozubik, Lenka Radova, Jakub Trizuljak, Sarka Pospisilova et Michael Doubek. « Characterization of Pathogenic Variants Associated with Hereditary Thrombocytopenias in Families from the Czech Republic ». Blood 134, Supplement_1 (13 novembre 2019) : 2343. http://dx.doi.org/10.1182/blood-2019-122696.

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Introduction Inherited thrombocytopenias (IT) are a heterogeneous group of 33 different forms of monogenic disorders caused by molecular defects affecting 40 genes at least. The pathogenic germline variants play an important role in the development and maintenance of hematopoietic system (megakaryopoesis and thrombopoesis). These changes lead to disruption of these processes and are presented as the thrombocytopenia phenotype (low platelet count, blood-examination). However, patients are occasionally misdiagnosed with the immune thrombocytopenia and unsuccessfully treated with steroid therapy and splenectomy. In some patients, accurate diagnosis of IT can only be established based on the results of molecular genetic testing. Furthermore, it has also been shown that some hematological conditions with Mendelian type of hereditability precede the development of hematooncological disease. Patients and Methods DNA samples from peripheral blood or buccal swabs of four unrelated families were isolated. The whole exome sequencing (WES) was performed using the NextSeq 500 Illumina instrument with adequate chemistry and sequencing libraries were prepared according to the SeqCap EZ Human Exome Probes v3 protocol. The generated data were processed using in-house bioinformatics pipelines. The detected pathogenic variants were confirmed by Sanger sequencing. Moreover, the novel variant was analyzed in silico using analytical procedures including protein modelling, too. Germline DNA analysis was performed on all available samples and somatic DNA analysis was done for the oncological patient. Within each family, the obtained pathogenic variants were compared between the individuals with IT phenotype and their disease-free relatives. Results The pathogenic variants were characterized in four families with different forms of IT. Moreover, the additional genetic variants were detected in three of them which predispose to the development of hematological malignancies. In the first family, a novel heterozygous variant c.320C>T; p.(Thr107Met) in TUBB1 gene is probably responsible for essential thrombocytopenia disease because all rare TUBB1 variants until now have been detected in patients with macrothrombocytopenia. The known pathogenic variant c.1402G>T; p.(Val468Phe) in JAK2 gene (10.9% frequency) was identified in a family member suffering from the myeloproliferative disease. In the second family, heterozygous pathogenic variants c.3076C>T; p.(Arg1026Trp) in ITGA2B gene and c.3188G>A; p.(Arg1063His) in JAK2 gene were detected, associated with platelet-type bleeding disorders and hereditary erythrocytosis with megakaryocytic atypia and predisposition for hematological malignancy, respectively. It is known that stomach tumor occurred in patient´s family before. In the third family, heterozygous pathogenic variant c.3493C>T; p.(Arg1165Cys) in MYH9 gene was identified in a patient with macrothrombocytopenia. This variant was associated with Sebastian syndrome, macrothrombocytopenia and granulocyte inclusions and predisposition to kidney failure, hearing loss, and cataracts. In the fourth family, ANKRD26-related thrombocytopenia with predisposition to myeloid malignancy was probably identified in a patient with detected heterogeneous known variant c.-140C>G in 5´ UTR of ANKRD26 gene. Moreover, the novel c.682C>T; p.(Arg228Trp) variant in SYTL3 gene with uncertain significance was detected in this patient. Conclusions The pathogenic variants were detected in unrelated affected families with macrothrombocytopenia, platelet-type bleeding disorders and hereditary erythrocytosis with megakaryocytic atypia, Sebastian syndrome, and ANKRD26-related thrombocytopenia. Moreover, the genetic variants predispose to myeloid malignancy were identified. Molecular genetic testing helped the clinicians to determine the correct diagnosis in these patients. This study was supported by Ministry of Health of the Czech Republic (grant No 16-29447A), and TA CR (TE02000058). Disclosures No relevant conflicts of interest to declare.

