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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.

Simple Summary Microsatellite instability (MSI) assessment has become a major issue in the management of colorectal cancer, with the recent approval of anti-PD1 immunotherapies in MSI-metastatic colorectal cancer. The reference PCR method (MSI-PCR) can be costly, time and tissue-consuming. However, NGS could facilitate the assessment of MSI status while simultaneously screening for targetable oncogenic mutations (KRAS, NRAS, BRAF) for any colorectal cancer, but the algorithms developed to date use a large number of microsatellites that have not been approved by international guidelines and which are generally incompatible with small NGS panels. We present the MEM algorithm, which mimics the interpretation of MSI-PCR data by a human operator to reliably assess MSI status using only five validated microsatellites (BAT-25, BAT-26, NR-21, NR-24 and NR-27). We demonstrated that the MEM algorithm was in perfect agreement with MSI-PCR results, in terms of both MSI status and individual microsatellite status, in a cohort of 146 patients. Abstract Purpose: MEM is an NGS algorithm that uses Expectation-Maximisation to detect the presence of unstable alleles from the NGS sequences of five microsatellites (BAT-25, BAT-26, NR-21, NR-24 and NR-27). The purpose of this study was to compare the MEM algorithm with a reference PCR method (MSI-PCR) and MisMatch Repair protein immunohistochemistry (MMR-IHC). Methods: FFPE colorectal cancer samples from 146 patients were analysed in parallel by MSI-PCR and NGS using the MEM algorithm. MMR-IHC results were available for 133 samples. Serial dilutions of an MSI positive control were performed to estimate the limit of detection. Results: the MEM algorithm was able to detect unstable alleles of each microsatellite with up to a 5% allelic fraction. Of the 146 samples, 28 (19.2%) were MSI in MSI-PCR. MEM algorithm results were in perfect agreement with those of MSI-PCR, at both MSI status and individual microsatellite level (Cohen’s kappa = 1). A high level of agreement was noted between MSI-PCR/MEM algorithm results and MMR-IHC results (Cohen’s kappa = 0.931). Conclusion: the MEM algorithm can determine the MSI status of colorectal cancer samples on a small NGS panel, using only five microsatellites approved by international guidelines, and can be combined with screening for targetable mutations.

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.

Up to 80% of BRCA1 and BRCA2 genetic variants remain of uncertain clinical significance (VUSs). Only variants classified as pathogenic or likely pathogenic can guide breast and ovarian cancer prevention measures and treatment by PARP inhibitors. We report the first results of the ongoing French national COVAR (cosegregation variant) study, the aim of which is to classify BRCA1/2 VUSs. The classification method was a multifactorial model combining different associations between VUSs and cancer, including cosegregation data. At this time, among the 653 variants selected, 101 (15%) distinct variants shared by 1,624 families were classified as pathogenic/likely pathogenic or benign/likely benign by the COVAR study. Sixty-six of the 101 (65%) variants classified by COVAR would have remained VUSs without cosegregation data. Of note, among the 34 variants classified as pathogenic by COVAR, 16 remained VUSs or likely pathogenic when following the ACMG/AMP variant classification guidelines. Although the initiation and organization of cosegregation analyses require a considerable effort, the growing number of available genetic tests results in an increasing number of families sharing a particular variant, and thereby increases the power of such analyses. Here we demonstrate that variant cosegregation analyses are a powerful tool for the classification of variants in the BRCA1/2 breast-ovarian cancer predisposition genes.

AimsTo map the colorectal carcinoma (CRC) diseases with significant Wnt signalling pathway activation for delineating their clinicopathological and molecular profiles.MethodsMapping is based on hierarchical clustering analyses of a series of 283 CRCs. Data tabulated were histopathological patterns, immunophenotypic differentiation, RAS, RAF, CTNNB1 mutations and microsatellite instability status, tumour-infiltrating lymphocytes (TILs) and genetic setting. Beta-catenin expression in more than 10% of cell nuclei in the centre of tumour serves as a surrogate marker of significant activation of Wnt signalling pathway.ResultsNuclei beta-catenin expression was present in 95% of CRCs; 56% of them met the criteria of high level of nuclei beta-catenin expression (≥10%). Proportion of beta-catenin positive nuclei was significantly higher in younger patients, rectal and left-sided colonic carcinomas. CRCs with high level of nuclei beta-catenin expression were regrouped into three clusters: (1) microsatellite stability (MSS) CRCs with no constitutive MAPK pathway activation including 90% of low-grade adenocarcinoma, NOS, with intestinal differentiation without TILs; (2) RAS-mutated MSS CRCs including low-grade adenocarcinoma, NOS, with intestinal differentiation and mucinous adenocarcinoma without TILs; (3) MSI-H CRCs including both BRAF-mutated CRCs evolving from serrated pathway and CTNNB1-mutated CRCs associated with Lynch syndrome.ConclusionsMSS low-grade adenocarcinoma, NOS, with intestinal differentiation without TILs (‘crypt-like adenocarcinoma’) might be the morphological pending of canonical molecular subtype of CRC defined as displayed molecular epithelial differentiation and upregulation of WNT in consensus molecular classification of CRC.

