Articles de revues : « Compound mutation »

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Li, Jin, Zhenyu Yan, Agus Darwanto, Peng Fang, Weihua Liu, Kristy Drafahl, Julie Toplin, Cindy Spittle et Chad Galderisi. « Phasing Analysis Of TKI Resistance Mutations In The BCR-ABL1 Kinase Domain and Neighboring Domains Using Next-Generation Sequencing ». Blood 122, n^o 21 (15 novembre 2013) : 3817. http://dx.doi.org/10.1182/blood.v122.21.3817.3817.

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Abstract Background Many CML patients treated with tyrosine kinase inhibitors (TKIs) eventually develop resistance as a result of ABL1 kinase domain (KD) mutations, and sequential treatment with different TKIs may select for multiple BCR-ABL1 mutations. Whether multiple mutations arise in distinct clones (in trans, or polyclonal mutations) or instead are present within the same BCR-ABL1 molecule (in cis, or compound mutations), has been shown to have important implications with respect to TKI sensitivities (Eide, C.A. et al., Blood 2011). Distinguishing between polyclonal and compound mutations, or mutation phasing, for the ABL1 KD has not been clinically practical with standard mutation detection methods. Here we have developed a highly sensitive next-generation sequencing (NGS) assay on the Ion Torrent PGM along with a proprietary data analysis pipeline that together enable deep sequencing of the BCR-ABL1 KD and neighboring domains with a 1% limit of detection and quantitative reporting of mutation phasing. Methods RT and long range PCR was performed to amplify BCR-ABL1 e1a2/3, e13a2/3, and e14a2/3 fusion transcripts and the PCR products were enzymatically randomly fragmented and ligated with Ion Torrent sequencing adaptors. Size-selected libraries were quantified, pooled, amplified with the OneTouch system and sequenced with the Ion Torrent PGM using 400 bp sequencing chemistry. Sequencing data were analyzed with Torrent Suite 3.4.2 with variant frequency cutoff adjusted to 1%. Variants were further annotated with a proprietary analysis pipeline and variant report was produced after manually reviewing variants by Integrative Genomics Viewer. If more than one non-synonymous variant was reported in a sample, a proprietary phasing analysis pipeline was applied to report the mutation spectrum of all of the combinations of multiple mutations in the sample. Results To validate the accuracy of the sequencing method which employs 400 bp sequencing chemistry, we compared this assay with our previously validated BCR-ABL1 NGS assay based on Ion Torrent 200 bp sequencing chemistry for a set of clinical specimens from CML patients previously treated with TKI. Results were highly concordant and similarly sensitive, with 11/11 variants (frequencies ranging from 2% to 100%) identified with comparable frequencies by both methods. To evaluate the specificity of the phasing analysis, an artificial sample was created by mixing two samples (b2_10 and b2_6) with 6 distinct variants present at ratio of 1:19. Because variants are unique in each sample, any compound mutation composed of b2_10 variant and b2_6 variant identified would be false positive. The false positive error rate (percentage of b2_6 variant as compound mutation with b2_10 variant and vice versa) ranged from 0 – 0.6%, which was consistent with sequencing error rate. We conservatively define a compound mutation as true if it is present in at least 5% of any one of the component variants in the compound mutation. Mutation detection and phasing analysis were reproducible on different chips (314 v2 and 318 v2) and different library preps from the same long range PCR product of BCR-ABL1. Table 1 shows the mutation spectrum from sample b2_6. Of the four variants detected, L248V and G250E were mutually exclusive (in trans), while T315I and M351T were present as compound mutations with each other and, separately, with either L248V or G250E. Notably, >86% of the molecules harbored single mutations, and no compound mutations containing more than 2 variants were observed. Conclusions We have developed and validated a sensitive NGS assay that enables deep sequencing of the BCR-ABL1 KD and neighboring domains along with quantitative mutational phasing. This method has been applied in evaluating >250 clinical specimens for a clinical trial of a third-generation TKI(results reported separately). The ability to easily determine the mutation phasing of a CML patients’ mutation profile using this assay will allow for investigations into compound mutation-based resistance mechanisms and may be used to better guide treatment decisions. Disclosures: Li: MolecularMD: Employment. Yan:MolecularMD: Employment. Darwanto:MolecularMD: Employment. Fang:MolecularMD: Employment. Liu:MolecularMD: Employment. Drafahl:MolecularMD: Employment. Toplin:MolecularMD: Employment. Spittle:MolecularMD: Employment. Galderisi:MolecularMD: Employment.

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Bhatwadekar, Seema S., et Parth Shah. « Mutational Phasing : Clinical Relevance in Tyrosine Kinase Domain Mutations Using Next Generation Sequencing in Chronic Myeloid Leukemia ». Blood 132, Supplement 1 (29 novembre 2018) : 4269. http://dx.doi.org/10.1182/blood-2018-99-114130.

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Abstract Background: Tyrosine kinase mutation analysis in BCR/ABL1 gene is important for management of patients with chronic myeloid leukemia. Sanger Sequencing has been the mainstay for testing with Next Generation Sequencing (NGS) now becoming the primary technology. In this study we show a comparison between NGS versus Sanger Seqencing based ABL kinase domain mutation analysis with a likely trend of clinical relevance based on a compound versus polyclonal state of mutational distribution which may also need to be considered for patient management and therapy. Methodology: A total of 213 Imatinib-resistant patients with CML for BCR-ABL1 mutation analysis were processed on both technologies.Initial blood counts were assessed and RNA was extractedfollowed by cDNA conversion. NGS libraries were prepared with 400bp multiplexed amplicons to allow optimal phasing. Results: 179 samples were negative by both technologies. A total of only 20 samples were positive and concordant by both technologies(58.2%). Mutations in 14 other samples however were only detected in NGS(41.17%). In these 14 samples (41.17%), NGS was able to detect 23 mutations with mutation frequencies of 3-28%, which were missed by Sanger. Conclusions: Moreover 11/34 patients had 2 or >2 mutations. An inhouse script delineated mutations as compound or polyclonal from NGS data. 2/11 cases demonstrated compound mutations (Mutations in the same clone) while 7/11 cases were polyclonal per NGS. Sanger sequencing cannot differentiate between polyclonal and compound mutations. 2/11 cases appeared to have polyclonal and compound mutations. 4/11 patients presented in a blast crisis or accelerated phase CML. Interestingly, most of these patients hadat leasttwo mutations and were polyclonal(3/4). Significantly previously archived samples patients with polyclonal mutations showed polyclonality at extremely low frequency percentages in initial samples. None of the single mutation patients had presented in a blast crisis or an accelerated phase. Disclosures No relevant conflicts of interest to declare.

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Chen, Jiaqi, Hongxing Liu, Fang Wang, Yang Zhang, Xue Chen, Daijing Nie, Yu Li, Yincheng Tan, Yuanli Xu et Xiaoli Ma. « Dynamic Evolution of Ponatinib Resistant BCR-ABL1 T315 and Compound Mutations ». Blood 134, Supplement_1 (13 novembre 2019) : 3796. http://dx.doi.org/10.1182/blood-2019-129579.

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The third-generation tyrosine kinase inhibitor (TKI) ponatinib exhibits activity against all common BCR-ABL1 kinase domain (KD) single mutations, including the highly resistant gatekeeper T315I. However, the drug response is variable and the clinical resistance mutations may still befall with few reports to date. We performed next-generation sequencing (NGS) detection of BCR-ABL1 KD mutations in sequential samples of three BCR-ABL1-positive leukemia patients who developed clinical resistance to ponatinib, to explore the dynamic evolution of ponatinib mutations. Case 1 was diagnosed as chronic myeloid leukemia (CML) chronic phase when he was 29 years old in 2009. His maintenance therapies were imatinib and dasatinib for seven years and then replaced with ponatinib due to the blast crisis of CML and the T315I mutation with variant allele frequency (VAF) of 46%. He did not achieved molecular remission after attempting multiple combined chemotherapy and TKIs including ponatinib, with the T315I mutation persists and eventually increases to VAF of 97%. He was then was medicated with a combined chemotherapy plus dasatinib and ponatinib. But NGS KD mutation investigation showed multiple T315I compound mutations, T315M and T315I mutations six months later. The patients went through salvage allogenic hemopoietic stem cell transplantation (allo-HSCT), and the above polyclonal and compound mutations were still carried after transplantation. Finally, Q252H/T315I (VAF,50%) became the dominant clone, and ponatinib and HQP1351 (domestic TKI designed for T315I) were ineffective for this compound mutation (Figure a). A similar dynamic evolution to the BCR-ABL1 KD mutation of Case1 also occurred in case 2. The 7-year-old boy was diagnosed with BCR-ABL1-positive acute B-lymphocytic leukemia (Ph+B-ALL) in April 2014. NGS results showed that he had D276G (7%), F311I (27%) and F317L (31%) polyclonal mutations in BCR-ABL1 KD after 18 months of imatinib administration which were subsequently treated with ponatinib. After 5 months of treatment with ponatinib, F311I and F317L mutations disappeared, but G250E (5%) and D276G/T315L (4%) compound mutation appeared; subsequent progression to D276G/T315L (32%), G250W/E255V/F311I (4%) and F311I/T315I (58%) polyclonal compound mutations; long induction chemotherapy combined with ponatinib treatment remained unresolved, and finally there was only D276G/T315L compound mutation (VAF, 100%, figure b and d). Notably, a rare mutation T315L (c.943_945delinsCTC/p.T315L) appeared in the BCR-ABL1 KD. D276G is known to be sensitive to various TKIs, so we speculate that ponatinib is ineffective for the T315L mutation. Case 3 was a 46-year-old woman who diagnosed with Ph+B-ALL in July 2018. The Q252H (20%) and T315I (44%) double mutations appeared after oral administration of imatinib for 2 months, and then switched to ponatinib for remission. After 8 months, the bone marrow and peripheral blood samples showed not only the T315I/F359V compound mutation (VAF, 90% and 94%), but also the T315L mutation (VAF, 5% and 6%, c.943_944AC>CT/p.T315L). The clinician combined her BCR-ABL1 KD mutation and condition, ponatinib was discontinued. After 1 month of chemotherapy combined with dasatinib, the patient's condition improved, but the BCR-ABL1 KD mutation progressed to T315L (18%, figure c and e) and T315I/F359V compound mutation (76%). She eventually died from severe pulmonary infection and sepsis. NGS analysis identify KD mutation with sensitivity about 2%, and can also distinguish between compound and polyclonal mutations. All of the ultimately dominant ponatinib resistant mutations (Q252H/T315I, D276G/T315L, and T351I/F359V) in these three cases were T315 compound mutations derived from the T315I or other original mutation with additional mutation event. The T315L/M mutations and compound mutations collaborated by T315 and other KD mutations may confer the major component of ponatinib resistance. The dynamic resistant mutation in these three patients adds to the currently less content compendium of ponatinib clinical resistance. All of the three patients encountered ponatinib toxic side effects and had to discontinue or reduce the dose, which also confer favorable opportunity for the development of drug-resistant mutations. Figure Disclosures No relevant conflicts of interest to declare.

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Mian, Afsar Ali, Hadiqa Raees, Sujjawal Ahmad, Oliver Ottmann et El-Nasir M. A. Lalani. « Arsenic Trioxide Suppresses Growth of BCR-ABL1 Positive Cells with "Gatekeeper" or Compound Mutation ». Blood 138, Supplement 1 (5 novembre 2021) : 4346. http://dx.doi.org/10.1182/blood-2021-154511.

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Abstract Introduction: Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph +), having a (9;22) chromosomal translocation. The BCR-ABL1 fusion protein is the hallmark of Ph + leukemia. BCR-ABL1 is characterized by constitutively activated ABL1 tyrosine kinase activity that determines its transformation potential. Tyrosine kinase inhibitors (TKI) have greatly improved the overall prognosis of these diseases. However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR-ABL1 inhibitors represent major clinical problems. The most important resistance mechanism against TKIs is the acquisition of point mutations within the BCR-ABL1 kinase domain that impair drug binding, restoring the oncoprotein's constitutively active tyrosine kinase activity. The selection of leukemic clones driven by BCR-ABL1 harboring point mutations, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). Second- and third generation TKIs such as nilotinib, dasatinib, and ponatinib effectively overcome point mutation-mediated resistance. Ponatinib is the only U.S. Food and Drug Administration approved TKI with activity against all known BCR-ABL1 point mutations, including BCR-ABL1-T315I. However, the emergence of compound mutations (two mutations within the same BCR-ABL1 allele) has been linked to resistance to all approved TKIs, including ponatinib, posing a clinical challenge with limited treatment options. The anti-cancer agent arsenic trioxide (ATO) has been used to treat patients with acute promyelocytic leukemia (APL). APL patients respond very well to ATO therapy and achieve complete remission, possibly through induction of apoptosis and differentiation. In addition, it has been demonstrated that combined treatment of ATO with interferon or nilotinib significantly suppressed cell proliferation. However, the potential effects of ATO on BCR-ABL1 mutations and especially on compound mutation is not apparent. This study aimed to investigate the role of ATO in BCR-ABL1 resistant mutations, including compound mutation in Ph + leukemias. Methods: We undertook preclinical evaluation of ATO and compared it with approved TKIs e.g. imatinib, nilotinib, dasatinib, ponatinib and ABL inhibitor asciminib, in vitro models of CML and primary patient-derived long term cultures (PD-LTC) of Ph + ALL patients with or without mutation. The effects on mutational resistance were investigated in Ba/F3 cells expressing BCR-ABL1 with T315I mutation and T315I-E255K mutation. For non-mutational resistance, we used PD-LTCs from Ph + ALL patients with different levels of non-mutational drug resistance. Cell proliferation was assessed by XTT. Results: ATO efficiently inhibited the growth of all PD-LTCs in cellular assays at dosages of 200-500nM. It also suppressed the growth of Ph + PD-LTC with non- mutational resistance (BV) and the BCR-ABL1-T315I positive PD-LTC (KO) in this dosage range. In all modelsWe treated Ba/F3 cells expressing native BCR-ABL1, BCR-ABL1-T315I mutation and BCR-ABL1-T315I-E255K (compound mutation) with increasing concentrations of imatinib (250, 500 and 1000nM), nilotinib (100, 200 and 400nM), dasatinib (10, 25 and 50nM), ponatinib (10, 50 and 100nM), asciminib) (ABL allosteric inhibitor) (5, 10 and 20nM) and ATO (0.5, 1.0 and 2.0 µM). We found that all the inhibitors significantly inhibited the proliferation of Ba/F3 cells expressing wild type BCR-ABL1 in a dose-dependent manner. In contrast, the growth of Ba/F3 cells expressing BCR-ABL1-T315I was inhibited by increasing concentration of ponatinib, asciminib and ATO. ATO potently inhibited the most challenging mutation (T315I-E255K) with a clinically relevant concentration (IC50 250nM). All approved ABL kinase inhibitors (AKIs) and allosteric inhibitors like asciminib could not inhibit the growth of Ba/F3 cells expressing BCR-ABL1 compound mutation. Conclusions: Our findings indicate that ATO significantly suppressed the proliferation of cells expressing non-mutated BCR-ABL1, single and compound mutated BCR-ABL1. These results support including ATO in treating patients with Ph + leukemias having BCR-ABL1 resistant single or compound mutati Disclosures Ottmann: Novartis: Honoraria; Amgen: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Fusion: Honoraria; Incyte: Honoraria, Research Funding.