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Vantyghem, Sophie, Pierre Peterlin, Sylvain Thepot, Audrey Ménard, Viviane Dubruille, Camille Debord, Thierry Guillaume et al. « Multicentric Real Life Evaluation of the Impact of Next-Generation Sequencing on the Clinical Management of Chronic Myeloid Malignancies ». Blood 134, Supplement_1 (13 novembre 2019) : 5771. http://dx.doi.org/10.1182/blood-2019-122958.

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Introduction Next generation sequencing (NGS) has allowed to improve knowledge about the genomic landscape of hematological malignancies. Somatic mutations (SM) are valuable new biomarkers but the utility of incorporating routine sequencing to guide diagnosis and therapeutic decisions remains challenging. We report here an observational multicentric study aimed at assessing the impact of SM testing by NGS in a real-life setting on the diagnosis and treatment of chronic myeloid malignancies (CMM). Patients and Method All patients who benefited from molecular assessment, between 10/2014 and 03/2019 in our University Hospital were included. All provided informed consent for data collection. All NGS requests were validated during a regional multidisciplinary concertation meeting. A custom targeted panel of 34 genes (145kbp i.e. ASXL1,BCOR, BCORL1, CBL, CSF3R, DNMT3A, ETV6, EZH2, GATA2, IDH1, IDH2, JAK2, KDM6A, KIT, KRAS, MPL, NPM1, NRAS, PIGA, PTEN, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TNFAIP3, TP53, U2AF1, ZRSR2) was applied on DNA extracted from peripheral blood or bone marrow samples. DNA libraries, built with the Haloplex® target enrichment protocol (Agilent Technologies, Santa Clara, CA), were paired-end sequenced (150bp reads) with a MiSeq® Instrument (Illumina, San Diego, CA). Data analysis used an in-house pipeline including three variant callings (GATK HaplotypeCaller, VarScan and SAMTools). In a first group (A), NGS indication was to search for clonal hematopoiesis (CH), defined by the presence of at least one SM, in order to confirm or rule out a diagnosis of Idiopathic Cytopenia of Undetermined Significance (ICUS), Clonal Cytopenia of Undetermined Significance (CCUS), myelodysplastic syndrome (MDS), mixed myelodysplastic/myeloproliferative neoplasm (MDS/MPN), aplastic anemia (AA)/hypoplastic myelodysplasia (hMDS) or myeloproliferative neoplasm (MPN), based on recommendations of the WHO classification. In a second group (B), the theranostic impact of SM was studied. Prognostic SMs according to Bejar (2011) were used for MDS and MDS/MPN excluding chronic myelomonocytic leukemia that were analyzed with Itzykson score (2013) and/or CPSS-Mol score (Elena 2016). Prognostic SMs according to Vannucchi (2013) were used for myelofibrosis. Results The median age of the cohort was 60 years old (range: 10-87) with a median follow up of 1.1 years from molecular assessment to last follow-up. Within group A (94 patients), the most frequent blood count anomalies were cytopenia (68%), thrombocytosis (16%), and monocytosis (13%). The karyotype was normal in 77% and failed in 5% of the cases. Non-specific abnormalities (i.e. loss of chr Y, del 20q), were found in 8% of the cases. Before molecular assessment, the diagnoses proposed were ICUS (n=37), suspicion of MDS/MPN (n=16), AA/hMDS (n=16), or MPN (n=25). CH was detected in 31 patients comforting the diagnosis of CMM for 33% of group A (8 CCUS, 3 MDS, 7 MDS/MPN, 6 medullary hypoplasia, 7 MPN) patients. Considering the patients for whom no CH was detected (n=63), the initial suspected diagnosis of CMM was ruled out in 47 patients (i.e. 50% of group A). For the 16 remaining (i.e. 17% of group A), no firm diagnosis could be retained. Within group B (95 patients), NGS identified prognosis SM in 33% of the patients, i.e. poor prognosis SM in 24, including 8/40 MDS, 10/29 MDS/MPN and 6/17 myelofibrosis and good prognosis SM(SF3B1) in 7 of them, respectively 6/40 MDS and 1/29 MDS/MPN. Prognostic SMs had a therapeutic impact in 18/95 pts (19%). Indeed 13 patients with poor prognosis SM had a therapeutic change including 12 allogeneic stem-cell transplantation and 1 hypomethylating agent. Conversely, 5 patients with a good prognosis SM or absence of poor prognosis SM had a de-escalation of treatment intensity. Conclusion The use of NGS in daily practice had a clinical impact in both diagnostic and therapeutic decisions provided that the prescription is made in a critically explored context and not as a systematic test. In this "real life" cohort, the presence or absence of SM was a useful complement for integrated diagnoses in 83% of the patients, allowing to confirm (33%), or exclude (50%) a suspected condition. Moreover, in this cohort 34% of the patients had a SM with a reported prognostic impact and the treatment was modified in 19% of the cases. Yet, it remains necessary to integrate these results with other diagnostic criteria. Disclosures Peterlin: AbbVie Inc: Consultancy; Jazz Pharma: Consultancy; Astellas: Consultancy; Daiichi-Sankyo: Consultancy. Moreau:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Le Gouill:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche-Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support. Chevallier:Daiichi Sankyo: Honoraria; Incyte: Consultancy, Honoraria; Jazz Pharmaceuticals: Honoraria.