The aim of this study was to interrogate the heterogeneity of colorectal mucinous adenocarcinomas. This study is based on hierarchical clustering approach combining clinicopathological and molecular patterns known to be relevant to oncogenesis and therapeutic management of patients with colorectal carcinoma, ie, microsatellite instability, O6-methylguanine-DNA methyltransferase (MGMT) status, KRAS, and BRAF mutations and wnt signaling pathway activation. Comparison of the study group of 60 mucinous adenocarcinomas defined according to World Health Organization classification with control group of 136 colorectal adenocarcinomas successively removed shows higher frequency of BRAF and KRAS mutations and microsatellite instability-high status and lower frequency of wnt signaling pathway activation in mucinous adenocarcinomas. Hierarchical clustering isolated three relevant clusters: (i) cluster of microsatellite stable mucinous adenocarcinomas (54%) with KRAS mutation, and frequent MGMT changes, more frequently located in the left colon, often associated with contiguous precursor adenoma; (ii) cluster of BRAF-mutated mucinous adenocarcinomas (28%) with either microsatellite instability-high or microsatellite stable status, occurring in elderly female patients, nearly all located in the right colon, having the signature of serrated pathway of carcinomas; and (iii) a heterogeneous cluster of microsatellite instability-high mucinous carcinomas (18%), including inherited colorectal carcinomas, displaying a high-grade histological pattern. Age, TNM stage, and BRAF mutation had prognostic value. Hierarchical clustering analysis led to the identification of several clinicopathological entities of colorectal mucinous adenocarcinomas with epidemiologic, prognostic, and therapy relevance. Both KRAS and BRAF mutations appear as drivers in the alternate oncogenetic pathways governing the development of sporadic colorectal mucinous adenocarcinomas.

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 (

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.

This study addresses the extent of the heterogeneity of KRAS status, present in a minority of metastatic colorectal carcinomas (mCRCs), on the basis of a thorough analysis of surgical resection specimens. Eighteen patients with mCRC were included. KRAS mutations (exon 2, codons 12 and 13) were determined using PCR and subsequent direct sequencing. This analysis included primary tumours (n=21), synchronous (n=10) and metachronous (n=18) matched metastases, and pelvic recurrence (n=1). Heterogeneity of KRAS status consisted in KRAS mutated in (i) the primary tumour but not in its synchronous metastasis, (ii) the metastasis but not in the primary tumour, (iii) the pelvic recurrence but not in the primary tumour, (iiii) some metastases and not in others from the same patient. Finally, the KRAS status varied among different areas of the same metastatic focus. This study defines the concept of KRAS mosaicism that affects a minority of mCRCs.

ABO histo-blood group type and secretor status are two genetically determined factors that contribute to resistance and susceptibility to Norwalk virus (NV). Archived serum samples but not saliva samples are available from NV and many other norovirus challenge studies and outbreaks. A person's ABO phenotype is easily determined from their archived sera, but the individual's secretor phenotype cannot easily be ascertained without saliva. We now report that a person's secretor genotype can also be determined from the archived serum samples. Of the 51 volunteers who participated in a NV challenge study, all eight non-secretors were resistant to NV infection, all of the 42 NV-infected volunteers were secretor positive, and a single uninfected secretor was histo-blood group type B. In agreement with a previous report, secretor status was most predictive of risk of NV infection. The methods described in this report should rapidly improve our knowledge of the associations between carbohydrate antigen expression and susceptibility to different strains of the non-cultivatable noroviruses by enabling retrospective studies from previously collected volunteer challenge and outbreak sera.