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Kim, Soo-Hyun, Soo Young Choi, Sung-Eun Lee, Ju-Hee Bang, Ji-Young Byeun, Jin-Eok Park, Hye-Rim Jeon, Eun-Jung Jang, Saengsuree Jootar et Dong-Wook Kim. « Dynamics and Characteristics of BCR-ABL1 Multiple Mutations in Tyrosine Kinase Inhibitor Resistant CML ». Blood 120, n^o 21 (16 novembre 2012) : 1677. http://dx.doi.org/10.1182/blood.v120.21.1677.1677.

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Abstract Abstract 1677 Background: BCR-ABL1 kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL1 kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment. Methods: Since 2002, 414 CML patients were screened for mutation analysis due to sign of resistance to TKI including imatinib, nilotinib, dasatinib, bosutinib, radotinib or ponatinib at Seoul St Mary's Hospital using direct sequencing and ASO-PCR. Among them, 31 patients showed multiple mutations. We analyzed serial samples from the 31 patients using subcloning and sequencing to investigate whether the multiple mutations are on same clone (defined as compound clone), separated clones (defined as multiple clone) or co-existent clones (defined as mixed clone) and characterize its clinical relevance and dynamics. Results: Status of the patients with multiple mutations is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 31 patients. Cloning of cDNA region corresponding to BCR-ABL1 KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing. Multiple mutations of 65% patients (20 out of 31) existed compound mutation which means the individual mutant types are located on the same BCR-ABL1 molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL1 KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL1 mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL1 mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL1 gene in patients with point mutation are relatively unstable. Among 20 patients with compound mutation, 9 patients were available for serial timepoint samples under same TKI therapy. In all nine patients (100%), portion of compound clone was increased as treatment went on. With a median follow-up 53.3 months (range, 0–113.2 months), of 31 patients with multiple mutation, 7 patients remained alive; 4 of 11 (36%) in the multiple clone group vs 3 of 20 (15%) in the mixed clone group (P = 0.066). Conclusion: Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutations as treatment went on. Patients with compound clone showed poor outcomes compared with multiple clone group in our cohort, further investigation on a large patient cohort will be needed. Updated data with longer follow-up duration will be presented in the meeting. Disclosures: No relevant conflicts of interest to declare.

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Jung, Hyun Ae, Sehhoon Park, Jong-Mu Sun, Se-Hoon Lee, Jin Seok Ahn, Myung-Ju Ahn et Keunchil Park. « Treatment and Outcomes of Metastatic Non-Small-Cell Lung Cancer Harboring Uncommon EGFR Mutations : Are They Different from Those with Common EGFR Mutations ? » Biology 9, n^o 10 (7 octobre 2020) : 326. http://dx.doi.org/10.3390/biology9100326.

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Approximately 10% of the epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are uncommon EGFR mutations. Although the efficacy of second (2G) or third generation (3G) EGFR tyrosine kinase inhibitors (EGFR-TKIs) in the patients with uncommon EGFR mutation has been proven, further studies are warranted to define the optimal treatment approach for uncommon EGFR mutation-positive NSCLC. This study retrospectively investigated the treatment patterns and outcomes of patients with uncommon EGFR mutation-positive NSCLC from January 2011 to December 2019 at the Samsung Medical Center, Seoul, Korea. During the study, 2121 patients with EGFR mutation-positive NSCLC received first-generation (1G, gefitinib or erlotinib) or 2G EGFR-TKI (afatinib) as the first-line (1L) systemic therapy. Of this, 135 (6.4%) patients harbored uncommon EGFR mutations. Of 135, 54 (40%, 54/135) patients had overlapping mutations with major EGFR mutations. The objective response rate (ORR) for the 1L EGFR-TKI was 63.3%. The median progression-free survivals (PFSs) were 8.6 months (95% CI: 3.8–13.5), 11.7 months (95% CI: 6.6–16.7), 7.7 months (95% CI: 4.9–17.4), and 5.0 months (95% CI: 3.7–6.1) for major uncommon EGFR mutation (G719X, L861Q), compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), other compound mutation, and other uncommon mutations, respectively. The median overall survivals (OSs) were 25.6 months (16.9–34.2), 28.8 (95% CI: 24.4–33.4), 13.5 months (95% CI: 7.4–27.8), and 9.4 months (95% CI: 3.4–10.5) for major uncommon EGFR mutation (G719X), compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), other compound mutation, and other uncommon mutations, respectively. The response rate, median PFS, and OS were 63.3%, 16.3 months (95% CI: 15.6–16.9), and 37.5 months (95% CI: 35.4–39.6) for common EGFR mutation-positive NSCLC. After failing 1L EGFR-TKI, repeated tissue or liquid biopsy were carried out on 44.9% (35/78) of patients with T790M detected in 10/35 (28.6%) patients. With subsequent 3G EGFR-TKI after failing the first-line EGFR-TKI, the ORR and PFS for 3G EGFR-TKI were 80% and 8.9 months (95% CI: 8.0–9.8). These patients showed a median OS of 34.6 months (95% CI: 29.8–39.4). The ORR, PFS and OS were poorer in patients with uncommon (especially other compound and other uncommon mutation) than those with common EGFR mutations. T790M was detected in 28.6% of the uncommon EGFR mutation-positive patients for whom prior 1G/2G EGFR-TKIs failed and underwent repeat biopsy at the time of progression.

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Kim, Dong-Wook, Dongho Kim, Soo-Hyun Kim, Saengsuree Jootar, Hyun-Gyung Goh, Jeong Lee, Soo-Young Choi, Young-Seok Lee et Sang-Mi Oh. « Dynamics and Characteristics of BCR-ABL Multiple Mutations In Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia. » Blood 116, n^o 21 (19 novembre 2010) : 3443. http://dx.doi.org/10.1182/blood.v116.21.3443.3443.

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Abstract Abstract 3443 BCR-ABL kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment. From January 2002 to June 2010 at Seoul St Mary's Hospital, 277 CML patients were screened for mutation analysis due to sign of resistance to tyrosine kinase inhibitors including imatinib, nilotinib, dasatinib or bosutinib. We found that 95 patients have point mutation in BCR-ABL kinase domain through direct sequencing or ASO-PCR. Among them, 17 patients showed multiple mutation containing more than one type of point mutations in BCR-ABL KD. We investigated the patients with multiple mutations to characterize its clinical relevance and dynamics. Once mutation found, follow-up samples from the corresponding patients were collected and analyzed prospectively, or mutation status was analyzed retrospectively with cryopreserved samples if they were available. Status of the patients with multiple mutation is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 17 patients. Cloning of cDNA region corresponding to BCR-ABL KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing. Multiple mutations of 88% patients (15 out of 17) existed compound mutation which means the individual mutant types are located on the same BCR-ABL molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL gene in patients with point mutation are relatively unstable. Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutation as treatment went on. Currently investigation of clinical relevance of compound mutation and other analyses are being carried on and more results will be provided in detail at the conference. Table 1. Patients Tx at mutation detection (mg) Compound type Compound % 1 Nilotinib400 G250E+T315I 6.7 G250E+D444G 33.3 T315I+D444G 6.7 2 Nilotinib400 M244V+T315I 95.0 3 Dasatinib100 Y253H+T315I 95.0 4 Dasatinib140 T315I+E459K 55.6 5 Dasatinib200 T315I+M351T 66.7 6 Dasatinib100 NCM Dasatinib80 NCM Dasatinib100 M244V+F359V 16.7 7 Bosutinib500 NCM 8 Dasatinib140 T315I+F359C 35.3 9 Imatinib400 E255K+T315I 5.6 10 Dasatinib80 E255V+T315I 90.0 11 Imatinib800 E255K+T315I 10.5 12 Nilotinib800 E255K+T315I 12.5 13 Dasatinib100 F311I+T315I 35.0 F311I+F317Lb 10.0 Imatinib400 F311I+T315I 10.0 F311I+F317La 15.0 F311I+F317Lb 55.0 14 Nilotinib800 Y253H+F359I 5.6 15 Bosutinib500 V299L+E459K 95.0 Nilotinib400 + Dasatinib100 V299L+F359I 5.0 V299L+E459K 55.0 V299L+F317La+E459K 15.0 V299L+F359I+E459K 15.0 V299L+F317La+F359I+E459K 5.0 16 Imatinib600 NCM 17 Imatinib400 NCM NCM: no compound mutation. Disclosures: No relevant conflicts of interest to declare.

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Finkielstain, Gabriela P., Wuyan Chen, Sneha P. Mehta, Frank K. Fujimura, Reem M. Hanna, Carol Van Ryzin, Nazli B. McDonnell et Deborah P. Merke. « Comprehensive Genetic Analysis of 182 Unrelated Families with Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency ». Journal of Clinical Endocrinology & ; Metabolism 96, n^o 1 (1 janvier 2011) : E161—E172. http://dx.doi.org/10.1210/jc.2010-0319.

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Background: Genetic analysis is commonly performed in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Study Objective: The objective of the study was to describe comprehensive CYP21A2 mutation analysis in a large cohort of CAH patients. Methods: Targeted CYP21A2 mutation analysis was performed in 213 patients and 232 parents from 182 unrelated families. Complete exons of CYP21A2 were sequenced in patients in whom positive mutations were not identified by targeted mutation analysis. Copy number variation and deletions were determined using Southern blot analysis and PCR methods. Genotype was correlated with phenotype. Results: In our heterogeneous U.S. cohort, targeted CYP21A2 mutation analysis did not identify mutations on one allele in 19 probands (10.4%). Sequencing identified six novel mutations (p.Gln262fs, IVS8+1G>A, IVS9-1G>A, p.R408H, p.Gly424fs, p.R426P) and nine previously reported rare mutations. The majority of patients (79%) were compound heterozygotes and 69% of nonclassic (NC) patients were compound heterozygous for a classic and a NC mutation. Duplicated CYP21A2 haplotypes, de novo mutations and uniparental disomy were present in 2.7% of probands and 1.9 and 0.9% of patients from informative families, respectively. Genotype accurately predicted phenotype in 90.5, 85.1, and 97.8% of patients with salt-wasting, simple virilizing, and NC mutations, respectively. Conclusions: Extensive genetic analysis beyond targeted CYP21A2 mutational detection is often required to accurately determine genotype in patients with CAH due to the high frequency of complex genetic variation.

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Liu, Shiguo, Shasha Zhang, Wenjie Li, Aiqing Zhang, Fengguang Qi, Guohua Zheng, Shengli Yan et Xu Ma. « Clinical and Genetic Analysis of a Compound Heterozygous Mutation in the Thyroglobulin Gene in a Chinese Twin Family With Congenital Goiter and Hypothyroidism ». Twin Research and Human Genetics 15, n^o 1 (février 2012) : 126–32. http://dx.doi.org/10.1375/twin.15.1.126.

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Mutations in the thyroglobulin (TG) gene, which has an estimated incidence of approximately 1 in 100,000 new-borns, cause autosomal recessive congenital hypothyroidism. The mutational spectrum of the TG gene and the phenotype–genotype correlations have not yet fully been established. We report a compound heterozygous mutation in the TG gene in a Chinese twin family with congenital goiter and hypothyroidism. We also describe the gene mutation associated with the genotype–phenotype of these children with congenital goiter and hypothyroidism. The whole coding sequence of the TG gene was analyzed by direct sequence, and the identified changes in the sequence were tested for benign polymorphism by denaturing high-performance liquid chromatography screening of the mutation and sequencing 200 chromosomes from normal controls. Analysis of the TG gene of the affected twin revealed a compound heterozygous mutation, including a novel missense mutation G2687A, which is predicted to result in a glutamine to arginine substitution at codon 877, and a known nonsense mutation C7006T, predicted to result in an arginine to stop codon at codon 2317. Analysis of 200 normal chromosomes did not identify the same change in healthy subjects. This is the first report of a TG gene mutation in the Chinese Han population. Our study provides further evidence that mutations in the TG gene cause congenital goiter and hypothyroidism, demonstrates genetic heterogeneity of the mutation, and increases our understanding of phenotype–genotype correlations in congenital hypothyroidism.

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Rao, Vamshi K., Christine J. DiDonato et Paul D. Larsen. « Friedreich’s Ataxia : Clinical Presentation of a Compound Heterozygote Child with a Rare Nonsense Mutation and Comparison with Previously Published Cases ». Case Reports in Neurological Medicine 2018 (9 août 2018) : 1–5. http://dx.doi.org/10.1155/2018/8587203.

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Friedreich’s ataxia is a neurodegenerative disorder associated with a GAA trinucleotide repeat expansion in intron 1 of the frataxin (FXN) gene. It is the most common autosomal recessive cerebellar ataxia, with a mean age of onset at 16 years. Nearly 95-98% of patients are homozygous for a 90-1300 GAA repeat expansion with only 2-5% demonstrating compound heterozygosity. Compound heterozygous individuals have a repeat expansion in one allele and a point mutation/deletion/insertion in the other. Compound heterozygosity and point mutations are very rare causes of Friedreich’s ataxia and nonsense mutations are a further rarity among point mutations. We report a rare compound heterozygous Friedrich’s ataxia patient who was found to have one expanded GAA FXN allele and a nonsense point mutation in the other. We summarize the four previously published cases of nonsense mutations and compare the phenotype to that of our patient. We compared clinical information from our patient with other nonsense FXN mutations reported in the literature. This nonsense mutation, to our knowledge, has only been described once previously; interestingly the individual was also of Cuban ancestry. A comparison with previously published cases of nonsense mutations demonstrates some common clinical characteristics.

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Yu, Ziqiang, Jian Su, Xia Bai, Zhaoyue Wang et Changgeng Ruan. « New Compound Heterozygous Mutations of GPIIb in Patient with Glanzmann Thrombasthenia. » Blood 110, n^o 11 (16 novembre 2007) : 3921. http://dx.doi.org/10.1182/blood.v110.11.3921.3921.