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Fahimi, Hossein, Samira Behroozi, Sadaf Noavar et Farshid Parvini. « A novel recessive PDZD7 bi-allelic mutation in an Iranian family with non-syndromic hearing loss ». BMC Medical Genomics 14, n^o 1 (2 février 2021). http://dx.doi.org/10.1186/s12920-021-00884-4.

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Abstract Background Autosomal recessive non-syndromic hearing loss (ARNSHL) is genetically and phenotypically heterogeneous with over 110 genes causally implicated in syndromic and non-syndromic hearing loss. Here, we investigate the genetic etiology of deafness in two GJB2 and GJB6 negative patients presenting with pre-lingual, progressive, severe hearing loss. Methods Targeted exome sequencing (TES) using Next Generation Illumina Sequencing was used to analyze the exonic and some other important genomic regions of 154 genes in the proband. Subsequently, the mutation found was confirmed by Sanger sequencing in other affected sibling and healthy family members. The possible impact of the reported mutation on the corresponding protein was also evaluated by using bioinformatics tools. Moreover, the affected patients underwent audiological and ophthalmic evaluations. Results TES identified a novel homozygous missense mutation c.251T>C (p.I84T) in exon 3 of PDZD7 gene. In addition, segregation and phenotype-genotype correlation analysis as well as in-silico evaluations confirmed the autosomal recessive inheritance pattern and disease-causing nature of mutation found. Conclusions In overall, our finding could expand the pathogenic mutations spectrum and strengthens the clinical importance of the PDZD7 gene in ARNSHL patients. It can also aid to conduct genetic counseling, prenatal diagnosis and clinical management of these types of genetic disorders.

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Adeberg, Sebastian, Maximilian Knoll, Christian Koelsche, Denise Bernhardt, Daniel Schrimpf, Felix Sahm, Laila König et al. « DNA-methylome-assisted classification of patients with poor prognostic subventricular zone associated IDH-wildtype glioblastoma ». Acta Neuropathologica, 4 juin 2022. http://dx.doi.org/10.1007/s00401-022-02443-2.

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AbstractGlioblastoma (GBM) derived from the “stem cell” rich subventricular zone (SVZ) may constitute a therapy-refractory subgroup of tumors associated with poor prognosis. Risk stratification for these cases is necessary but is curtailed by error prone imaging-based evaluation. Therefore, we aimed to establish a robust DNA methylome-based classification of SVZ GBM and subsequently decipher underlying molecular characteristics. MRI assessment of SVZ association was performed in a retrospective training set of IDH-wildtype GBM patients (n = 54) uniformly treated with postoperative chemoradiotherapy. DNA isolated from FFPE samples was subject to methylome and copy number variation (CNV) analysis using Illumina Platform and cnAnalysis450k package. Deep next-generation sequencing (NGS) of a panel of 130 GBM-related genes was conducted (Agilent SureSelect/Illumina). Methylome, transcriptome, CNV, MRI, and mutational profiles of SVZ GBM were further evaluated in a confirmatory cohort of 132 patients (TCGA/TCIA). A 15 CpG SVZ methylation signature (SVZM) was discovered based on clustering and random forest analysis. One third of CpG in the SVZM were associated with MAB21L2/LRBA. There was a 14.8% (n = 8) discordance between SVZM vs. MRI classification. Re-analysis of these patients favored SVZM classification with a hazard ratio (HR) for OS of 2.48 [95% CI 1.35–4.58], p = 0.004 vs. 1.83 [1.0–3.35], p = 0.049 for MRI classification. In the validation cohort, consensus MRI based assignment was achieved in 62% of patients with an intraclass correlation (ICC) of 0.51 and non-significant HR for OS (2.03 [0.81–5.09], p = 0.133). In contrast, SVZM identified two prognostically distinct subgroups (HR 3.08 [1.24–7.66], p = 0.016). CNV alterations revealed loss of chromosome 10 in SVZM– and gains on chromosome 19 in SVZM– tumors. SVZM– tumors were also enriched for differentially mutated genes (p < 0.001). In summary, SVZM classification provides a novel means for stratifying GBM patients with poor prognosis and deciphering molecular mechanisms governing aggressive tumor phenotypes.