SCN5A Mutation and ST Segment Elevation in Inferior Leads. Mutations in the SCN5A gene can lead to the Brugada syndrome, a genetically inherited form of idiopathic ventricular fibrillation that has a characteristic ECG phenotype usually restricted to precordial leads V1–V3. We identified a novel G752R SCN5A missense mutation leading to various degrees of the Brugada ECG phenotype in members of a French family. In the proband, the G752R mutation produced ST segment elevation and prominent J wave in leads II, III, and aVF. In four other relatives, ST segment elevation in the right precordial but not in the inferior leads was observed either spontaneously or under flecainide challenge. Recombinant G752R mutant exhibited a markedly reduced Na+ current amplitude and a voltage shift in both activation and inactivation curves. The mutant was found in all affected but not in nonaffected family members. One additional gene-carrier had an almost normal ECG (silent gene-carrier). We provide genetic demonstration that Brugada ECG anomalies related to a unique SCN5A mutation can be observed either in the inferior or the right precordial leads. (J Cardiovasc Electrophysiol, Vol. 14, pp. 200-203, February 2003)

We have read with much interest the article Gottlieb et al. [2009] about BAK1 (MIM♯ 600516), as well as the epistolary exchange with Dr. Hatchwell that followed [Gottlieb et al., 2010; Hatchwell, 2010]. Our reading was all the more careful that the message delivered by the authors was strong, and was largely echoed and amplified in many scientific and nonscientific journals. Much noise was made about this article presented as a revolution in genetics, hanging a question mark over the dogma of a unique genome shared by all body cells. We were therefore eager to know all the details about this outstanding finding. In the first place, we thought that it was quite an original idea to simultaneously compare the genotypes of the BAK1 gene between abdominal aortic tissue, matched nondiseased tissue, and blood leukocytes. Yet, we were quickly annoyed by many inaccuracies and unclear explanations that seemed to distort the authors' message. The most confusing point fits in the commentary on the article by Hatchwell [2010]. We did not understand why the authors had not compared the same type of nucleic acid between blood (DNA) and matching aortic tissues (RNA). And yet, in their reply to Dr. Hatchwell, the authors state that “blood and tissue BAK1 genomic sequences were identical” [Gottlieb et al., 2010]. It is astounding that the authors forgot to mention this crucial observation in their original article, because it runs counter to the theory of a genome varying according to the tissue, that is, the very message widely spread by the media. Indeed, this observation means that the two different genotypes reported by Gottlieb et al. in Table 2 of their article do not correspond to differences between tissues but rather to differences between types of nucleic acid (DNA vs. RNA). It therefore excludes the authors' postulate of a mosaicism and of an allelic selection due to some environmental conditions. The alternative hypothetic mechanism of somatic mutations seems also to be discarded. Indeed, Gottlieb et al.'s Table 2 suggests that allelic variants at position 42 (rs1051911), 52 (rs1051912), and 81 (rs1051913) are likely in very strong or even complete linkage disequilibrium, because only two haplotype combinations are represented (major haplotype CAG/GCT/ATC vs. variant haplotype CAA/GCC/ATT). By the way, it is not clear whether the genotypes reported in Gottlieb et al.'s Table 2 were found in a homozygous state; it seems odd that the sequences of the aortic tissues do not exhibit traces of the major allelic variants, which are largely predominant in blood (of note, rs1051912 and rs1051913 variant alleles were never found in any of the HapMap or SNP500 cancer populations). Assuming that the genotypes are really homozygous anyway, the results in Table 2 of Gottlieb et al.'s article mean that all the aortic DNA samples tested would have to derive from a primary DNA that would have systematically undergone the same triple modification. To our knowledge, even in cancer, where recurrent somatic mutations in hot spots are well known (e.g., mutation V600E in BRAF or mutations in codons 12 and 13 of KRAS in colorectal cancer) [Oliveira et al., 2007], a triple nucleotidic modification that would systematically affect three distinct and nonadjacent codons has never been described. The explanation of the difference between DNA and RNA sequences observed by the authors must therefore lie at the RNA level. Yet, it cannot be explained by RNA editing. The work of Gottlieb et al. [2009] indeed highlights changes G>A (codon 42), T>C (codon 52), and C>T (codon 81), whereas in the mechanism of RNA editing described to date, ADARs and cytidine deaminases induce A>G and C>T change, respectively [Mattick and Mehler, 2008]. Thus, only the variant observed in codon 81, that is, the one corresponding to a synonymous change, could be affected by RNA editing, unless the authors point to a new mechanism. A more plausible hypothesis remains the crosscontamination between BAK1 transcript and genomic DNA of pseudogene BAK2 (BAK1P1) suggested by Hatchwell [2010]. Gottlieb et al. [2010] excluded that BAK1 cDNA sequences of abdominal aortic tissues may have been contaminated by BAK2 sequences, because of the RNAse treatment used for reverse transcription. They consider thereby that BAK2 exists in its genomic form only [Gottlieb et al., 2010]. However, according to the databank reference sequence XM_002348050, BAK2 would have been predicted according to similarities to 1 mRNA, 18 ESTs, and 5 proteins. The fact that one supporting evidence is an mRNA suggests that an mRNA transcript and even a translation is not unlikely for BAK2. This would confirm the hypothesis of a sequencing artifact [Hatchwell, 2010], given that the RNA sequences of BAK1 and BAK2 differ exactly at the points of variation (single nucleotide polymorphisms [SNPs]) observed by Gottlieb et al. [2009] (Fig. ​(Fig.1).1). Besides, in order to refute the hypothesis of a contamination by a sequence other than BAK1, the authors use two variants concerning codons 2 (ATG/GCC/TCG for BAK2 vs. ATG/GCT/TCG for BAK1) and 145 of both BAK1 and BAK2 that would enable to discriminate the two genes [Gottlieb et al., 2010]. The problem is that the variant at codon 2 is not present in all the BAK2 sequences of reference found in the NCBI databank (Fig. ​(Fig.2),2), which makes very doubtful the validity of the reference sequence for BAK2 and thus the demonstration of Gottlieb et al. [2009,2010]. Moreover, the authors do not comment on the six other variants that differ between the sequences of BAK1 (XM_002348050) and BAK2 ({"type":"entrez-nucleotide","attrs":{"text":"NG_000850","term_id":"149944627"}}NG_000850), which would be useful to make a discrimination between the two gene sequences. Figure 1 Comparison of BAK1 cDNA (NCBI reference: {"type":"entrez-nucleotide","attrs":{"text":"NM_001188.3","term_id":"109698605"}}NM_001188.3) and BAK2 genomic sequences (NCBI reference: {"type":"entrez-nucleotide","attrs":{"text":"NG_000850","term_id":"149944627"}} ... Figure 2 Comparison of the different sequences retrieved from NCBI for BAK2 gene. Focus on the two variants in codons 2 and 145 used by Gottlieb et al. [2010] to refute a crosscontamination between BAK1 and BAK2 sequences. We show here that two of the five BAK2 ... In conclusion, like Dr. Hatchwell, we are not convinced that the cDNA sequence presented by Gottlieb et al. [2009,2010] in the aortic tissues corresponds to BAK1 and not to pseudogene BAK2. Our doubts are all the more strong because the five controls tested by the authors all carry the three rare variants rs1051911 (codon 42), rs1051912 (codon 52), and rs105193 (codon 81). Whenever these rare variants were truly predisposing to aneurysm, the probability to find them in all five controls would be very low, unless there had been a significant bias in the recruitment of the controls. Yet, even if the contamination was confirmed there would still remain one enigma: why would the authors find a homozygous variant sequence, because the oligonucleotidic primers that they used for their experiments perfectly match both BAK1 and BAK2 mRNA sequences? One explanation could be an allele-specific amplification due to an undescribed polymorphism in one of the primer sequences. All in all, Gottlieb et al. [2009] contains an accumulation of inaccuracies and paradoxical results that put a doubt on their conclusions. We do not pretend that their interpretations are wrong, but we believe that there is too huge a contrast between the strong message delivered by the authors and the quality of the experiments and report that they proposed, which did not meet the high-level standard expected for a major work—considering also the obvious translation errors of original Table 2 of Gottlieb et al.'s article corrected in the Erratum published online on 22 November 2009. Consequently, we hope that the authors will extend their study in order to clarify the sequence variations. It is also noteworthy that the quite neutral title of the article, “BAK1 gene variation and abdominal aortic aneurysms,” does not reflect the revolutionary idea relayed by the media. This suggests that the authors probably did not expect such an impact for their work, and that their results might have been prematurely overrated.