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Abstract Glanzmann thrombasthenia (GT) is a homozygous or compound heterozygous autosomal recessive bleeding disorder caused by the qualitative or quantitative deficiency of integrin GPIIb-IIIa, which acts as the receptor of platelet fibrinogen. Here we report a case of GT with a compound heterozygous mutation in GPIIb according to the results of flow cytometry and genetic investigation.The flow cytometry was used to measure the average amounts of integrin GPIIb-IIIa on the patient’s platelets, and all 30 exons of GPIIb were amplified and sequenced with the corresponding primers.The average fluorescence intensity of integrin GPIIb-IIIa were 3.07 and 12.5, respectively, compared with 23.7 and 254, respectively, in the normal healthy individuals. And sequencing analysis of all exons of GPIIb demonstrated that there existed following compound heterozygous mutations in GPIIb gene: one heterozygote mutation (68 C→A) in the 1st exon, which resulted in Pro 23 His substitution in signal peptide domain; one nonsense heterozygous mutation (1750 C→T) in the 17th exon, which result in premature termination; one heterozygote mutation (2159 T→C) in the 21stexon, which resulted in Leu 720 Pro substitution. According to Glanzmann thrombasthenia database of ISTH (http://sinaicentral.mssm.edu/intranet/research/glanzmann/listmutations?mut=GPIIb), 68 C→A mutation and 2159 T→C mutation are novel mutations in the GPIIb heavy chain. These compound heterozygous mutations in GPIIb gene might be a novel pathogenetic mechanism of GT, which impaired the protein translation and co-expression with GPIIIa on the membrane of platelet.

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Costa, Jean-Marc, Dominique Vidaud, Ingrid Laurendeau, Michel Vidaud, Edith Fressinaud, Jean-Pierre Moisan, Albert David, Dominique Meyer et Jean-Maurice Lavergne. « Somatic mosaicism and compound heterozygosity in female hemophilia B ». Blood 96, n^o 4 (15 août 2000) : 1585–87. http://dx.doi.org/10.1182/blood.v96.4.1585.

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Abstract Sequencing the complete factor IX gene of 2 sisters with hemophilia B with different phenotypes and no family history of hemorrhagic diathesis revealed a common 5′ splice site mutation in intron 3 (T6704C) in both and an additional missense mutation (I344T) in one. The presence of dysfunctional antigen in the latter strongly suggested that these mutations are in trans. Neither mutation was found in leukocyte DNA from the asymptomatic parents, but the mother was in somatic mosaicism for the shared splice site mutation. This case illustrates the importance of defining the phenotype and considering somatic mosaicism in sporadic cases. It underlines the limitations of complete gene sequencing for the detection of mosaicism and has implication for genetic counseling.

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Costa, Jean-Marc, Dominique Vidaud, Ingrid Laurendeau, Michel Vidaud, Edith Fressinaud, Jean-Pierre Moisan, Albert David, Dominique Meyer et Jean-Maurice Lavergne. « Somatic mosaicism and compound heterozygosity in female hemophilia B ». Blood 96, n^o 4 (15 août 2000) : 1585–87. http://dx.doi.org/10.1182/blood.v96.4.1585.h8001585_1585_1587.

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Sequencing the complete factor IX gene of 2 sisters with hemophilia B with different phenotypes and no family history of hemorrhagic diathesis revealed a common 5′ splice site mutation in intron 3 (T6704C) in both and an additional missense mutation (I344T) in one. The presence of dysfunctional antigen in the latter strongly suggested that these mutations are in trans. Neither mutation was found in leukocyte DNA from the asymptomatic parents, but the mother was in somatic mosaicism for the shared splice site mutation. This case illustrates the importance of defining the phenotype and considering somatic mosaicism in sporadic cases. It underlines the limitations of complete gene sequencing for the detection of mosaicism and has implication for genetic counseling.

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Xu, Ying-Yang, et Yu-Xiang Zhi. « A Compound Mutation (c.953C ». Allergy, Asthma & ; Immunology Research 10, n^o 3 (2018) : 285. http://dx.doi.org/10.4168/aair.2018.10.3.285.

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Ma, Yongsheng, Shan Zeng, Dean D. Metcalfe, Cem Akin, Sasa Dimitrijevic, Joseph H. Butterfield, Gerald McMahon et B. Jack Longley. « The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors ; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations ». Blood 99, n^o 5 (1 mars 2002) : 1741–44. http://dx.doi.org/10.1182/blood.v99.5.1741.

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Mutations of c-KIT causing spontaneous activation of the KIT receptor kinase are associated with sporadic adult human mastocytosis (SAHM) and with human gastrointestinal stromal tumors. We have classified KIT-activating mutations as either “enzymatic site” type (EST) mutations, affecting the structure of the catalytic portion of the kinase, or as “regulatory” type (RT) mutations, affecting regulation of an otherwise normal catalytic site. Using COS cells expressing wild-type or mutant KIT, 2 compounds, STI571 and SU9529, inhibited wild-type and RT mutant KIT at 0.1 to 1 μM but did not significantly inhibit the Asp816Val EST mutant associated with SAHM, even at 10 μM. Using 2 subclones of the HMC1 mast cell line, which both express KIT with an identical RT mutation but which differ in that one also expresses the Asp816Val EST mutation, both compounds inhibited the RT mutant KIT, thereby suppressing proliferation and producing apoptosis in the RT mutant-only cell line. Neither compound suppressed activation of Asp816Val EST mutant KIT, and neither produced apoptosis or significantly suppressed proliferation of the cell line expressing the Asp816Val mutation. These studies suggest that currently available KIT inhibitors may be useful in treating neoplastic cells expressing KIT activated by its natural ligand or by RT activating mutations such as gastrointestinal stromal tumors but that neither compound is likely to be effective against SAHM. Furthermore, these results help establish a general paradigm whereby classification of mutations affecting oncogenic enzymes as RT or EST may be useful in predicting tumor sensitivity or resistance to inhibitory drugs.

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Khorashad, Jamshid, Todd W. Kelley, Philippe Szankasi, Lauren T. Adrian, Christopher A. Eide, Matthew S. Zabriskie, Thoralf Lange et al. « Frequency and Clonality of BCR-ABL Compound Mutations in Chronic Myeloid Leukemia »,. Blood 118, n^o 21 (18 novembre 2011) : 3744. http://dx.doi.org/10.1182/blood.v118.21.3744.3744.

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Abstract Abstract 3744 Background: BCR-ABL kinase domain (KD) mutations are a common mechanism of chronic myeloid leukemia (CML) resistance to tyrosine kinase inhibitor (TKI) therapy. It is well known that some patients harbor more than one KD mutation in the same sample, but the frequency of true compound mutations (defined as two or more mutations in the same allele) and the clonal relationships between mutant clones have not been established. Methods: The first group of samples (Group 1) was selected based on evidence of more than one BCR-ABL KD mutation by Sanger sequencing. Samples from a second group of patients (Group 2) who had one mutation in BCR-ABL KD by Sanger sequencing were also analyzed. The BCR-ABL KD was amplified using nested RT-PCR, the final amplicon was shotgun-cloned and 10 clones from each sample were sequenced. Results: Thus far, 18 samples from group 1 have been analyzed. Sequencing of 180 colonies from this group revealed >80 different mutations. Compound mutations were confirmed in 13 samples (Table 1), while 5 samples revealed the two mutations were in separate clones. T315I, F359V, V299L and M351T were over-represented among compound mutations and the T315I/F359V and V299L/M351T compound mutations were each detected in samples from two patients. On average, each patient sample had 7 different mutations per 10 clones (range: 3–14), some were silent or not previously shown to be associated with TKI resistance in biochemical or cell-based assays. In the 13 samples from Group 1 with confirmed compound mutations (Table 1), all respective individual mutations had been shown to confer TKI resistance in biochemical and/or in vivo studies. In 4 patients, the compound mutant clone was non-dominant (≤40% of clones). In 3 of these 4 patients (11, 13, 16; Table 1), both mutations were also detected independently in co-existent clones, while 1 patient (18) showed 2 compound mutant clones (T315/F359V and V299L/M351T) at comparable levels (40% each). In the remaining 9 patients from Group 1, the compound mutant represented >70% of clones. More than 2 mutations were observed in 41/102 screened compound mutant clones from Group 1. The number of mutations in these clones ranged from 3 to 6. Except V299L/M351T/E329G, which was observed in 5/10 screened clones of one patient (1), no clone with more than 2 mutations was detected in more than 1 clone per sample (≤10%). Notably, none of these additional mutations has yet been associated with TKI resistance in biochemical or cell-based assays. So far, samples from 7 patients in Group 2 have been analyzed. Sequencing of the clones from these patients also revealed the presence of low-level compound mutants. On average 30% of the clones had compound mutations (range: 20%-40%) and an average of 5 different mutations were detected among the 10 sequenced clones per patient (range: 3–6), some of which were silent or not previously shown to be associated with TKI resistance in biochemical or cell-based assays. Conclusions: 1. True compound mutations are common in patients with evidence of multiple mutations by direct sequencing. 2. Mutant clones evolve sequentially as well as in parallel, suggesting complex clonal relationships, in which the identical phenotype (mutation) may be acquired independently by multiple clones. 3. Clones harboring compound mutations comprised of more than 2 mutations rarely achieve dominance, suggesting that the number of different mutations compatible with maintenance of BCR-ABL kinase activity may ultimately be limited. 4. Low-level compound mutations are also detected by cloning and sequencing in the samples from patients who have had one detected mutation in screening (Group 2). Disclosures: Lange: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Yamazaki, Tomio, Akira Katsumi, Yoshihiro Okamoto, Toshio Takafuta, Shinobu Tsuzuki, Kazuo Kagami, Isamu Sugiura, Tetsuhito Kojima, Kingo Fujimura et Hidehiko Saito. « Two Distinct Novel Splice Site Mutations in a Compound Heterozygous Patient with Protein S Deficiency ». Thrombosis and Haemostasis 77, n^o 01 (1997) : 014–20. http://dx.doi.org/10.1055/s-0038-1655729.

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SummaryGenetic analysis revealed two distinct novel splice site mutations in a compound heterozygous patient with protein S deficiency. The paternal mutation was a G-to-T transition at position -1 of the acceptor splice site of intron N (Mutation I), and the maternal mutation was a G-to-C transversion at position -1 of the donor splice site of intron C (Mutation II). Both splice site mutations decreased the mutated mRNA accumulation to the same extent, approximately 40% of the normal mRNA. However, the mutations were associated with different phenotypical expressions: the paternal mutant protein S was not detected in vivo, while the maternal mutant protein S was present in the plasma in reduced quantity. Because Mutation I caused a cryptic splicing in the mutated mRNA, resulting in a reading frameshift and premature termination, the predicted mutant protein S might be highly unstable. In contrast, Mutation II led to the substitution of Val46 by Leu, which might be much less deleterious for the synthesis, secretion and stability of the predicted mutant protein S. It was supposed that the different post-translational metabolisms produced the distinct phenotypical expressions of the mutations.

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Smith, Catherine C., Michael Brown, Jason Chin, Corynn Kasap, Sara Salerno, Lauren E. Damon, Kevin Travers et al. « Single Molecule Real Time (SMRT™) Sequencing Sensitively Detects Polyclonal and Compound BCR-ABL in Patients Who Relapse on Kinase Inhibitor Therapy »,. Blood 118, n^o 21 (18 novembre 2011) : 3752. http://dx.doi.org/10.1182/blood.v118.21.3752.3752.

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Abstract Abstract 3752 Background: Secondary kinase domain (KD) mutations are the most well-recognized mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and other cancers. In some cases, multiple drug resistant KD mutations can coexist in an individual patient (“polyclonality”). Alternatively, more than one mutation can occur in tandem on a single allele (“compound mutations”) following response and relapse to sequentially administered TKI therapy. Distinguishing between these two scenarios can inform the clinical choice of subsequent TKI treatment. There is currently no clinically adaptable methodology that offers the ability to distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD where TKI-resistant mutations are detected, next-generation platforms are unable to generate reads of sufficient length to determine if two mutations separated by 500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) circular consensus sequencing technology is a novel third generation deep sequencing technology capable of rapidly and reliably achieving average read lengths of ∼1000bp (Travers et al, 2010) and frequently beyond 3000bp, allowing sequencing of the entire ABL KD on single strand of DNA. We sought to address the ability of SMRT sequencing technology to distinguish polyclonal from compound mutations using clinical samples obtained from patients who have relapsed on BCR-ABL TKI treatment. Results: We analyzed an 863bp area of the BCR-ABL KD in 6 patients who had clinically relapsed on ABL kinase inhibitor therapy. SMRT sequencing detected mutations at a sensitivity of ∼1–2% of the total sequenced population, and successfully distinguished polyclonal from compound BCR-ABL KD mutations in several patient samples. Results were largely consistent with those obtained by PCR subcloning and sequencing, although SMRT sequencing detected additional mutations and/or mutation combinations. In the most complex case, 7 distinct mutation-bearing alleles were detected in an individual patient after sequential relapse on imatinib and dasatinib. Mutant clones contained single and compound mutations combining distinct mutations (Y253H, T315F, T315A, T315I, T319A, E355G). Three distinct substitutions at residue T315 were detected: T315A, T315I and T315F. Notably, these findings are clinically important as the T315A mutation confers resistance to dasatinib but not imatinib, while the T315F and T315I mutations are resistant to all three clinically approved BCR/ABL inhibitors (imatinib, dasatinib, and nilotinib). Phospho-flow analysis for p-Crkl, a direct substrate of BCR-ABL, was conducted following ex vivo exposure of patient cells from the same time point to all three BCR-ABL inhibitors, and demonstrated the existence of distinct populations of cells with varying sensitivity to each drug (i.e. polyclonal drug sensitivity), underscoring the potential clinical importance of distinguishing polyclonal from compound mutations. Additionally, SMRT sequencing routinely detected alleles harboring compound mutations not detectable by conventional direct sequencing. Data analysis of samples from additional patients is ongoing and will be presented. Conclusions: Pacific Biosciences RS SMRT sequencing sensitively detects KD mutations in patient samples and can distinguish TKI-resistant clones containing compound mutations to reveal a complex mutational landscape in an individual patient not detectable by conventional sequencing. SMRT sequencing of the BCR-ABL KD can feasibly be developed into a rapid and economical clinical test with the additional advantages of increased sensitivity and reliability over current methods. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles promises to further facilitate a personalized approach to patient management. Disclosures: Brown: Pacific Biosciences: Employment. Chin:Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.