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Jeppesen, Line Dahl, Lotte Hatt, Ripudaman Singh, Palle Schelde, Lotte Andreasen, Sara Markholt, Dorte L. Lildballe et Ida Vogel. « Screening for Fetal Aneuploidy and Sex Chromosomal Anomalies in a Pregnant Woman With Mosaicism for Turner Syndrome—Applications and Advantages of Cell-Based NIPT ». Frontiers in Genetics 12 (14 septembre 2021). http://dx.doi.org/10.3389/fgene.2021.741752.

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Background: Cell-free NIPT and cell-based NIPT are risk-free testing options using maternal blood samples to screen for fetal aneuploidies, but the methods differ. For cell-free NIPT, the fetal fraction of cell-free DNA in plasma is analyzed with a high background of maternal DNA. In contrast, for cell-based NIPT, a limited number of the rare, intact fetal cells are isolated for the genetic analysis. This case demonstrates the differences regarding testing for fetal sex-chromosomes anomalies (SCAs) between these two tests.Materials and Methods: A pregnant woman with mosaicism for Turner syndrome opted for NIPT in first trimester. For the cell-free NIPT analysis, DNA extraction, genome-wide massive parallel sequencing, and data analysis were carried out as described by the kit manufacturer (Illumina©, San Diego, CA, USA). For cell-based NIPT, the first sample gave no result, but the woman consented to repeat cell-based NIPT. After whole genome amplification and STR analysis, fetal DNA from three individual fetal cells was subjected to chromosomal microarray (aCGH, Agilent oligoarray, 180 kb).Results: Fetal fraction was 7%, and cell-free NIPT showed 2 copies of chromosomes 13, 18, and 21 and a decreased proportion of chromosome X, suggestive of fetal Turner syndrome. In contrast, the cell-based NIPT result showed no aneuploidy and two X-chromosomes in the fetus.Conclusion: cell-based NIPT may provide a non-invasive testing option to screen for SCAs in women with mosaicism for monosomy-X in blood, where cell-free NIPT cannot discriminate whether the X-loss is maternal or fetal.

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Klee, Philippe, Mirjam Dirlewanger, Valérie McLin, Maria Teresa Carminho et Valerie M. Schwitzgebel. « SUN-602 Weight Loss After Glucagon-Like Peptide-1 Receptor Agonist Treatment in Childhood Obesity with Diabetes and Cirrhosis Associated with a Homozygous MC4R Mutation ». Journal of the Endocrine Society 4, Supplement_1 (avril 2020). http://dx.doi.org/10.1210/jendso/bvaa046.611.