Introduction: Erythrocytoses are characterized by an elevated red cell mass. The most widely studied disease is Polycythemia Vera (PV), a myeloproliferative neoplasm due to the acquired JAK2-V617F mutation. However, other types of erythrocytoses exist and are of major importance. They can be either inherited (Congenital Erythrocytosis-CE) or diagnosed in adult patients with no family history (Idiopathic Erythrocytosis-IE). CE/IE are not associated with myeloproliferation but they can be associated with severe thrombo-embolic or haemorrhagic events, pulmonary arterial hypertension and, rarely, tumours. The 8 genes identified so far as causing CE lie at the crossroads of major biological pathways (metabolism, inflammation, oncogenesis) and are implicated in multiple diseases. These genes are involved (i) in the regulation of the hypoxia pathway, PHD2 (also called EGLN1), HIF-2A (EPAS1), VHL, (ii) in proliferation and differentiation of erythroid progenitors (EPOR), or (iii) in mature cell function, haemoglobins (HBB, HBA1, HBA2) or bisphosphoglyceratemutase (BPGM). However, in 80% of cases the cause remains unknown meaning that no proper diagnosis can be made, no prognosis or advice can be provided to CE/IE patients and their families, and no curative treatment exists. Method: We created and developed a national network in France to (i) identify, (ii) collect and (iii) analyze the genomic abnormalities in patients suspected of CE/IE. The selection of patients was performed using a clinical and biological data sheet including mandatory further tests in order to exclude patients with PV or obvious secondary erythrocytosis related to lung, cardiac or renal disorder. Next generation sequencing (NGS) has been used to analyse the presence of mutations in 17 genes (VHL, PHD1, PHD2 and PHD3, HIF1A, HIF2A, HIF3A, FH, BPGM, and 8 other candidate genes). SureDesign software (Agilent, Santa Clara, CA) was used to design the custom HaloPlex capture assay. For sequence capture, HaloPlex Target Enrichment System Kit (AgilentR), for Illumina sequencing was used, according to the manufacturer's instructions. Results: To date, samples from 103 patients have been recorded, among whom 46 have been tested using NGS approach. Variants in 10 (21%) patients [9 males and 1 female ; median age 50 y. (12-71)] with unknown significance have been detected, including 4 in PHD genes, 5 in HIF genes, and 1 in JAK2 gene. In patients with variants, a familial history of erythrocytosis was noted in 2. No independent thrombotic complication was reported in the 10 patients. The proportion of variants detected (21%) was close to the classical rate of genomic abnormalities usually observed in CE/IE. In 2 patients (one with a PHD2 and one with a JAK2 variants), the erythropoietin was low, whereas for the others, the erythropoietin was normal. Of note, the median age of the patients was surprisingly high, suggesting that the diagnostic was not previously performed due to the absence of available tests. Indeed, the diagnostic approaches using NGS techniques led to a considerable time gain and facilitated the identification of certain molecular abnormalities associated with CE/IE Conclusion: NGS is a useful tool to explore mutations in CE/IE, but identifies genetic variants in only 20% of patients with such disorder. In vitro, in cellulo and in vivo (including zebrafish models) functional studies are currently performed to validate the clinical relevance of these variants. Further exams including whole exome sequencing are planned to achieve a right diagnosis in the 80% remaining CE/IE patients without identified genomic abnormalities. Disclosures No relevant conflicts of interest to declare.