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Sakuma, Naoko, Hideaki Moteki, Hela Azaiez, Kevin T. Booth, Masahiro Takahashi, Yasuhiro Arai, A. Eliot Shearer et al. « Novel PTPRQ Mutations Identified in Three Congenital Hearing Loss Patients With Various Types of Hearing Loss ». Annals of Otology, Rhinology & ; Laryngology 124, n^o 1_suppl (18 mars 2015) : 184S—192S. http://dx.doi.org/10.1177/0003489415575041.

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Objectives: We present 3 patients with congenital sensorineural hearing loss (SNHL) caused by novel PTPRQ mutations, including clinical manifestations and phenotypic features. Methods: Two hundred twenty (220) Japanese subjects with SNHL from unrelated and nonconsanguineous families were enrolled in the study. Targeted genomic enrichment with massively parallel DNA sequencing of all known nonsyndromic hearing loss genes was performed to identify the genetic cause of hearing loss. Results: Four novel causative PTPRQ mutations were identified in 3 cases. Case 1 had progressive profound SNHL with a homozygous nonsense mutation. Case 2 had nonprogressive profound SNHL with a compound heterozygous mutation (nonsense and missense mutation). Case 3 had nonprogressive moderate SNHL with a compound heterozygous mutation (missense and splice site mutation). Caloric test and vestibular evoked myogenic potential (VEMP) test showed vestibular dysfunction in Case 1. Conclusion: Hearing loss levels and progression among the present cases were varied, and there seem to be no obvious correlations between genotypes and the phenotypic features of their hearing loss. The PTPRQ mutations appeared to be responsible for vestibular dysfunction.

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Wang, Chunli, Ying Chen, Bixia Zheng, Mengshu Zhu, Jia Fan, Juejin Wang, Zhanjun Jia, Songming Huang et Aihua Zhang. « Novel compound heterozygous CLCNKB gene mutations (c.1755A>G/c.848_850delTCT) cause classic Bartter syndrome ». American Journal of Physiology-Renal Physiology 315, n^o 4 (1 octobre 2018) : F844—F851. http://dx.doi.org/10.1152/ajprenal.00077.2017.

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Inactivated variants in CLCNKB gene encoding the basolateral chloride channel ClC-Kb cause classic Bartter syndrome characterized by hypokalemic metabolic alkalosis and hyperreninemic hyperaldosteronism. Here, we identified two cBS siblings presenting hypokalemia in a Chinese family due to novel compound heterozygous CLCNKB mutations (c.848_850delTCT/c.1755A>G). Compound heterozygosity was confirmed by amplifying and sequencing the patientʼs genomic DNA. The synonymous mutation c.1755A>G (Thr585Thr) was located at +2 bp from the 5′ splice donor site in exon 15. Further transcript analysis demonstrated that this single nucleotide mutation causes exclusion of exon 15 in the cDNA from the proband and his mother. Furthermore, we investigated the expression and protein trafficking change of c.848_850delTCT (ΔTCT) and exon 15 deletion (ΔE15) mutation in vitro. The ΔE15 mutation markedly decreased the expression of ClC-Kb and resulted in a low-molecular-weight band (~55 kDa) trapping in the endoplasmic reticulum, while the ΔTCT mutant only decreased the total and plasma membrane ClC-Kb protein expression but did not affect the subcellular localization. Finally, we studied the physiological functions of mutations by using whole cell patch-clamp and found that the ΔE15 or ΔTCT mutation decreased the current of the ClC-Kb/barttin channel. These results suggested that the compound defective mutations of the CLCNKB gene are the molecular mechanism of the two cBS siblings.

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Kesarwani, Meenu, Zachary Kincaid et Mohammad Azam. « DUSP1 Confers Oncogene Dependence in CSF3R Induced Leukemia ». Blood 132, Supplement 1 (29 novembre 2018) : 1341. http://dx.doi.org/10.1182/blood-2018-99-119092.

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Abstract In 2013, Maxson and colleagues reported mutations in colony stimulating factor-3 (CSF3R) from chronic neutrophilic leukemia (CNL), and atypical CML (aCML) patients. These mutations are clustered into two different regions of the receptor protein; membrane proximal region (proximal mutation) and frameshift or non-sense mutations in the cytoplasmic tail (truncation mutation). Further analysis revealed a significant majority of patients harbor both membrane-proximal and truncation mutations on the same allele (compound mutations). In vivo analysis of these mutants using mouse models revealed that both membrane proximal and compound mutations induce aggressive leukemia while truncation mutations are non-leukemic. Further studies showed that proximal mutation is sensitive to both JAK2 and MAPK inhibitors while compound mutation is only sensitive to MAPK inhibition but resistant to JAK2 inhibition. Unfortunately, unlike imatinib (BCR-ABL inhibitor) both clinically approved JAK2 inhibitor (ruxolitinib) and MEK1/2 inhibitor (trametinib) are not cytotoxic. Instead, they are cytostatic. In other words, both of these inhibitors lack clonal selectivity for the leukemic cells. While treatment with Jak2 or Mek1/2 inhibitors provide some relief to patients, but the responses are short-lived, as leukemic cells in a very short period of time (4-6 months) adapt to thrive under Jak2 and Mek1/2 inhibition resulting to loss of treatment response and disease relapse. In addition, prolonged inhibition of JAK2 and MEK/ERK are seemingly detrimental for normal hematopoietic and adult tissue homeostasis. These observations warrant identifying additional therapeutic targets that must be safe and able to eliminate the mutant clone. A recent study using CML as a model system to understand the mechanisms driving therapeutic response (clonal selectivity) to TKI (tyrosine kinase inhibitor) treatment demonstrated that the AP1 transcription factor c-Fos and dual specificity phosphatase, Dusp1, regulate the therapeutic response to drug treatments. Perhaps more interestingly, these studies provided evidence that the expression levels of c-Fos and Dusp1 determine apoptotic thresh-hold in cancer cells such that lower levels confer sensitivity, while higher levels drive resistance to TKI treatments (Kesarwani M, et al. Nature Medicine, 2017). Results We hypothesized that CSF3R induced CNL might have higher expression of c-Fos and Dusp1 resulting to a higher apoptotic threshold, and thus, abrogating the cytotoxic effect of Jak2 and Mek1/2 inhibitors. To test this, we performed gene expression analysis of bonemarrow cells expressing proximal, truncation, compound mutations and pMSCV-Ires-GFP (as a control). These analyses revealed elevated 4-8-fold overexpression of Dusp1 in cells expressing membrane proximal and compound CSF3R mutations in comparison to vector control and non-leukemic truncation mutations. Further genetic studies using Dusp1 knock-out mice showed dependence of oncogenic CSF3R (proximal and compound) on the Dusp1. Genetic deletion of Dusp1 in cells expressing oncogenic CSF3R (proximal and compound) is synthetic lethal (Fig. 1). Mice transplanted with CSF3RT618I (proximal mutation) or CSF3RT618I/Q741X (compound mutation) expressing cells lacking Dusp1 show gradual depletion of leukemic cells where leukemic cells are completely cleared within 4-7 weeks (Fig. 1E). Rate of clearance is much faster in compound mutation, cells are cleared within 4 weeks after the transplantation (Fig. 1E). In contrast, mice transplanted with cells expressing pMSCV-Ires-GFP (vector) and MPLW515L (another receptor-oncogene induces myeloproliferative neoplasm) show stable engraftment. These data provide evidence that the oncogenic CSF3R is uniquely addicted to Dusp1 and crucial for disease development. Therefore, Dusp1 targeting in CSF3R mutated leukemias may exert a stable and curative response. Conclusion. We observed Dusp1 overexpression in CNL expressing oncogenic CSF3R (proximal and compound), but not in cells expressing non-oncogenic CSF3R (truncation mutations). Oncogenic CSF3R is uniquely addicted to the DUSP1 expression, where genetic deletion of DUSP1 is lethal to CSF3R driven leukemias but not to the other leukemic drivers, such as, MPLW515L. Thus, providing a rationale for targeting the DUSP1 in CSF3R driven CNL to eliminate the mutant clones for curative response. Disclosures No relevant conflicts of interest to declare.

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Zhang, Jia, et Zhao Wang. « Pedigree Gene Investigation and Parameters of NK Cell Activity, CD107a Degranulation Amd HLH Related Defective Protein Play Significant Role in the Diagnosis of Primary HLH ». Blood 128, n^o 22 (2 décembre 2016) : 4876. http://dx.doi.org/10.1182/blood.v128.22.4876.4876.

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Abstract Abstract This study aims to evaluate the significance of pedigree genetic screening and rapid immunological parameters in the diagnosis of primary HLH and explore their correlation. Twelve Chinese families of primary HLH with mutations in PRF1, UNC13D, LYST, RAB27A, SH2D1A, BIRC4 were recruited and conducted pedigree investigation, including family genetic screening, NK cell activity, CD107a degranulation and expression of HLH related defective protein. Ten patients were identified with homozygous, compound heterozygous, or hemizygous mutations in PRF1, UNC13D, RAB27A, SH2D1A and BIRC4, including one case of homozygous mutation (c.1349C>T:p.T450M) and four cases of compound heterozygous mutations (frameshift deletion c.65delC:p.P22fs and c.G503A:p.S168N; c.1168C>Tp.R390X and c.1349C>T:p.T450M; frameshift mutation c.1090_1091delCT:p.T364fsX93 and c.1349C>T:p.T450M; non-frameshift deletions c.1083_1094del:p.361_365del and c.T172C :p.S58P) in PRF1, one case of homozygous mutation (c.G2588A:p.G863D) and one compound heterozygous (c.C640T:p.R214X and c.G407A:p.C136Y) in UNC13D, one case of homozygous mutation (c.C244T:p.R82C) in RAB27A, and two male cases of hemizygous mutations in SH2D1A(c.32T>G:p.I11S) and BIRC4(c.G592A:p.V198M). Meanwhile, two patients were identified mutations in two or three different gene, including a male with a hemizygous mutation in SH2D1A(c.7G>T:p.A3S) and a heterozygous mutation(c.127C>A: p.L43M) in PRF1, and a female with three hemizygous mutations respectively in UNC13D(c.G680A:p.R227H), LYST(c.A8368C:p.K2790Q) and BIRC4(c.C962G:p.A321G).The primary HLH patients and their family members presented different levels of decreased NK cell activity. Individuals who were found mutations in PRF1, SH2D1A and BIRC4 showed low expression of perforin, SAP and XIAP. And the patients with homozygous and compound heterozygous mutations in UNC13D and RAB27A showed significant reducing of cytotoxic degranulation function and their family members presented normal or different levels of decreased CD107a. The patient with combination defects involving two genes(UNC13D and LYST) in the degranulation pathway showed decreased of CD107a degranulation which implied a digenic inheritance and diagnosed primary HLH. Pedigree genetic screening and rapid detection of immunological parameters play an important role in the diagnosis of primary HLH and demonstrate good consistency. Disclosures No relevant conflicts of interest to declare.

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Ray, Rudra, Ankita Biswas, Sunistha Bhattacharjee et Maitreyee Bhattacharyya. « Phenotypes of Hb Okayama Mutation ». Blood 132, Supplement 1 (29 novembre 2018) : 4898. http://dx.doi.org/10.1182/blood-2018-99-118079.

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Abstract Introduction: Since its first detection in the year of 1983 very little has been reported about Hb Okayama mutation. Hb Okayama, reported as a silent mutation (Globin Gene Server Hb Var ID: 220, dbSNP rs713040), happened to be detected in the process of HPLC analysis for the measurement of HbA1c in Japanese diabetic patient [1,2,3]. Till date only few Hb Okayama has been reported from Japanese and Austrian ethnicity [2,3,4]. Its phenotypes and co-inheritance with other beta globin gene mutation is not yet known. In this study we report phenotypes of Hb Okayama in heterozygous as well as compound heterozygous state when it is co-existing with a common beta globin gene mutation. To the best of our knowledge no such study has been reported in world literature. Methodology: Two groups of patients- beta carriers requiring blood transfusion; and patients with low MCV, MCH but normal in HPLC were further investigated by molecular analysis. ARMS PCR analysis for beta globin gene mutation detection, GAP PCR for alpha deletion and triplication analysis and DNA sequencing (ABI 3500 Genetic Analyzer) to identify rare beta mutations were carried out. Result: Among around 300 patients subjected to molecular investigation over last three years there were 118 cases of thalassaemia trait (by HPLC and confirmed to carry a heterozygous common beta mutations by ARMS PCR analysis), but were requiring blood transfusion or behaving like intermedia. They were subjected to alpha globin gene triplication analysis by GAP PCR. There were 84 cases found to carry absence of alpha triplication with heterozygous beta mutation. Beta globin gene sequencing analysis of these 84 patients revealed that there were 4 patients carrying Okayama heterozygous mutation [ Figure 1, Figure 2 ] along with a common heterozygous beta gene mutation ( IVS 1-5 G>C mutation). HPLC report of these patients carrying Hb Okayama along with IVS 1-5 mutations in compound heterozygous state showed increased HbA2 values like that of beta trait [Table 1 ]. Another group of patients with low MCV, MCH but Normal in HPLC were subjected to alpha deletion analysis after ruling out low Ferritin level. There were 72 patients showing absence of alpha deletion by GAP PCR analysis and carried low MCV, MCH value with Normal HPLC parameters were also subjected to beta globin gene sequencing which revealed the presence of Hb Okayama mutation among two patients in heterozygous state [Figure- 1, Figure 2], who had HbA2 values in normal or border line range with low MCV, MCH levels [Table-2]. Discussion: HbA2 measurement is used as the marker for screening of beta trait. Silent beta-thalassaemia carriers represent normal HbA2 level which makes their identification difficult. Okayama mutation or Hb Okayama is known to be a silent mutation [1-4] with normal HPLC. Hb Okayama is structural beta variant with a change of amino acid ( His > Gln) at Codon 2 (CD 2). Change in the nucleotide sequence at 70603 position from T to A or C (CAT>CAA or CAG) of beta globin gene (NG_000007.3) causes this mutation. The phenotypic associations of Hb Okayama in thalassaemia have very little been known till date. There has been report of very high expression of HbF(70%) value in HPLC resulting from compound heterozygous mutations one of which being silent (Cap+1) [ 5] ; where as there are also reports where the phenotypes of compound heterozygous including a silent mutation showing normal HPLC parameters [ 6 ]. However, in those studies no information about the clinical history and blood transfusion is described. In this study Hb Okayama heterozygous co-inheriting with IVS1-5(G>C) heterozygous mutation showed beta trait like HPLC parameters though all the patients carrying these compound heterozygous mutations required blood transfusion. The silent feature of Hb Okayama was evident in the case of the patients carrying only Hb Okayama mutation who showed absolutely normal HPLC parameters with low MCV, MCH and none of them requiring blood transfusion. Conclusion: Beta globin gene expression analysis to understand the association of Hb Okayama mutation in heterozygous and compound heterozygous states will enable to explain the mechanism of its phenotypes. How this mutation interferes with the expression of HbF or switching of delta globin gene is also to be understood as the HbF levels in HPLC was found to be like beta traits in this study. Disclosures No relevant conflicts of interest to declare.