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Abstract Background Mutations in the melanocortin-4 receptor (MC4R) represent the most common cause of monogenic obesity. Treatment options are limited but glucagon-like peptide-1 receptor agonists (GLP-1 RA) may be of use to induce weight loss. Methods Exome of the patient was captured using the Agilent SureSelect QXT Human All Exon V5 kit and sequenced on Illumina. Clinical findings and results We report obesity-associated diabetes and cirrhosis in a 13-year girl born from consanguineous parents of Afghan origin. Past medical history revealed mild mental retardation and excessive weight gain since infancy. Linear growth was normal. Her father was obese and no diabetes was found in the family. The girl was initially investigated for hoarseness and found to have pulmonary hypertension, later accepted to be secondary to cirrhosis and portal hypertension. Physical examination revealed obesity (BMI 34.9kg/m2) and acanthosis nigricans. Blood exams showed leucopenia and thrombocytopenia without anemia, compatible with portal hypertension. Chest CT revealed important dilatation of the pulmonary arteries, a nodular liver and splenomegaly. Liver biopsy confirmed cirrhosis. An extensive workup including whole exome sequencing identified a homozygous MC4R variant [NM_005912.2 (MC4R): c.63_64del, p.(Tyr21*)], classified as pathogenic according to the ACMG guidelines. Both parents were heterozygous for this variant. An endocrinological workup showed insulin resistance with a HOMA-IR index of 7.27 and diabetes with peak blood glucose of 11.5mmol/l. HbA1c was 5.1% (32mmol/mol). Thyroid tests, leptin, proinsulin levels (3.5pmol/l, n <11.0pmol/l) were normal. The mutation being homozygous with a predicted complete loss of function (https://www.mc4r.org.uk/), no treatment with a MC4R agonist was tried. At the age of 15 years (BMI 36.0kg/m2), the patient underwent liver transplantation because of progressive portal hypertension and to halt the progression of pulmonary hypertension. At the age of 16 years (BMI 33.2kg/m2, HbA1c 4.9% (30.0 mmol/mol), HOMA-IR 5.3) a treatment with GLP-1 RA (liraglutide) was started at a dosage of 0.6mg and progressively increased to 3mg, in an attempt to induce weight loss, avoid the accumulation of liver fat and to protect the graft. GLP-1 RA is supposed to exerts its effects on appetite independently of the MC4R pathway. 2 months after liraglutide introduction, no side effects, a weight loss of 4kg and a decrease of appetite were observed (BMI 31.6kg/m2, HbA1c 4.5% (26mmol/mol), HOMA-IR 3.14). Conclusion Obesity-associated MCR4 mutations, in homozygous state, may lead to diabetes, liver cirrhosis and porto-pulmonary hypertension. Treatment options are scarce, but GLP-1 RA seem to have a rapid, positive effect on weight and metabolic control. Would earlier treatment have prevented progression to end-stage-liver disease and need for liver transplantation?

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Collins, Jason M., Rahul Gondalia, Anne E. Justice, Katelyn Holliday, James Stewart, Eugenia Wong, Yun Li et al. « Abstract P143 : Methylome-Wide Association Of DNA Methylation And Aircraft Noise Exposure In The Women’s Health Initiative ». Circulation 141, Suppl_1 (3 mars 2020). http://dx.doi.org/10.1161/circ.141.suppl_1.p143.

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Background: Noise pollution is common and can affect health, but mechanisms underlying this relationship remain incompletely characterized. We therefore examined the novel association between aircraft noise and DNA methylation (DNAm), an environmentally modifiable epigenetic phenomenon that affects gene expression and is associated with cardiovascular and neurological disease. Methods: We conducted a methylome-wide association study in race/ethnicity-stratified subpopulations of 4,535 post-menopausal Women’s Health Initiative participants (mean age: 64.4 years; 59.6% white; 24.7% African American; 15.7% Hispanic/Latina). We estimated DNAm in whole blood as a proportion of methylated cytosines at each Cytosine-phosphate-Guanine (CpG) site using the Illumina 450k BeadChip. In cooperation with the Federal Aviation Administration, we generated day-night-average sound level (DNL) contours around 90 major U.S. airports using the Aviation Environmental Design Tool. The DNL applies a penalty for nighttime exposure and is the primary metric for informing policy in the U.S. We estimated geocoded participant address-specific, annual, DNL aircraft noise exposures in decibels (dB) from the contours on the day of blood draw. We categorized aircraft noise exposures as non-exposed or exposed (DNL < 45 or ≥ 45dB) and used linear mixed-effects models to estimate CpG site-specific DNAm-noise associations adjusted for sociodemographic, behavioral, and technical covariates. We combined site-specific associations across race-ethnicity strata in fixed-effects, inverse variance-weighted meta-analyses and then identified the significant ( P < 1.0x10 -7 ), non-heterogeneous ( P Cochran’s Q > 0.10) associations among them. Results: Overall, DNAm was inversely associated with aircraft noise at cg21525369 (Chromosome 15; P = 6.40x10 -8 ). Cg21525369 is 56 kb upstream from ACAN , a gene encoding an extracellular matrix protein (aggrecan). Aggrecan is expressed in response to ultrasound; forms perineural nets around cochlear hair cells and auditory brainstem neurons; affects afferent extension, synaptic transmission, and neuronal vulnerability; deposits in tympanic membranes of patients with sensorineural hearing loss, and redistributes in auditory brainstem after deafferentation. Conclusions: The biologically plausible DNAm-noise association among postmenopausal U.S. women suggests aircraft noise pollution may affect methylation of DNA near a gene previously associated with hearing, although its cardiovascular, otologic, and neurological implications remain unclear pending sensitivity analysis, external replication, mediation analysis, and functional characterization.

