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The discovery in 2005 of the JAK2 V617F gain-of-function mutation in myeloproliferative neoplasms and more particularly in polycythemia vera has deeply changed the diagnostic and therapeutic approaches to polycythemia. More recently, the use of NGS in routine practice has revealed a large number of variants, although it is not always possible to classify them as pathogenic. This is notably the case for the JAK2 E846D variant for which for which questions remain unanswered. In a large French national cohort of 650 patients with well-characterized erythrocytosis, an isolated germline heterozygous JAK2 E846D substitution was observed in only two cases. For one of the patients, a family study could be performed, without segregation of the variant with the erythrocytosis phenotype. On the other hand, based on the large UK Biobank resource cohort including more than half a million UK participants, the JAK2 E846D variant was found in 760 individuals, associated with a moderate increase in hemoglobin and hematocrit values, but with no significant difference to the mean values of the rest of the studied population. Altogether, our data as well as UK Biobank cohort analyses suggest that the occurrence of an absolute polycythemia cannot be attributed to the sole demonstration of an isolated JAK2 E846D variant. However, it must be accompanied by other stimuli or favoring factors in order to generate absolute erythrocytosis.

Lynch syndrome (LS) is a condition which predisposes individuals primarily to early-onset colorectal and endometrial cancer. LS is characterized by a germline pathogenic variant in one of the MMR (MisMatch Repair) gene, inducing a phenotype of microsatellite instability in the tumor, which may be associated with a loss of expression of MMR proteins detected by standard immunohistochemistry on tumor tissue. Most of the time, LS is inherited from a parent in whom the condition may not be known due to incomplete penetrance, but de novo pathogenic variant is a rare occurrence. Here, we describe the case of a 52-year-old woman with no family history of LS, referred to the genetics department for colorectal cancer at the age of 50. Genetic analysis revealed a de novo germline pathogenic variant in the MSH6 gene. To date, this case is only the second report of a de novo pathogenic variant in the MSH6 gene in Lynch syndrome. De novo mutations have been extensively studied over the past years, but little is known about their origin and mechanism of occurrence in MMR genes. However, knowledge of mutation status allows better cancer risk management for the patient and an appropriate genetic testing and counseling for her family.

A JAK2V617F‐negative polycythemia associated with low serum epo needs to be tested for an exon 12 JAK2 mutation. When negative, due to potential serious complications in PV, a next generation sequencing is necessary to rule out false negative results.

Background Correct identification of the EGFR c.2369C>T p.(Thr790Met) variant is key to decide on a targeted therapeutic strategy for patients with acquired EGFR TKI resistance in non-small cell lung cancer. The aim of this study was to evaluate the correct detection of this variant in 12 tumor tissue specimens tested by 324 laboratories participating in External Quality Assessment (EQA) schemes. Methods Data from EQA schemes were evaluated between 2013 and 2018 from cell lines (6) and resections (6) containing the EGFR c.2369C>T p.(Thr790Met) mutation. Adequate performance was defined as the percentage of tests for which an outcome was available and correct. Additional data on the used test method were collected from the participants. Chi-squared tests on contingency tables and a biserial rank correlation were applied by IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA). Results In 26 of the 1190 tests (2.2%) a technical failure occurred. For the remaining 1164 results, 1008 (86.6%) were correct, 151 (12.9%) were false-negative and 5 (0.4%) included incorrect mutations. Correct p.(Thr790Met) detection improved over time and for repeated scheme participations. In-house non-next-generation sequencing (NGS) techniques performed worse (81.1%, n = 293) compared to non-NGS commercial kits (85.2%, n = 656) and NGS (97.0%, n = 239). Over time there was an increase in the users of NGS. Resection specimens performed worse (82.6%, n = 610 tests) compared to cell line material (90.9%, n = 578 tests), except for NGS (96.3%, n = 344 for resections and 98.6%, n = 312 for cell lines). Samples with multiple mutations were more difficult compared to samples with the single p.(Thr790Met) variant. A change of the test method was shown beneficial to reduce errors but introduced additional analysis failures. Conclusions A significant number of laboratories that offer p.(Thr790Met) testing did not detect this relevant mutation compared to the other EQA participants. However, correct identification of this variant is improving over time and was higher for NGS users. Revising the methodology might be useful to resolve errors, especially for resection specimens with low frequency or multiple variants. EQA providers should include challenging resections in the scheme.