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Porzio, O., V. Cunsolo, M. Malaponti, E. De Nisco, A. Acquafredda, L. Cavallo, M. Andreani et al. « Divergent Phenotype of Two Siblings Human Leukocyte Antigen Identical, Affected by Nonclassical and Classical Congenital Adrenal Hyperplasia Caused by 21-Hydroxylase Deficiency ». Journal of Clinical Endocrinology & ; Metabolism 91, n^o 11 (1 novembre 2006) : 4510–13. http://dx.doi.org/10.1210/jc.2006-0779.

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Abstract Context: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders most often caused by enzyme 21-hydroxylase deficiency. Most mutations causing enzymatic deficiency are generated by recombinations between the active gene CYP21 and the pseudogene CYP21P. Only 1–2% of affected alleles result from spontaneous mutations. The phenotype of CAH varies greatly, usually classified as classical or nonclassical, depending on variable degree in 21-hydroxylase activity. Here we report a divergent phenotype of two human leukocyte antigen identical siblings, affected by nonclassical and classical CAH caused by 21-hydroxylase deficiency due to different genotype. Patients and Methods: Using direct sequencing method and Southern blot, we studied two children (one male and one female), affected, respectively, by nonclassical and classical CAH and their parents. Results: The mother was heterozygous for the Q318X mutation, and the father was heterozygous for the V281L mutation. The brother was a compound heterozygote for the mutations V281L and Q318X, whereas the proband was compound heterozygote for the Q318X mutation and a large conversion. The two children are human leukocyte antigen identical (A*02;B*14;DRB1*01/A*33;B*14;DRB1*03). Conclusions: Different phenotype of the proband is the result of compound heterozygosity for the maternal mutation Q318X and a de novo large conversion.

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Van Wesenbeeck, L., E. Rondelez, M. Feyaerts, A. Verheyen, K. Van der Borght, V. Smits, C. Cleybergh, H. De Wolf, K. Van Baelen et L. J. Stuyver. « Cross-Resistance Profile Determination of Two Second-Generation HIV-1 Integrase Inhibitors Using a Panel of Recombinant Viruses Derived from Raltegravir-Treated Clinical Isolates ». Antimicrobial Agents and Chemotherapy 55, n^o 1 (18 octobre 2010) : 321–25. http://dx.doi.org/10.1128/aac.01733-09.

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ABSTRACTThe integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from raltegravir-treated patients (baseline and follow-up samples), were used to study the cross-resistance profile of two second-generation integrase inhibitors, MK-2048 and compound G. Samples with Q148H/R mutations had elevated fold change values with all compounds tested. Although samples with the Y143R/C mutation had reduced susceptibility to RAL, they remained susceptible to MK-2048 and compound G. Samples with the N155H mutation had no reduced susceptibility to compound G. In conclusion, our results allowed ranking of the INIs on the basis of the antiviral activities using recombinant virus stocks from RAL-treated patient viruses. The order according to decreasing susceptibility is compound G, MK-2048, and EVG.

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Shao, Hongxia, Jingna Hua, Qi Wu, Xiaoge Li, Ming Zhang, Herong Wang, Junping Wu et al. « Identification of a Mutation in the Novel Compound Heterozygous CFTR in a Chinese Family with Cystic Fibrosis ». Canadian Respiratory Journal 2020 (7 mai 2020) : 1–5. http://dx.doi.org/10.1155/2020/6507583.

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Cystic fibrosis (CF) is one of the most common autosomal recessive disorders among Caucasians of Northern European descent but is uncommon in the Chinese population. Objectives. To elucidate the mutation in the novel compound heterozygous CFTR causing CF in Chinese family. Materials and Methods. Clinical samples were obtained from a Chinese family, the brother and sister with recurrent airway infections, hypoxemia and obstructive ventilatory impairment, sinusitis, clubbed fingers, salty sweat, and nasal polyposis. We performed whole-exome sequencing on the family and validated all potential variants by Sanger sequencing. Results. Next-generation sequencing showed a novel compound heterozygous CFTR mutation (c.400 A > G p.Arg134Gly and c.3484 C > T p.Arg1162∗) which resulted in CF in the family. Conclusions. As this mutation is consistent with the observed clinical manifestations of CF and no other mutations were detected after scanning the gene sequence, we suggest that their CF phenotypes are caused by the compound heterozygous mutation, c.400 A > G p.Arg134Gly and c.3484 C > T p.Arg1162∗. As c.400 A > G is not currently listed in the Cystic Fibrosis Mutation Database, this information, regarding the CF-causing mutations in two Chinese patients, is of interest.

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Ura, Hiroki, Sumihito Togi et Yo Niida. « Targeted Double-Stranded cDNA Sequencing-Based Phase Analysis to Identify Compound Heterozygous Mutations and Differential Allelic Expression ». Biology 10, n^o 4 (24 mars 2021) : 256. http://dx.doi.org/10.3390/biology10040256.

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There are two combinations of heterozygous mutation, i.e., in trans, which carries mutations on different alleles, and in cis, which carries mutations on the same allele. Because only in trans compound heterozygous mutations have been implicated in autosomal recessive diseases, it is important to distinguish them for clinical diagnosis. However, conventional phase analysis is limited because of the large target size of genomic DNA. Here, we performed a genetic analysis on a patient with Wilson disease, and we detected two heterozygous mutations chr13:51958362;G>GG (NM_000053.4:c.2304dup r.2304dup p.Met769HisfsTer26) and chr13:51964900;C>T (NM_000053.4:c.1841G>A r.1841g>a p.Gly614Asp) in the causative gene ATP7B. The distance between the two mutations was 6.5 kb in genomic DNA but 464 bp in mRNA. Targeted double-stranded cDNA sequencing-based phase analysis was performed using direct adapter ligation library preparation and paired-end sequencing, and we elucidated they are in trans compound heterozygous mutations. Trio analysis showed that the mutation (chr13:51964900;C>T) derived from the father and the other mutation from the mother, validating that the mutations are in trans composition. Furthermore, targeted double-stranded cDNA sequencing-based phase analysis detected the differential allelic expression, suggesting that the mutation (chr13:51958362;G>GG) caused downregulation of expression by nonsense-mediated mRNA decay. Our results indicate that targeted double-stranded cDNA sequencing-based phase analysis is useful for determining compound heterozygous mutations and confers information on allelic expression.

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Byeon, Yeji, Seung Hee Jung, Daseul Yoon, Seock Yong Kang, Jiyeon Park, Hyeim Jo, Seong-Il Choi et al. « Abstract 5477 : Compound A, a fourth-generation allosteric inhibitor, a potent and highly selective EGFR with L858R activating and C797S resistance mutations for the treatment of NSCLC ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 5477. http://dx.doi.org/10.1158/1538-7445.am2022-5477.

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Abstract The L858R activating mutation in Epidermal Growth Factor Receptor (EGFR) accounts for approximately 40% of EGFR-mutant non-small cell lung cancer (NSCLC). These patients receive targeted therapy targeting EGFR mutation. To date all approved EGFR tyrosine kinase inhibitors (TKIs) are ATP competitive inhibitors, inevitably lead to drug resistance and to disease progress. Osimertinib is a third-generation EGFR TKI that selectively inhibits both EGFR TKI-sensitizing (L858R) mutation and T790M mutation that confers acquired resistance to first- and second-generation EGFR-TKIs. The patients develop secondary resistance L858R/C797S and L858R/T790M/C797S to this treatment in some cases. There have been strong unmet needs for specific therapies targeting C797S resistance mutations. Additionally, toxicities caused by inhibition of wild-type (WT) EGFR are frequently reported with the first-generation inhibitors (e.g. Gefitinib and Erlotinib). Our Compound A is a highly mutant-selective fourth-generation allosteric inhibitor that targets L858 activating mutations and without affecting EGFR WT that compares favorably with all ATP competitive inhibitors, indicating that it might avoid potential toxicities from targeting EGFR WT. Compound A has a single-digit nanomolar potency against L858R activating mutation and those harboring T790M and C797S resistance mutations. Oral administration of Compound A to tumor-bearing mice showed significant antitumor activity as a single-agent at well-tolerated doses via inhibiting EGFR pathway in the engineered osimertinib-resistance EGFRL858R/T790M/C797S and EGFRL858R/C797S CDX models. Furthermore, Compound A demonstrated excellent efficacy in EGFRL858R/T790M CDX model as a single-agent. Collectively, Compound A has a robust potency against L858R activating mutations including T790M, C797S, and T790M/C797S that cause acquired resistance to ATP competitive EGFR TKIs in vitro and in vivo. Based on the preclinical data, Compound A is a potent, orally available, and mutant-selective fourth-generation allosteric inhibitor of EGFR with L858R activating mutations for the treatment of NSCLC, suggesting that it might have the potential to demonstrate activity in the first line and resistance settings as a single agent. The pre-clinical data described here supports the clinical development of Compound A in NSCLC patients with L858R activating mutations of EGFR. Citation Format: Yeji Byeon, Seung Hee Jung, Daseul Yoon, Seock Yong Kang, Jiyeon Park, Hyeim Jo, Seong-Il Choi, Somyi Park, Seung-chul Lee, Yang Hun Tae, Tae Min Kim, Sung-Yup Cho, Soyeon Kim, Donghyun Ko, Dong-Kyu Kim, Dong-Wan Kim. Compound A, a fourth-generation allosteric inhibitor, a potent and highly selective EGFR with L858R activating and C797S resistance mutations for the treatment of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5477.

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Qiu, Yue, Sen Chen, Le Xie, Kai Xu, Yi Lin, Xue Bai, Hui-Min Zhang et al. « Auditory Neuropathy Spectrum Disorder due to Two Novel Compound Heterozygous OTOF Mutations in Two Chinese Families ». Neural Plasticity 2019 (18 novembre 2019) : 1–7. http://dx.doi.org/10.1155/2019/9765276.

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Auditory neuropathy spectrum disorder (ANSD), also called auditory neuropathy (AN), is a unique type of prelingual hearing impairment. Up to 10% of deaf infants and children are affected by this disease. Mutation of the OTOF gene which encodes otoferlin is the common cause of congenital nonsyndromic ANSD. To date, over 110 mutations have been identified in the OTOF gene according to the Human Gene Mutation Database (HGMD). Here, next-generation sequencing (NGS) revealed that the compound heterozygous mutations c.4748G>A/c.2523+1G>T and c.5248G>C/c.5098G>C of the OTOF gene were present in two Chinese ANSD patients. Each patient had a known pathogenic mutation (c.4748G>A or c.5098G>C) and a novel mutation (c.2523+1G>T or c.5248G>C). Comparative amino acid sequence analysis across different species revealed that the residues at these novel mutation sites are evolutionarily highly conservative. This indicated that the novel mutations were possible causes of the disorder in the patients. Our findings extend the OTOF mutation spectrum and further confirm the role of the OTOF gene in ANSD.

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Bauer, Lisa, Roberto Manganaro, Birgit Zonsics, Daniel L. Hurdiss, Marleen Zwaagstra, Tim Donselaar, Naemi G. E. Welter et al. « Rational design of highly potent broad-spectrum enterovirus inhibitors targeting the nonstructural protein 2C ». PLOS Biology 18, n^o 11 (6 novembre 2020) : e3000904. http://dx.doi.org/10.1371/journal.pbio.3000904.

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There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine—which also targets 2C—but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.

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Krone, Nils, Andreas Braun, Adelbert Anton Roscher, Dietrich Knorr et Hans Peter Schwarz. « Predicting Phenotype in Steroid 21-Hydroxylase Deficiency ? Comprehensive Genotyping in 155 Unrelated, Well Defined Patients from Southern Germany ». Journal of Clinical Endocrinology & ; Metabolism 85, n^o 3 (1 mars 2000) : 1059–65. http://dx.doi.org/10.1210/jcem.85.3.6441.

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Abstract Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders. CAH is most often caused by deficiency of steroid 21-hydroxylase. The frequency of CYP21-inactivating mutations and the genotype-phenotype relationship were characterized in 155 well defined unrelated CAH patients. We were able to elucidate 306 of 310 disease-causing alleles (diagnostic sensitivity, 98.7%). The most frequent mutation was the intron 2 splice site mutation (30.3%), followed by gene deletions (20.3%), the I172N mutation (19.7%) and large gene conversions (7.1%). Five point mutations were detected that have not been described in other CAH cohorts. Genotypes were categorized in 4 mutation groups (null, A, B, and C) according to their predicted functional consequences and compared to the clinical phenotype. The positive predictive value for null mutations (ppvnull) was 100%, as all patients with these mutations had a salt-wasting phenotype. In mutation group A (intron 2 splice site mutation in homozygous or heterozygous form with a null mutation), the ppvA to manifest with salt-wasting CAH was 90%. In group B predicted to result in simple virilizing CAH (I172N in homozygous or compound heterozygous form with a more severe mutation), ppvB was 74%. In group C (P30L, V281L, P453S in homozygous or compound heterozygous form with a more severe mutation), ppvC was 64.7% to exhibit the nonclassical form of CAH, but 90% when excluding the P30L mutation. Thus, in general, a good genotype-phenotype relationship is shown in patients with either the severest or the mildest mutations. A considerable degree of divergence is observed within mutation groups of intermediate severity. As yet undefined factors modifying 21-hydroxylase gene expression and steroid hormone action are likely to account for these differences in phenotypic expression.

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Ji, Yinghua, Jin Wang, Xiangli Meng, Jinling Xie, Ping Lu, Mengmeng Li, Ningning Luo, Yingxue Qi et Xiaofeng Zhu. « Molecular characteristics of EGFR exon20 mutations in NSCLC patients. » Journal of Clinical Oncology 40, n^o 16_suppl (1 juin 2022) : e21011-e21011. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e21011.