The binding of Norwalk virus (NV) recombinant capsids was tested in a panel of saliva samples collected from 96 donors with different ABO, secretor, and Lewis phenotypes. As previously reported, binding occurred specifically to saliva from secretors, regardless of their Lewis phenotype status. Blood group B saliva was poorly recognized, whereas binding to blood group O saliva was higher and binding to blood group A saliva was highest. Transfection of either blood group A or B enzyme into H epitope-expressing cells showed that masking of H epitopes by the A and B antigens blocked the attachment of NV capsids. The high level of binding to blood group A secretor saliva could be explained by an optimal H type 1 ligand density, which was lower than that in blood group O saliva and much higher than that in blood group B saliva. Indeed, despite a higher ligand density, saliva from homozygotes with 2 functional FUT2 alleles was less strongly recognized than saliva from heterozygotes with 1 functional and 1 inactivated FUT2 allele. Partial fucosidase treatment of duodenal tissue sections and binding to a synthetic probe with varying densities of H type 1 trisaccharide indicated that optimal attachment occurred at medium ligand density.

Mutations in the SCN5A gene can lead to the Brugada syndrome, a genetically inherited form of idiopathic ventricular fibrillation that has a characteristic ECG phenotype usually restricted to precordial leads V1-V3. We identified a novel G752R SCN5A missense mutation leading to various degrees of the Brugada ECG phenotype in members of a French family. In the proband, the G752R mutation produced ST segment elevation and prominent J wave in leads II, III, and aVF. In four other relatives, ST segment elevation in the right precordial but not in the inferior leads was observed either spontaneously or under flecainide challenge. Recombinant G752R mutant exhibited a markedly reduced Na+ current amplitude and a voltage shift in both activation and inactivation curves. The mutant was found in all affected but not in nonaffected family members. One additional gene-carrier had an almost normal ECG (silent gene-carrier). We provide genetic demonstration that Brugada ECG anomalies related to a unique SCN5A mutation can be observed either in the inferior or the right precordial leads.

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.