As next-generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share™ has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.

International audience; Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with von Hippel-Lindau disease, which is characterized by the development of highly vascularized tumors. Here, we identified a new VHL cryptic-exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identified mutations in E1' in seven families with erythrocytosis (one homozygous case and six compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in one large family with typical VHL disease but without any alteration in the other VHL exons. In this study we have shown that the mutations induced a dysregulation of the VHL splicing with excessive retention of E1' and are associated with a downregulation of VHL protein expression. In addition, we have demonstrated a pathogenic role for synonymous mutations in VHL-Exon 2 that alter splicing through E2-skipping in five families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially impact splicing, correlating with phenotype severity. This study demonstrates that cryptic-exon-retention or exon-skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research into the VHL-related-hypoxia-signaling pathway.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (

Background Congenital thrombotic thrombocytopaenic purpura (TTP) or Upshaw–Schulman syndrome (USS) is a rare, life-threatening, inherited thrombotic microangiopathy (TMA). USS is mostly due to bi-allelic recessive sequence variations of the a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13 (ADAMTS13) gene inducing a severe ADAMTS13 deficiency (activity Objective This article evaluates the influence of ADAMTS13 sequence variations on both clinical/biological phenotype and ADAMTS13 conformation in USS. Patients and Methods All USS patients from the French registry for TMAs (1 January 2000 to 1 June 2017) were investigated for ADAMTS13 genotype, phenotype (activity, antigen and autoantibodies) and conformation. Clinical records were analysed (inaugural acute TTP and follow-up). Child-onset USS was compared with adult-onset USS. Results Fifty-six USS patients from 51 families (34 child-onset and 22 adult-onset cases) were enrolled. Child-onset USS was characterized by a large panel of ADAMTS13 sequence variations (n = 43), spread all over ADAMTS13 gene and not correlated with either clinical features or plasmatic ADAMTS13 parameters. In contrast, adult-onset USS, consisting exclusively in pregnancy-induced TTP, included a smaller and distinct panel of ADAMTS13 sequence variations (n = 20) because of one mutation (p.Arg1060Trp) present in 82% of patients. ADAMTS13 conformation was studied in 16 USS patients (5 child-onset and 11 adult-onset USS, encompassing 16 distinct ADAMTS13 sequence variations) whose ADAMTS13 antigen levels were detectable: 14 of 16 patients (87.5%) exhibited abnormalities of ADAMTS13 conformation. Conclusion In USS, age-onset defines two entities and ADAMTS13 sequence variations modify ADAMTS13 conformation.

Erythrocytosis is a rare disorder characterized by increased red cell mass and elevated hemoglobin concentration and hematocrit. Several genetic variants have been identified as causes for erythrocytosis in genes belonging to different pathways including oxygen sensing, erythropoiesis and oxygen transport. However, despite clinical investigation and screening for these mutations, the cause of disease cannot be found in a considerable number of patients, who are classified as having idiopathic erythrocytosis. In this study, we developed a targeted next-generation sequencing panel encompassing the exonic regions of 21 genes from relevant pathways (~79 Kb) and sequenced 125 patients with idiopathic erythrocytosis. The panel effectively screened 97% of coding regions of these genes, with an average coverage of 450×. It identified 51 different rare variants, all leading to alterations of protein sequence, with 57 out of 125 cases (45.6%) having at least one of these variants. Ten of these were known erythrocytosis-causing variants, which had been missed following existing diagnostic algorithms. Twenty-two were novel variants in erythrocytosis-associated genes (EGLN1, EPAS1, VHL, BPGM, JAK2, SH2B3) and in novel genes included in the panel (e.g. EPO, EGLN2, HIF3A, OS9), some with a high likelihood of functionality, for which future segregation, functional and replication studies will be useful to provide further evidence for causality. The rest were classified as polymorphisms. Overall, these results demonstrate the benefits of using a gene panel rather than existing methods in which focused genetic screening is performed depending on biochemical measurements: the gene panel improves diagnostic accuracy and provides the opportunity for discovery of novel variants.