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e21011 Background: EGFR exon 20 mutations are associated with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). EGFR exon 20 mutations are usually not present alone, suggesting a more complex subtype of exon 20 mutations. Methods: In this study, we retrospectively analyzed the different subtypes and compound mutation profiles of EGFR 20 mutations. A total of 1,233 NSCLC patients’ tumors were collected and the mutations were detected by next-generation sequencing (NGS) from May 2020 to June 2021. Results: A total of 415 NSCLC patients' tumors harbored EGFR exon 20 mutations, accounting for 33.66% of the entire EGFR mutation cohort. Among these 415 patients, most of mutations were single-nucleotide variant (SNV) (379; 91.33%), and a few were indel or insertion mutations (39; 9.40%). The insertions were all in the back of exon 20, which were resistant to EGFR-TKIs. In addition, the subtypes of SNV were concentrated in T790M (270; 71.24%), S768I (48; 12.66%) and C797S (33; 8.71%), and all T790M mutations were accompanied by other mutations. Notably, the compound mutations of exon 20 have also been analyzed in this study. EGFR exon 20 combined with other EGFR mutations were detected in 345 patients. The compound mutations of exon 20-18, exon 20-19, exon 20-20 and exon 20-21 were detected in tumors from 52 (15.07%), 163 (47.25%), 43 (12.46%) and 140 (40.58%) patients respectively. Remarkably, the compound mutation subtype of exon 20-20 was T790M/C797X (29; 67.55%) and exon 20-21 was T790M/L858R (124; 88.57%). Conclusions: Diverse patterns of SNV mutations are seen in EGFR exon 20 mutations tumors. EGFR 20 mutations often accompany other mutations, which are associated with resistance to EGFR-TKIs. These data may help avoid the resistance of future adjuvant targeted therapy.

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Smith, Catherine C., Michael Brown, Wendy T. Parker, Kimberly Lin, Kevin Travers, Susana Wang, Susan Branford et Neil Shah. « Single Molecule Real Time (SMRT™) Sequencing Sensitively Detects the Evolution of Polyclonal and Compound BCR-ABL Mutations in Patients Who Relapse On Kinase Inhibitor Therapy ». Blood 120, n^o 21 (16 novembre 2012) : 917. http://dx.doi.org/10.1182/blood.v120.21.917.917.

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Abstract Abstract 917 Background: Secondary kinase domain (KD) mutations represent the most well-documented mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). In CML, multiple TKIs with different mutation profiles are approved and the ability to detect KD mutations at the time of disease progression can impact therapy choice. To optimize clinical impact, second generation TKI selection must consider the majority TKI-resistant mutant population as well as smaller mutant sub-populations that may be selected with subsequent treatment. Sequential TKI therapy is associated with additional complexity: multiple mutations can coexist separately in an individual patient (“polyclonality”) or can occur in tandem on a single allele (“compound mutations”). Multiple mutations are associated with poor clinical outcome (Parker et al., Blood 2012). Compound mutations can cause in vitro resistance to ponatinib, the only TKI clinically active against the highly resistant T315I mutation (Eide, et. al, ASH 2012 abstract #1416). Currently, no clinically adaptable technology can distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD, most next-generation sequencing platforms cannot generate reads of sufficient length to determine if mutations separated by ≥500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) sequencing technology is a third generation deep sequencing technology capable of achieving average read lengths of ∼1000bp and frequently >3000bp, enabling sensitive and accurate sequencing of the entire ABL KD on a single strand of DNA. Though allele-specific detection methods such as MassARRAY offer sensitivity as low as ∼0.5%, these assays are designed to detect a limited number (∼31) of mutations whereas SMRT sequencing offers an unbiased approach capable of detecting novel variants. We sought to (1) develop a potential clinically-applicable SMRT sequencing assay for the detection of BCR-ABLKD mutations capable of distinguishing polyclonal and compound mutations, and (2) compare the accuracy and sensitivity of this method to standard sequencing and MassARRAY. Results: We assessed 54 samples from 36 CML patients who had clinically relapsed on ABL kinase inhibitor therapy and were previously analyzed by standard sequencing, and in a subset, by MassARRAY. We amplified an 863bp area of the BCR-ABLKD from patient-derived cDNA with primers containing 5' barcodes, enabling sequencing of 6 to 8 patient samples on a single SMRT cell on a single run. On average, 2519 reads were obtained for each sample per run (range 330 to 10,240). All of 131 known mutations detected by MassARRAY were identified by SMRT sequencing using a p-value threshold of 1.03e–03. SMRT sequencing also identified all 107 known mutations detected by direct sequencing with a p-value threshold of 6.0e–08. In addition to these known mutations, SMRT sequencing detected an additional 1320 non-silent mutations across all patient samples using a strict p-value threshold cut-off of 6e–08, ranging in abundance from 0.2% to 17% (median 0.75%). Among 47 samples where >1 mutation was detectable by direct sequencing or MassARRAY, SMRT sequencing revealed that 40 (85%) had compound mutations detectable at a frequency of ≥1. In total, we detected 73 different compound mutations at a frequency of ≥1%. In all cases where compound mutations were detected and more than one treatment timepoint was available, at least one compound mutation clearly evolved from a mutation detectable at a prior timepoint. In the most complex case, 4 separate mutations yielded 8 different mutant alleles. Conclusions: Pacific Biosciences RS SMRT sequencing detects KD mutations in patient samples with sensitivity comparable to or better than MassARRAY and can distinguish compound from polyclonal mutant clones. Among patient samples with multiple mutations, compound mutations were detectable in the vast majority of samples by SMRT sequencing, revealing a complex mutational landscape not demonstrable by other clinically viable sequencing methods and previously unappreciated. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles by SMRT sequencing promises to further facilitate a personalized approach to patient management and inform models of disease evolution. Disclosures: Brown: Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Branford:Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cepheid : Consultancy. Shah:ARIAD, Bristol Myers-Squibb: Consultancy, Research Funding; Novartis: Consultancy.

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Le, Phan Tuong Quynh, Thanh Nha Uyen Le, Thi Thanh Binh Nguyen, Minh Thao Nguyen et Thi Minh Thi Ha. « An Extremely Rare SRD5A2 Gene c.485A>C Mutation in a Compound Heterozygous Newborn with Disorders of Sex Development First Identified in Vietnam ». Case Reports in Endocrinology 2022 (27 mars 2022) : 1–5. http://dx.doi.org/10.1155/2022/6025916.

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SRD5A2 (steroid 5-alpha-reductase 2) mutation, which impairs 5α-reductase-2 enzyme activity, is among the causes of 46,XY disorders of sex development (DSD). Here, we report a rare pathogenic mutation NM_000348.4:c.485A>C (NP_000339.2:p.His162Pro) of SRD5A2 gene in a compound heterozygous state first identified in a Vietnamese newborn with 5α-reductase-2 enzyme deficiency. We also first submitted this rare mutation to ClinVar database (VCV000973099.1). The patient presented with hyperpigmented labia-majora-like bifid scrotum, clitoris-like phallus, perineoscrotal hypospadias, and blind-ending vagina. The other mutation NM_000348.4:c.680G>A (NP_000339.2:p.Arg227Gln) was reported previously. This compound heterozygous mutation was first detected by next-generation sequencing. By Sanger sequencing, we confirmed that the c.485A>C mutation was maternal inherited, whereas the c.680G>A mutation was paternal inherited. Up to date, this is the first report of this rare compound heterozygous state of SRD5A2 c.485A>C and c.680G>A mutations in patients with 46,XY DSD generally as well as in Vietnamese population particularly and is also the second report in the world carrying the pathogenic mutation NM_000348.4:c.485A>C (NP_000339.2:p.His162Pro). Our finding has enriched the understanding of the spectrum of SRD5A2 variants and phenotypic correlation in Asian patients with 46,XY DSD.

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Yang, Y., X. Bai, H. Liu, L. Li, C. Cao et L. Ge. « Novel Mutations of Cathepsin C Gene in Two Chinese Patients with Papillon-Lefèvre Syndrome ». Journal of Dental Research 86, n^o 8 (août 2007) : 735–38. http://dx.doi.org/10.1177/154405910708600809.

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Papillon-Lefèvre syndrome (PLS) is an inherited human disease characterized by early-onset periodontitis and palmoplantar hyperkeratosis. Mutations of the lysosomal protease cathepsin C ( CTSC) gene have been shown to be the genetic cause of Papillon-Lefèvre syndrome. There are several case reports in China, while there has been no study on the genetic analysis of PLS. We studied two Chinese patients carrying Papillon-Lefèvre syndrome and showing premature tooth loss and palmoplantar hyperkeratosis. Mutation screening and sequence analysis of the CTSC gene revealed a compound heterozygous mutation (c.415 G>A and c.778 T>C) in one patient, and two novel compound heterozygous mutations (c.851G>A and c.112delCCTG) in the other patient. Our novel discovery indicates that the phenotypes observed in these two patients are due to the CTSC gene mutation.

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Tai, E. Shyong, Evelyn S. C. Koay, Edmund Chan, Tzer Jing Seng, Lih Ming Loh, Sunil K. Sethi et Chee Eng Tan. « Compound Heterozygous Familial Hypercholesterolemia and Familial Defective Apolipoprotein B-100 Produce Exaggerated Hypercholesterolemia ». Clinical Chemistry 47, n^o 3 (1 mars 2001) : 438–43. http://dx.doi.org/10.1093/clinchem/47.3.438.

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Abstract Background: Familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB) represent ligand-receptor disorders that are complementary. Individuals with both FH and FDB are unusual. We report a family with both disorders and the impact of the mutations on the phenotypes of the family members. Methods: We used single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) for genetic analysis of all 18 exons and the promoter region of the LDL receptor and DGGE for genetic analysis of the apolipoprotein B-100 (apo B-100) gene. The functional significance of the apo B-100 mutation was studied using a U937 cell proliferation assay. Fasting serum lipid profiles were determined for the index case and seven first-degree relatives. Results: One of the patient’s sisters had a missense mutation (Asp407→Lys) in exon 9 of the LDL receptor and a serum LDL-cholesterol concentration of 4.07 mmol/L. Four other first-degree relatives had hyperlipidemia but no LDL-receptor mutation. However, these subjects had a mutation of the apo B-100 gene (Arg3500→Trp). The cell proliferation rate of U937 cells fed with LDL from other subjects with the same mutation was fourfold less than that of controls. The index case had both FH- and FDB-related mutations. Her serum LDL-cholesterol (9.47 mmol/L) was higher than all other relatives tested. Conclusions: Existence of both FH and FDB should be considered in families with LDL-receptor mutations in some but not all individuals with hypercholesterolemia or when some individuals in families with FH exhibit exaggerated hypercholesterolemia.

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Shahbazi, S., R. Mahdian, K. Karimi et A. Mashayekhi. « Molecular characterization of iranian patients with inherited coagulation factor VII deficiency ». Balkan Journal of Medical Genetics 20, n^o 2 (29 décembre 2017) : 19–25. http://dx.doi.org/10.1515/bjmg-2017-0027.

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AbstractCoagulation factor VII (FVII) is a key enzyme of the extrinsic coagulation cascade that is predominantly produced by hepatocytes. TheF7gene mutations cause FVII deficiency with considerable molecular and phenotypic heterogeneity. We characterized the molecular alterations of theF7gene and their corresponding mRNA transcripts in Iranian patients from eight unrelated families. The mutations were detected by polymerase chain reaction (PCR)-sequencing of allF7gene exons, their flanking intronic sequences, as well as their corresponding cDNA fragments. Homozygous P303T, C91S and R304Q mutations were detected in patient 2, patient 5, and patient 6, respectively. Patient 7 was a compound heterozygote for S282R and H348R and patient 8 was a compound heterozygote for R304Q and IVS7+7A>G mutations. Furthermore, our investigation revealed three heterozygous individuals, patient 1 and patient 3 with the A244V mutation who were symptomatic and patient 4 with V(–39)I mutation who was also asymptomatic. TheF7mRNA expression analysis revealed that, except the transcript of V(–39)I, other mutation-harboring transcripts were expressed at detectable levels. In conclusion, this report reinforces the genetic and phenotypic heterogeneity of FVII deficiency. The findings of the mRNA study implied that decreased FVII protein activity subsequent to missense mutations does not completely reflect the degradation of mutation-harboring mRNA.

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Liu, Hongxing, Jiaqi Chen, Fang Wang, Jiancheng Fang, Wen Teng, Xue Chen, Yang Zhang et al. « NGS-Based Screening to Comprehensively Decipher TKIs Resistant Mutations in BCR-ABL1 Positive Leukemias ». Blood 136, Supplement 1 (5 novembre 2020) : 30. http://dx.doi.org/10.1182/blood-2020-140917.

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In BCR-ABL1 positive leukemia, mutations in the BCR-ABL1 kinase domain (KD) are the most common TKIs resistance mechanism. Sanger sequencing (SS) is currently the gold standard for detecting ABL1 KD mutations, but its low detection sensitivity cannot reveal mutations below 20% VAFs (variant allele frequency), and it is challenging to distinguish compound or polyclonal mutations. In recent years, the NGS-based BCR-ABL1 KD mutation screening protocol provides a powerful tool for deciphering complex patterns (compound or polyclonal mutations) and higher detection sensitivity (~2%, hotspot mutation 1%). Based on an inhouse designed NGS-based BCR-ABL1 KD mutation screening protocol, we retrospectively analyzed 348 specimens that undergone SS (ALL, n=164, CML, n=128, CML-BP, n=33, AML, n=3, other, n=20, other includes MAL, B-LBL). The BCR-ABL1 transcripts of 109 cases were 0, 184 cases were >0.1%, and the remaining 55 cases were between 0-0.1%. NGS screening results showed that the average VAF was 32.81% (range, 1.0%-100%), which was not provided by SS. Compared with SS, NGS screening provided a higher mutation-positive rate (22.13% vs. 33.05%, P =0.0013), and Gatekeeper T315I remained the most common BCR-ABL1 KD mutation (12.24% vs. 14.06%, P =0.4513). When the BCR-ABL1 transcript was 0, no mutation was detected in SS; while NGS ABL1-KD discovered mutations in 12 cases (0 vs. 11.01%, P =0.0004) with VAFs lower than 10% except for one S438F (VAF 14.9%) mutation,among them are the well-known F359V and E450G, as well as ten rare mutations, including T277A and E494K. There were also significant differences in mutations between 0 and 0.1% in BCR-ABL1 transcripts (16.36% vs 32.73%, P =0.046). However, when the BCR-ABL1 transcript >0.1%, there was no difference in mutation-positive rate between the two groups (36.96% vs. 46.20%, P=0.072). NGS screening disclosed more multiple mutations (59 vs 82, P =0.03), with significant advantages in detecting >3 mutations (3 vs 18, P = 0.0009). Compound mutations (CMs) were determined in 8 cases (8/384, 2.08%), with the incidence of 24.24% (8/33) in cases which carrying two or more mutations, of which T315I-including CMs were the most common (6/8, 75%). CMs or polyclonal mutation analysis is particularly crucial for severe patients who have received multiple consecutive TKIs treatments, and those with multiple background mutations and a higher risk of progression to clinical resistance. Our results showed that the inhouse designed NGS-based screening protocols could decipher TKIs resistant mutations more comprehensively than SS(Fig 1), and worthy of being implicated in clinical practice. Figure 1 Disclosures No relevant conflicts of interest to declare.