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Galan Carrillo, Isabel, Liliana Galbis, Víctor Martínez Jiménez, Pedro Pablo Ortuño, Susana Roca, Juan David Castro, Fernanda Ramos, Lidia Rodríguez et Encarnación Guillén. « MO039 : Multidisciplinary approach improves genetic diagnosis of Alport syndrome in the next-generation sequencing ERA ». Nephrology Dialysis Transplantation 37, Supplement_3 (mai 2022). http://dx.doi.org/10.1093/ndt/gfac062.020.

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Abstract BACKGROUND AND AIMS Alport Syndrome (AS; ORPHA 63) is one of the most frequent hereditary kidney diseases (HKD), caused by COL4A5 mutations in X-linked AS (XLAS) and COL4A3-4 mutations in the autosomal forms, dominant (ADAS) and recessive (ARAS). Definitive diagnosis of AS is essential for starting treatment, prognosis and genetic counseling. Next-generation sequencing (NGS) has been implemented in our lab and integrated in a multidisciplinary approach with nephrologists, pediatricians, and clinical and molecular geneticists from four hospitals in the Spanish Region of Murcia (1.5 million inhabitants). Our aim is to evaluate the yield of this approach, the genetic spectrum in AS and the predicting factors for a positive genetic result. METHOD During 3-year activity, 181 individuals (164 families) with diagnostic suspicion of AS have been analyzed by a coordinated clinical multidisciplinary protocol and NGS after informed consent. A customized Agilent panel was designed to capture 113 genes, including those related to AS: COL4A3-5. Interpretation of sequence variants was performed according to the American College of Medical Genetics and Genomics Guidelines. Sanger sequencing was performed to confirm variants identified by NGS and to segregate them in the families. After a negative result with a high suspicion of HKD, multiplex ligation-dependent probe amplification (MLPA), amplified NGS panel or clinical exome were performed. RESULTS Genetic variants were detected in 73 patients (40.9%). A total of 49 patients (27.1%) from 46 families showed pathogenic and probably pathogenic variants associated with AS. Additionally, nine patients (5.5%) were reclassified as other HKD (Table 1). After all, 58 patients (32.0%) had an HKD genetic diagnosis. There were 47 incidental findings. Comparing patients with final diagnosis of AS with patients with negative diagnosis, the former were 71.2% women (P = 0.013), mean age 49 ± 13 y.o. (no statistical differences). Among genetic AS patients, 88.1% had microhematuria (three patients had only persistent 0–5 RBC/HPF), 20.3% had hearing loss and 6.8% had ophthalmologic disturbances, with no statistical differences between groups. Hematuria quantification, characteristic glomerular hematuria or proteinuria did not associate with AS genetic diagnosis (P = 0.310, 0.056 and 0.598, respectively). Mean eGFR (CKD-EPI) and albumin-to-creatinine ratio at diagnostic were 71.89 ± 32.52 mL/min/1.73 m2 and 670 ± 870 mg/gr, without statistical differences. Positive family history was clearly present in 76.3% of final AS group (P = 0.009). Eleven patients had undergone a kidney biopsy before genetic test, and in 28 patients it was avoided because of NGS results. CONCLUSION NGS in a multidisciplinary approach has enabled the diagnosis of a collagen IV disease in 27.1% of patients with suspicion of AS and the identification of additional 5.5% with other HKD, leading to a total yield in molecular characterization of 32.6%. Additionally, the need of kidney biopsy has decreased. The most frequent AS gene in our cohort is COL4A4 followed by COL4A3 linked to ADAS, which is the predominant AS type. A positive family history was the only predicting factor for a positive genetic result, whereas glomerular hematuria, hearing loss or ophthalmologic disturbances did not predict the result. This multidisciplinary approach, with active participation of nephrologists and clinical geneticists, allows better interpretation of genetic variants, management of patients and families and accurate genetic counseling.