Next-generation sequencing (NGS) has enabled new approaches for detection of mutations in the BRCA1 and BRCA2 genes responsible for hereditary breast and ovarian cancer (HBOC). The search for germline mutations in the BRCA1 and BRCA2 genes is of importance with respect to oncogenetic and surgical (bilateral mastectomy, ovariectomy) counselling. Testing tumor material for BRCA mutations is of increasing importance for therapeutic decision making as the poly ADP ribose polymerase (PARP) inhibitor, olaparib, is now available to treat patients with specific forms of ovarian cancer and BRCA mutations. Molecular genetics laboratories should develop reliable and sensitive techniques for the complete analysis of the BRCA1 and BRCA2 genes. This is a challenge due to the size of the coding sequence of the BRCA1/2 genes, the absence of hot spot mutations, and particularly by the lower DNA quality obtained from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. As a result, a number of analyses are uninterpretable and do not always provide a result to the clinician, limiting the optimal therapeutic management of patients. The availability of Fresh Frozen Tissue (FFT) for some laboratories and the excellent quality of the DNA extracted from it offers an alternative. For this reason, we evaluated Multiplicom's BRCA MASTR Dx assay on a set of 97 FFT derived DNA samples, in combination with the MID for Illumina MiSeq for BRCA1 and BRCA2 mutation detection. We obtained interpretable NGS results for all tested samples and showed > 99,7% sensitivity, specificity and accuracy., SCOPUS: ar.j, info:eu-repo/semantics/published

Pregnancy may be complicated by a rare but life-threatening disease called thrombotic thrombocytopenic purpura (TTP). Most cases of TTP are due to an acquired autoimmune or hereditary (Upshaw-Schulman syndrome [USS]) severe deficiency of a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13 (ADAMTS13). In the present study, we performed a cross-sectional analysis of the national registry of the French Reference Center for Thrombotic Microangiopathies from 2000-2010 to identify all women who were pregnant at their initial TTP presentation. Among 592 adulthood-onset TTP patients with a severe ADAMTS13 deficiency, 42 patients with a pregnancy-onset TTP were included. Surprisingly, the proportion of USS patients (n = 10 of 42 patients [24%]; confidence interval, 13%-39%) with pregnancy-onset TTP was much higher than that in adulthood-onset TTP in general (less than 5%) and was mostly related to a cluster of ADAMTS13 variants. In the present study, subsequent pregnancies in USS patients not given prophylaxis were associated with very high TTP relapse and abortion rates, whereas prophylactic plasmatherapy was beneficial for both the mother and the baby. Pregnancy-onset TTP defines a specific subgroup of patients with a strong genetic background. This study was registered at www.clinicaltrials.gov as number NCT00426686 and at the Health Authority, French Ministry of Health, as number P051064.

AIM: To evaluate the risk associated with variants of the UNC5C gene recently suspected to predispose to familial colorectal cancer (CRC). METHODS: We screened patients with familial CRC forms as well as patients with sporadic CRCs. In a first time, we analyzed exon 11 of the UNC5C gene in 120 unrelated patients with suspected hereditary CRC, 58 patients with suspected Lynch-associated cancer or polyposis, and 132 index cases of Lynch syndrome families with a characterized mutation in a DNA mismatch repair (MMR). Next, 1023 patients with sporadic CRC and 1121 healthy individuals were screened for the variants identified in patients with familial cancer. RESULTS: Of 120 patients with familial CRC of unknown etiology, one carried the previously reported mis-sense mutation p.Arg603Cys (R603C) and another exhibited the unreported variant of unknown significance p.Thr617Ile (T617I). The p.Ala628Lys (A628K) mutation previously described as the main UNC5C risk variant for familial CRC was not detected in any cases of familial CRC of unknown etiology, but was present in a patient with familial gastric cancer and in two Lynch syndrome patients in co-occurrence with MMR mutations. A statistically non-significant increase in cancer risk was identified in familial CRC and/or other Lynch-associated cancers (1/178 patients vs 2/1121 healthy controls, OR = 3.2, 95%CI: 0.29-35.05, P = 0.348) and in sporadic CRCs (4/1023 patients vs 2/1121 healthy controls, OR = 2.2, 95%CI: 0.40-12.02, P = 0.364). CONCLUSION: We confirm that UNC5C mutations are very rare in familial and sporadic CRCs, but further investigations are needed to justify routine UNC5C testing for diagnostic purposes.