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Dunlop, Felicity M., Patricia A. Crock, Joseph Montalto, John W. Funder et Kathleen M. Curnow. « A Compound Heterozygote Case of Type II Aldosterone Synthase Deficiency ». Journal of Clinical Endocrinology & ; Metabolism 88, n^o 6 (1 juin 2003) : 2518–26. http://dx.doi.org/10.1210/jc.2003-030353.

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An infant with failure to thrive, persistent hyponatremia and episodic vomiting and diarrhea was admitted to hospital at 9 months of age, and the diagnosis of type II aldosterone synthase deficiency was confirmed by plasma and urinary steroid determinations. The entire coding sequence of the aldosterone synthase gene (CYP11B2) was determined (both strands) in the affected infant, an unaffected sibling, and both parents. An exon 3 mutation (C554T, leading to amino acid T185I) was found in the father and both siblings, and an exon 9 mutation (A1492G, leading to T498A) was found in the affected infant and the mother. Expression of the mutant sequences in COS cells showed steroidogenic patterns typical of aldosterone synthase type II deficiency, including very low levels of aldosterone synthesis (≤0.5% of wild-type enzyme) consistent with the low aldosterone levels in the patient’s plasma. Both mutations in this compound heterozygote localize to the β3-sheet in the cytochrome P450 enzyme structure, as does the previously characterized R181W mutation. This region of the enzyme is not part of the putative structural core, but mutations to this region suggest that it is important for conferring the unique ability of aldosterone synthase to catalyze efficient oxygenation of the C18 carbon of steroid substrates.

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Wang, Li, Jingjing Li, Ge Wu et Xiangdong Kong. « A novel compound heterozygous variant in SMARCAL1 leading to mild Schimke immune-osseous dysplasia identified using whole-exome sequencing ». Journal of International Medical Research 49, n^o 4 (avril 2021) : 030006052110106. http://dx.doi.org/10.1177/03000605211010644.

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Schimke immuno-osseous dysplasia (SIOD) is a rare autosomal recessive inherited disorder that is caused by the SMARCAL1 mutation. The phenotype can vary from mild to severe on the basis of the patient’s age at onset. Herein, we report the case of a 14-year-old Chinese boy who presented with short stature, focal segmental glomerulosclerosis (FSGS), and facial dysmorphism. Genetic analysis revealed two compound heterozygous missense mutations, including a well-known mutation (c.1933C>T, p.R645C) and a novel mutation (c.2479G>A, p.V827M) in the SMARCAL1 gene, which were inherited from his parents. In silico analyses showed that the c.2479G>A (p.V827M) variant affects a highly conserved residue within the ATPase catalytic domain. Finally, we established the diagnosis of mild SIOD and treated the patient with diuretics and angiotensin receptor blockers. This report expands the mutational spectrum of SMARCAL1 and reinforces the importance of a detailed clinical evaluation, molecular detection, and appropriate genetic counseling.

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Kim, Youn Jung, Hong Zhang, Yejin Lee, Figen Seymen, Mine Koruyucu, Yelda Kasimoglu, James P. Simmer, Jan C. C. Hu et Jung-Wook Kim. « Novel WDR72 Mutations Causing Hypomaturation Amelogenesis Imperfecta ». Journal of Personalized Medicine 13, n^o 2 (14 février 2023) : 326. http://dx.doi.org/10.3390/jpm13020326.

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Amelogenesis imperfecta (AI) is a heterogeneous collection of hereditary enamel defects. The affected enamel can be classified as hypoplastic, hypomaturation, or hypocalcified in form. A better understanding of normal amelogenesis and improvements in our ability to diagnose AI through genetic testing can be realized through more complete knowledge of the genes and disease-causing variants that cause AI. In this study, mutational analysis was performed with whole exome sequencing (WES) to identify genetic etiology underlying the hypomaturation AI condition in affected families. Mutational analyses identified biallelic WDR72 mutations in four hypomaturation AI families. Novel mutations include a homozygous deletion and insertion mutation (NM_182758.4: c.2680_2699delinsACTATAGTT, p.(Ser894Thrfs*15)), compound heterozygous mutations (paternal c.2332dupA, p.(Met778Asnfs*4)) and (maternal c.1287_1289del, p.(Ile430del)) and a homozygous 3694 bp deletion that includes exon 14 (NG_017034.2:g.96472_100165del). A homozygous recurrent mutation variant (c.1467_1468delAT, p.(Val491Aspfs*8)) was also identified. Current ideas on WDR72 structure and function are discussed. These cases expand the mutational spectrum of WDR72 mutations causing hypomaturation AI and improve the possibility of genetic testing to accurately diagnose AI caused by WDR72 defects.

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Erickson-Miller, Connie L., Evelyn Delorme, Maya Iskander, Leslie Giampa, Christpher B. Hopson, Juan Luengo, Kevin Duffy et al. « Species Specificity and Receptor Domain Interaction of a Small Molecule TPO Receptor Agonist. » Blood 104, n^o 11 (16 novembre 2004) : 2909. http://dx.doi.org/10.1182/blood.v104.11.2909.2909.

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Abstract Compounds in the diazo/hydrazino acid class require expression of Tpo receptor (TpoR) for activity. These compounds have demonstrated a remarkable species specificity in their Tpo receptor agonist activity, i.e., the activation of signalling pathways by this class of compounds has only been demonstrated in human and chimpanzee platelets. Platelets of other species demonstrate signalling in response to rhTpo, however, no signalling in response to SB-497115 is detectable by electrophoretic mobility shift assays using platelets of cynomolgus macaques, cat, mouse, mouse, rag, pig, ferret or tree shrew. Additionally, HepG2 cells transiently transfected with human TpoR, but not murine or cynomolgus monkey receptor results in STAT activated-reporter gene activity. To elucidate the mechanism by which this occurs, HepG2 cells were transiently transfected with a STAT-activated reporter gene and various chimeric and mutated receptors and treated with SKF-57626, a tool compound in this diazo class. A series of cyno and human TpoR chimeric receptors were constructed in which the complement receptor homology region 1 (CRH1), CRH2 and the transmembrane (TM) and cytoplasmic domains were interchanged. The functionality of all the chimeric receptors was confirmed by response to rhTpo. The minimal human composition of the chimeric TpoR activated by SKF-57626 was composed of human sequence within the membrane proximal region of the CRH2 domain and the TM domain. Two amino acids in this region are different between cyno and human, a Thr to Ala change in the extracellular CRH2 domain and a Leu at residue 499 in cynomolgus monkeys rather than His in humans in the TM domain. Sequencing of TpoR transmembrane domains of mice, dogs and ferrets, species in which these compounds are inactive, confirmed that the receptor contains Leu499, similar to cynomolgus macaques, while chimpanzee TpoR is similar to human with His at residue 499. To verify the requirement for His499 in the TM domain, a point mutation replacing only Leu499 with His in the cyno TpoR conferred activity when treated with compound. Replacement of His499 with Leu in the human TM domain resulted in an inactive human TpoR in response to compound, but not rhTpo. Further experiments involved mutations in the murine G-CSF receptor (mGCSFR). Human TpoR and mGCSFR have little homology in their TM domains and there is no detectable signalling, proliferation or differentiation responses following treatment of GCSFR expressing cells with either compound or rhTpo. A mGSFR point mutation was tested that contained a His residue nine amino acids into the hydrophobic TM domain of GCSFR, corresponding to His499 in the human and chimpanzee TpoR. HepG2 cells transiently transfected with this receptor and the reporter gene construct responded to compound. A double mutation in which an additional residue three amino acids N-terminal to the His was replaced with Thr, as in the hTpoR exhibited an increase in activity over the single mutation. These results suggest a model in which these TpoR agonist compounds interact with His499, in addition to Thr496, to either change conformation of TpoR or induce dimerization, resulting in activation of the signal transduction pathways of TpoR and imparting biologically relevant function.

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Niu, Ao, Yang Wang, Yushe Yang, Jianhai Wei, Jian Ding, Yi Chen, Linjiang Tong et Hua Xie. « Synthesis and Biological Evaluation of Oxopyrido[2,3-d] Pyrimidine-7- ones Derivatives as Covalent L858R/T790M Mutant Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors ». Letters in Drug Design & ; Discovery 16, n^o 8 (8 août 2019) : 826–34. http://dx.doi.org/10.2174/1570180815666180523090558.

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Background: None small cell cancer (NSCLC) is one of the most common cancer around the globe. First generation EGFR-TKI such as gefitinib and erlotinib are now documentated a prolonged PFS in NSCLC patients with EGFR activating mutation. However, upon continuous treatment, patients become resistant due toCEE T790M mutation in most cases.Second generation covalent EGFR inhibitors like afatinib have a moderate inhibition to EGFRT790M in preclinical models,but it is lacking efficacy in the clinical use for patients with T790M mutation due to the dose-limiting EGFRWT-driven toxicities.Third generation EGFR inhibitors have the potential to overcome EGFRT790M resistance mutations while reducing EGFRWT-driven toxicities and are now under active research. Methods: We took compound 6 as our lead compound. We focused on structural modifications around the hydrophile side chain, the linker, and the Micheal addition receptor moiety of AMG. A novel series of Oxopyrido[2,3-d]pyrimidine-7-ones derivatives have been designed and synthesized. Their kinase inhibition activity against EGFRWT and EGFRL858R/T790M were tested by ELISA assays. SRB test was used for cellular anti-proliferation evaluation. Results: A total of 21 novel Oxopyrido[2,3-d]pyrimidine-7-ones derivatives have been designed and synthesized. The compounds were characterized with 1H-NMR and HRMS. Their structureactivity relationships have been preliminaryly investigated. As a result, compound 7k showed comparable activity in kinase inhibition assay and cell growth inhibition assay with our lead compound 6. Higher activity and selectivity over EGFRWT were observed in the in vitro antitumour assay comparing compound 7k to AZD-9291. Compound 7a exhibited higher selectivity over EGFRWT in kinase inhibition assay, but poor cell inhibition to NCI-1975 cell line. The in vivo pharmacokinetic studies in rats showed that compound 9a exhibited improved pharmacokinetic profiles comparing to 6. Compound 9a was also efficacious in an NCI-H1975 murine xenograft model 30 mg/kg QD. Conclusion: Compound 9a has a potent kinase inhibition to EGFRT790M and has a high selectivity over EGFRWT. It’s also efficacious in an in vivo pharmacodynamic evaluation assay. Significant advantages were observed in pharmacokinetic evaluation comparing 9a to 6, which provide us a reference to further drug design and research.

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Miura, Satoru, Te-Chun Hsia, Jen-Yu Hung, Hyun Ae Jung, Jin-Yuan Shih, Cheol-Kyu Park, Seung Hyeun Lee et al. « Activity of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in patients (pts) with NSCLC with uncommon EGFR mutations : A real-world cohort study (UpSwinG). » Journal of Clinical Oncology 39, n^o 15_suppl (20 mai 2021) : 9072. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.9072.

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9072 Background: EGFR TKIs are an established treatment (tx) option for pts with EGFR mutation-positive NSCLC with common mutations (Del19 or L858R); however, 7–23% of NSCLC tumors harbor uncommon EGFR mutations, where EGFR TKI efficacy is less established. These mutations are highly heterogeneous, and developments in detection by NGS are helping to identify mutations with little or no clinical data. Methods: In this non-interventional, global, multi-center study (NCT04179890), existing medical or electronic health records were identified for consecutive EGFR TKI-naïve pts with uncommon EGFR mutations (T790M, ex20ins, major uncommon [G719X, L861Q or S768I], ‘other’ or compound mutations) treated with erlotinib, gefitinib, afatinib, osimertinib or other systemic therapy. Endpoints were time to tx failure (TTF), ORR, OS and duration of response (DoR). Results: Overall, 246 pts (median age: 69.5 yrs; Asian: 84%; brain metastases: 8%; ECOG PS ≥2: 16%) were recruited from 9 countries. Most pts (n=226; 92%) received an EGFR TKI as 1st-line therapy; 132 (54%), 105 (43%) and 7 (3%) received afatinib, 1st-gen TKIs and osimertinib, respectively. 57% of pts received >1 line of therapy. Most pts (73%) had a major uncommon mutation, 9% had other mutations and 33% had a compound mutation; these were detected using PCR (75%) or sequencing (25%), mainly based on tissue biopsy (86%). Pathology reports varied in quality, often lacking detail on specific mutations e.g. 21% of ex18 and 72% of ex20ins were undefined. Median TTF and OS with EGFR TKIs were 9.9 and 24.4 mos; ORR was 42%. In pts treated with 1st-line chemotherapy (n=20), median TTF and ORR were 6.6 mos and 41%. Outcomes were most favorable in major uncommon and compound mutations (Table). TTF appeared to be higher with afatinib vs 1st-gen EGFR TKIs. In most mutation categories, median OS was >2 yrs, possibly reflecting high subsequent therapy uptake. Conclusions: In a real-world setting, EGFR TKIs were the preferred tx option in pts with uncommon EGFR mutations; strongest outcomes were seen in major uncommon and compound mutations, and in pts treated with afatinib. Data were in line with recent analyses of afatinib in uncommon mutations. Tx with an EGFR TKI should be considered as standard for most pts with uncommon mutations. Optimal tx for pts with uncommon mutations requires improvements in pathology reports, with more emphasis on NGS methodology and precise definition of mutations. Clinical trial information: NCT04179890. [Table: see text]

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Ichinose, Aya, Hideaki Moteki, Mitsuru Hattori, Shin-ya Nishio et Shin-ichi Usami. « Novel Mutations in LRTOMT Associated With Moderate Progressive Hearing Loss in Autosomal Recessive Inheritance ». Annals of Otology, Rhinology & ; Laryngology 124, n^o 1_suppl (18 mars 2015) : 142S—147S. http://dx.doi.org/10.1177/0003489415575043.

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Objective: We present a patient who was identified with novel mutations in the LRTOMT gene and describe the clinical features of the phenotype including serial audiological findings. Methods: One hundred six Japanese patients with mild to moderate sensorineural hearing loss from unrelated and nonconsanguineous families were enrolled in the study. Targeted genomic enrichment and massively parallel sequencing of all known nonsyndromic hearing loss genes were performed to identify the genetic cause of hearing loss. Results: Compound heterozygotes with a novel frame-shift mutation and a missense mutation were identified in the LRTOMT gene. The mutated residues were segregated in both alleles of LRTOMT, present within the LRTOMT2 protein coding region. The patient had moderate sloping hearing loss at high frequencies, which progressed at 1000 Hz and higher frequencies over a period of 6 years. Conclusion: Hearing loss caused by mutations in the LRTOMT gene is extremely rare. This is the first case report of a compound heterozygous mutation in a nonconsanguineous family.