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França, Monica Malheiros, Xiao Hui Liao, Gustavo Werpel Fernandes, Alina German, Antonio C. Bianco, Samuel Refetoff et Alexandra Dumitrescu. « OR01-01 Human Type 1 Iodothyronine Deiodinase (DIO1) Mutations Cause Abnormal Thyroid Hormone Metabolism ». Journal of the Endocrine Society 4, Supplement_1 (avril 2020). http://dx.doi.org/10.1210/jendso/bvaa046.966.

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Abstract Iodothyronine deiodinases (Ds) mediate thyroid hormone (TH) action. They catalyze triiodothyronine (T3) production and degradation via, respectively; outer (ORD) and inner (IRD) ring deiodination. Type 1 D (D1) has a relatively high Km for substrates thyroxine (T4) and reverse T3 (rT3), catalyzes both ORD and IRD and is inhibited by propylthiouracil (PTU). Although no mutations in DIO1 gene have been reported so far in humans, based on the D1-deficient C3H mouse and the heterozygous Dio1 knockout mice, the expected phenotype of D1 loss-of-function is higher serum rT3 and slightly elevated T4. Our objective was to identify new gene defects causing unexplained and discrepant thyroid function tests using whole-exome sequencing (WES). Exons and splice sites are captured with Agilent SureSelectXT and sequenced in Illumina HiSeq2500. Data are analyzed with BWA, GATK, and ANNOVAR applications for alignment, variant calling and annotation, respectively. Sanger sequencing is used to confirm and segregate WES variants in families. A heterozygous pathogenic missense variant in the DIO1 gene (c.282C>A:p.N94K; N-linker) was identified in four family members with relatively higher serum rT3, T4, and free T4 than unaffected relatives, and normal TSH. We identified a second heterozygous pathogenic mutation in the DIO1 (c.603G>A:p.M201I; thioredoxin-fold) in a second family. Two affected individuals presented slightly elevated TSH, higher serum rT3, normal T4 levels, while the T3/T4 ratio was lower compared to unaffected members. To assess the functional activity of the mutant D1 protein, human embryonic kidney epithelial cells (HEK293) were transiently co-transfected with pcDNA3 plasmids expressing pD1-WT, pD1-N94K or pD1-M201I, and pGFP as transfection control. In assays performed with 1μM 125I-T4, the catalytic activities of pD1-N94K and pD1-M201I were 44.7% and 54.1% lower as compared to pD1-WT, respectively. To study the enzyme kinetics, the D1 assay was repeated in the presence of increasing 125I-T4 concentrations (0.1-20μM). The enzymatic activity assays revealed similar Vmax for pD1-N94K and pD1-M201I mutants compared to pD1-WT (VmaxpD1-N94K=53.7 vs VmaxpD1-WT=40.9 and VmaxpD1-M201I=58.8 vs VmaxpD1-WT=42), but higher Michaelis constant (Km) than pD1-WT (KmpD1-N94K=16.4 vs KmpD1-WT=6 and KmpD1-M201I=21.4 vs KmpD1-WT=6.9), which demonstrates a reduced affinity for T4. In conclusion, we report the first DIO1 mutations in humans with serum thyroid tests suggestive of TH metabolism defects. These mutations affect the catalytic activity of the D1, demonstrating impaired functional activity of the mutant enzymes and consequently altering TH metabolism.

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