Academic literature on the topic 'Hearing Loss,illumina,Agilent'

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.

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.

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.

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.

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.

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.

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&gt;T MT-ND4 missense variants in three tumors resected at different time points from the same patient, and another heteroplasmic m.1023C&gt;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&gt;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.

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.

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.

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.

2006/2007
Secondo le stime del World Health Organization, le perdite uditive colpiscono circa 278 milioni di persone in tutto il mondo. Approssimativamente 1 bambino ogni 100, nasce con problemi d’udito. Nonostante l’identificazione negli ultimi 10 anni di più di 100 loci genetici associati a fenotipi di perdita uditiva, non tutti i corrispettivi geni causativi sono stati identificati. Normalmente utilizzando un approccio sperimentale di linkage tradizionale non è sempre possibile identificare un intervallo genomico sufficientemente corto da essere analizzato per la ricerca di mutazioni. Il lavoro presentato in questa tesi ha lo scopo di selezionare un set limitato di geni potenzialmente coinvolti nelle perdite uditive non sindromiche, utilizzando la combinazione di un approccio biologico e bioinformatico. Il punto di partenza dell’analisi è stato il gene GJB2. Il gene GJB2 codifica la Connessina 26, proteina coinvolta nella formazione delle gap junction tra le cellule, ma anche implicata in più del 50% dei casi di perdite uditive non sindromiche. Per questa ragione è stato suggerito un ruolo chiave nella biologia dell’orecchio, che va oltre la sua funzione di proteina canale. In questa tesi è stato esaminato il profilo d’espressione genica di cellule HeLa transfettate con la forma naturale e con delle forme mutate della Connessina26. Le analisi dei dati hanno identificato numerosi geni differenzialmente espressi e si è quindi deciso di passare ad un approccio informatico per ridurne il numero. Questa analisi ha permesso di identificare 19 geni in 11 loci privi di geni causativi selezionandoli in base alla loro espressione rispetto librerie di cDNA prodotte da orecchio. Sono stati quindi identificati i geni omologhi in topo per 5 dei 19 geni, con lo scopo di verificare la loro rilevanza con la perdita uditiva. Per tutti questi 5 geni è stata confermata l’espressione nell’organo di corti in topo e con Real-time RT-PCR nelle linee cellulari transfettate impiegate negli esperimenti di microarray. Il progetto proseguirà ora con lo screening di mutazioni nei geni candidati in famiglie di pazienti selezionate.
According to WHO estimates hearing impairment affects 278 million people worldwide. Approximately 1/1000 children are born with a significant hearing impairment. To date approximately 100 genetic loci involved in deafness have been described. Despite the fact that such a large number of genetic locations associated with deafness phenotypes are known, not all the genes involved have been identified yet. Using a traditional linkage approach, however, it is not always possible to map a locus to intervals short enough to be amenable for costly mutation analysis. So far no more than 40 deafness genes have been identified and these encode very heterogeneous proteins. The work presented in this thesis aims to identify a limited set of candidate genes with high potential to be involved in Non-Syndromic Hearing Loss using a combination of biological and bioinformatics approaches. The starting point of the analysis was the GJB2 gene. The GJB2 gene encodes for the gap junction protein Connexin26 and is responsible for more than half of the non-syndromic hearing loss cases. For this reason it has been proposed that this protein might play a wider role in the biology of the ear, beyond its mere channel function. I therefore performed whole genome expression profiles of HeLa cells transfected with the wild type form of the GJB2 gene and compared them to that of cells transfected with mutant forms of this gene to shed light on its function. Initially this experiment yielded a bewildering number of differentially expressed genes (4,984). Thus I devised an in silico strategy to narrow down this number, focusing on genes which were positionally linked to specific non-syndromic hereditary hearing loss conditions, as well as found within human ear cDNA libraries, thus potentially causative of the disease. This further analysis yielded 19 genes within 11 loci. In order to assess their relevance to hearing loss, the mouse homologs of these genes were identified for 5 of them and indeed they were all found to be expressed in the mouse organ of corti. These five genes were also validated by Real-time RT-PCR in the human cell line used for the microarray experiments.
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