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Popat, Sanjay, Te-Chun Hsia, Jen-Yu Hung, Hyun Ae Jung, Jin-Yuan Shih, Cheol Kyu Park, Seung Hyeun Lee et al. « Tyrosine Kinase Inhibitor Activity in Patients with NSCLC Harboring Uncommon EGFR Mutations : A Retrospective International Cohort Study (UpSwinG) ». Oncologist 27, n^o 4 (11 mars 2022) : 255–65. http://dx.doi.org/10.1093/oncolo/oyac022.

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Abstract Background Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are standard of care for patients with EGFR mutation-positive non–small-cell lung cancer (NSCLC) with common mutations (Del19 or L858R); however, 7%-23% of NSCLC tumors harbor uncommon EGFR mutations. These mutations are highly heterogeneous, and developments in detection techniques are helping to identify mutations with little or no clinical data. Patients and Methods In this retrospective, global, multi-center study (NCT04179890), existing health records were identified for consecutive EGFR TKI-naïve patients with uncommon EGFR mutations (T790M, ex20ins, major uncommon [G719X, L861Q, or S768I], or “other” mutations; compound mutations) treated with erlotinib, gefitinib, afatinib, or osimertinib in first or second line. Endpoints included time-to-treatment failure (TTF), objective response rate (ORR), and overall survival (OS). Results Overall, 246 patients (median age: 69.5 years; Asian: 84%) were included from 9 countries. Most patients (92%) received an EGFR TKI as first-line therapy; 54%, 43% and 3% received afatinib, first-generation TKIs, and osimertinib, respectively. Median TTF and OS with EGFR TKIs were 9.9 and 24.4 months; ORR was 43%. In patients treated with first-line chemotherapy (n = 20), median TTF and ORR were 6.6 months and 41%. Outcomes were most favorable in patients with major uncommon or compound mutations. Overall, TTF was 11.3 months with afatinib and 8.8 months with first-generation EGFR TKIs across mutation categories. In most mutation categories, median OS was >2 years. Conclusion In a real-world setting, EGFR TKIs were the preferred treatment option in patients with uncommon EGFR mutations; strongest outcomes were seen in patients with major uncommon and compound mutations.

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Deininger, Michael W., Neil P. Shah, Jorge E. Cortes, Dong-Wook Kim, Franck E. Nicolini, Moshe Talpaz, Michele Baccarani et al. « Impact Of Baseline (BL) Mutations, Including Low-Level and Compound Mutations, On Ponatinib Response and End Of Treatment (EOT) Mutation Analysis In Patients (Pts) With Chronic Phase Chronic Myeloid Leukemia (CP-CML) ». Blood 122, n^o 21 (15 novembre 2013) : 652. http://dx.doi.org/10.1182/blood.v122.21.652.652.

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Abstract Background In CML, the presence of BCR-ABL kinase domain (KD) mutations, including low-level mutations, can predict clinical responses to 2nd line BCR-ABL tyrosine kinase inhibitors (TKIs). In addition, sequential treatment with TKIs can lead to development of compound mutations (≥2 mutations in the same BCR-ABL allele) that can be highly TKI-resistant. Ponatinib is a potent BCR-ABL TKI that, preclinically, has demonstrated activity against all BCR-ABL mutations tested and suppresses the emergence of any single mutation at clinically achievable concentrations (40 nM with ≥30 mg/d). In vitro, higher ponatinib concentrations were required to suppress emergence of certain compound mutations on a background of T315I or E255V single mutations. We evaluated the impact of single, low-level, and compound mutations at BL on responses to ponatinib and EOT mutations in CP-CML pts in the phase 2 PACE trial. Methods Pts with CP-CML (93% received ≥2 prior TKIs, 60% ≥3) resistant or intolerant to dasatinib and/or nilotinib (N=203) or with T315I confirmed at BL (N=64) were enrolled. The primary endpt was major cytogenetic response (MCyR) by 12 mos. Median follow-up at analysis (1 Apr 2013) was 20 (0.1-28) mos, with a minimum follow-up of 18 mos for pts remaining on study. Next generation sequencing (NGS) and Sanger sequencing (SS) were done at a central laboratory. NGS was conducted on all BL samples (N=267) with the Ion Torrent PGM using chemistry that enabled read lengths up to 400 bp for detection of compound mutations; mutations observed at a frequency ≥1% are reported. SS was conducted on both BL and EOT samples. Results By NGS at BL, 267 mutations (amino acid substitutions in the ABL KD [M237-E507]) were detected among 163 (61%) pts; 106 (40%) mutations were low level mutations not detected by SS. 75 unique single mutations were observed: 27 were detected by SS and NGS, all 27 have been associated with resistance to TKIs other than ponatinib; 48 were low level mutations detected only by NGS, 5 have been associated with resistance to TKIs other than ponatinib. 12% of pts had only low level mutations. Overall, no mutations were detected in 39% of pts, 1 mutation in 37%, and ≥2 mutations in 24%. Compound mutations were detected in 65% of pts who had ≥2 mutations, representing 15% of pts overall (10% with 1, 5% with 2 to 4 compound mutations). 48 unique compound mutations were observed; T315I, F317L, and F359C/I/V were the most commonly observed mutations within compound mutations. Responses were seen in pts with each of the 20 unique single mutations present in >1 pt at BL by NGS, including Y253H, E255V/K, T315I, M351T, F359V. Responses were observed regardless of overall NGS BL mutation status (table). The high response rates and durability of response in pts with compound mutations suggest that, in general, the presence of compound mutations at BL did not adversely affect the activity of ponatinib. Of the 109 pts who discontinued, 84 had successful mutation assessments by SS at or near the EOT visit. 4 pts had mutations at EOT that were not detected by NGS at BL; all 4 involved compound mutations (T315I/F359V [100%/90%], T315I/M351T [100%/40%], Y253H/F359V [100%/100%; n=2]), with one or both of the involved mutations detected individually at BL or by history. Overall, 12 pts lost MCyR (none with T315I at BL); 6 of the 12 discontinued and had EOT mutations assessed, no changes from BL were observed. Conclusions Responses to ponatinib were observed regardless of BL mutation status. Interestingly, responses tended to be lower in pts without mutations, suggesting that BCR-ABL independent mechanisms may be involved. No single mutation conferring resistance to ponatinib in CP-CML has been observed to date. In general, ponatinib activity was not adversely affected by the presence of compound mutations at BL. Rarely, the development of compound mutations was observed at EOT in pts with one of the involved mutations at BL or by history. Early introduction of ponatinib may suppress the emergence of single BCR-ABL mutations, and, as a result, the development of compound mutations. NCT01207440 Disclosures: Deininger: BMS, ARIAD, NOVARTIS: Consultancy; BMS, NOVARTIS, CELGENE, GILEAD: Research Funding; ARIAD, NOVARTIS: Advisory Boards, Advisory Boards Other. Shah:Ariad, Bristol-Myers Squibb: Consultancy, Research Funding. Cortes:Ariad, Pfizer, Teva: Consultancy; Ariad, BMS, Novartis, Pfizer, Teva: Research Funding. Kim:BMS, Novartis, IL-Yang: Consultancy; BMS, Novartis, Pfizer, ARIAD, IL-Yang: Research Funding; BMS, Novartis, Pfizer, IL-Yang: Honoraria; BMS, Novartis, Pfizer: Speakers Bureau; BMS, Pfizer: Membership on an entity’s Board of Directors or advisory committees. Nicolini:Novartis, ARIAD, Teva: Consultancy; Novartis, BMS: Research Funding; Novartis, BMS, Teva, Pfizer, ARIAD: Honoraria; Novartis, BMS, TEva: Speakers Bureau; Novartis, ARIAD, Teva, Pfizer: Membership on an entity’s Board of Directors or advisory committees. Talpaz:Ariad, BMS, Sanofi, INCYTE: Research Funding; Ariad, Novartis: Speakers Bureau; Ariad, Sanofi, Novartis: Membership on an entity’s Board of Directors or advisory committees. Baccarani:ARIAD, Novartis, BMS: Consultancy; ARIAD, Novartis, BMS, Pfizer, Teva: Honoraria; ARIAD, Novartis, BMS, Pfizer, Teva: Speakers Bureau. Muller:Novartis, BMS, ARIAD: Consultancy; Novartis, BMS: Research Funding; Novartis, BMS, ARIAD: Honoraria. Lustgarten:ARIAD: employees of and own stock/stock options in ARIAD Pharmaceuticals, Inc Other, Employment. Clackson:ARIAD: employees of and own stock/stock options in ARIAD Pharmaceuticals, Inc Other, Employment. Turner:ARIAD: Employment. Haluska:ARIAD: employees of and own stock/stock options in ARIAD Pharmaceuticals, Inc Other, Employment. Hodgson:ARIAD: Employment, Equity Ownership. Rivera:ARIAD: Employment, Equity Ownership. Goldman:ARIAD: Honoraria. Kantarjian:ARIAD, Novartis, BMS, Phizer: Research Funding. Soverini:Novartis, BMS, ARIAD: Consultancy. Hochhaus:Ariad, Novartis, BMS, MSD, Pfizer: Research Funding; Novartis, BMS, Pfizer: Honoraria. Hughes:Novartis, BMS, ARIAD: Honoraria, Research Funding. Branford:Novartis, BMS, ARIAD: Research Funding; Novartis, BMS, ARIAD: Honoraria.

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Shahbaznejad, Leila, Sayed-Reza Raeeskarami, Raheleh Assari, Abbas Shakoori, Hamidreza Azhideh, Yahya Aghighi, Fatemeh Tahghighi et Vahid Ziaee. « Familial Mediterranean Gene (MEFV) Mutation in Parents of Children with Familial Mediterranean Fever : What Are the Exceptions ? » International Journal of Inflammation 2018 (1 octobre 2018) : 1–6. http://dx.doi.org/10.1155/2018/1902791.

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Objectives. Familial Mediterranean Fever (FMF) is one of the most prevalent periodic fever syndromes; MEFV, the responsible gene for the disease, is in the short arm of chromosome16. In the considerable count of the FMF patients, only one mutation is found in the MEFV and parents, who were the obligatory carriers for that mutation, were asymptomatic. The aim of this study was to evaluate these asymptomatic parents in regard to mutation in MEFV gene and similarity between parents and offspring patients. Methods. In this cross-sectional study, asymptomatic parents of FMF patients enrolled the study were referred to periodic fever clinic or pediatric rheumatology clinic of Tehran University of Medical Sciences. The patients should have at least one mutation in MEFV gene and none of them had any family history of autoinflammatory disease. Twelve mutations in MEFV gene were assessed in the parents by Vienna Lab FMF Strip Assay kit by MAS PCR/Reverse hybridization. Results. Forty-three patients and their parents participated in the study. Sixty-three percent (27) of patients were male. Onset of disease symptoms in 31 patients (72%) was before 4 years of old. Nine (21%) of the patients had homozygote, 16 (37%) compound heterozygote, and 17(40%) heterozygote for MEFV mutation; there was a case of complex alleles mutations (2%). M694V/M694V in 4 patients (9%) was the most homozygote genotype, and M694V/R761H in 4 (9%) and E148Q in 7 (16%) were the most compound heterozygote and heterozygote genotype, respectively. M694V, M680I, and E148Q were the most mutation in the parents. Overall, 41 patients had mutations similar to their parents’ mutation, except 2 whose parents had no mutation, but a patient did. Conclusion. It seems that occurrence of new mutations in offspring is not prevalent among FMF patients and there are other reasons for different clinical presentation in similar mutation carriers. On the other hand, in ethnicities with high prevalence of FMF, new mutation in descendant may occur, infrequently.

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Brown, Susan C., Marta Fernandez-Fuente, Francesco Muntoni et John Vissing. « Phenotypic Spectrum of α-Dystroglycanopathies Associated With the c.919T>a Variant in the FKRP Gene in Humans and Mice ». Journal of Neuropathology & ; Experimental Neurology 79, n^o 12 (26 novembre 2020) : 1257–64. http://dx.doi.org/10.1093/jnen/nlaa120.

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Abstract Mutations in the fukutin-related protein gene, FKRP, are the most frequent single cause of α-dystroglycanopathy. Rare FKRP mutations are clinically not well characterized. Here, we review the phenotype associated with the rare c.919T>A mutation in FKRP in humans and mice. We describe clinical and paraclinical findings in 6 patients, 2 homozygous, and 4-compound heterozygous for c.919T>A, and compare findings with a mouse model we generated, which is homozygous for the same mutation. In patients, the mutation at the homozygous state is associated with a severe congenital muscular dystrophy phenotype invariably characterized by severe multisystem disease and early death. Compound heterozygous patients have a severe limb-girdle muscular dystrophy phenotype, loss of ambulation before age 20 and respiratory insufficiency. In contrast, mice homozygous for the same mutation show no symptoms or signs of muscle disease. Evidence therefore defines the FKRP c.919T>A as a very severe mutation in humans. The huge discrepancy between phenotypes in humans and mice suggests that differences in protein folding/processing exist between human and mouse Fkrp. This emphasizes the need for more detailed structural analyses of FKRP and shows the challenges of developing appropriate animal models of dystroglycanopathies that mimic the disease course in humans.

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Khandelwal, G., S. Bhalla, M. Khullar et N. K. Panda. « High frequency of heterozygosity in GJB2 mutations among patients with non-syndromic hearing loss ». Journal of Laryngology & ; Otology 123, n^o 3 (23 juin 2008) : 273–77. http://dx.doi.org/10.1017/s0022215108002892.

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AbstractObjective:To determine the prevalence of GJB2 mutations among subjects with congenital, non-syndromic, sensorineural hearing loss, within a north Indian population.Materials and methods:This was a case–control study in which the frequencies of the three most prevalent GJB2 mutations (35delG, W24X and 167delT) were studied. Polymerase chain reaction restriction fragment length polymorphism assays were performed to detect these mutations. The entire coding region of the GJB2 gene was sequenced in all patients, and also in any of their family members who showed GJB2 mutations.Results:The 35delG mutation was found to be the most prevalent mutation (21 per cent), followed by the W24X mutation (7 per cent). This is the first report of the 35delG mutation in an Indian population. One patient was a compound heterozygote for 35delG/W24X. The 167delT mutation was not observed in any patient.Conclusions:These findings challenge the classical view that the W24X variant of the GJB2 gene represents a single ‘founder’ mutation.

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