Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

Germline JAK2 E846D Substitution as the Cause of Erythrocytosis?

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.

Comprehensive in silico and functional studies for classification of EPAS1/HIF2A genetic variants identified in patients with erythrocytosis.

Gain-of-function mutations in the EPAS1/HIF2A gene have been identified in patients with hereditary erythrocytosis that can be associated with the development of paraganglioma, pheochromocytoma and somatostatinoma. In the present study, we describe a unique European collection of 41 patients and 28 relatives diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified two infants with severe erythrocytosis associated with a mosaic mutation present in less than 2% of the blood, one of whom later developed a paraganglioma. The aim of this study was to determine the causal role of these genetic variants, to establish pathogenicity, and to identify potential candidates eligible for the new hypoxia-inducible factor-2 α (HIF-2α) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2b variants has been studied by using canonical and real-time reporter luciferase assays. These functional assays consisted of a novel edited vector containing an expanded region of the erythropoietin promoter combined with distal regulatory elements which substantially enhanced the HIF-2α-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 17 patients and 23 relatives. We described four new mutations (D525G, L526F, G527K, A530S) close to the key proline P531, which broadens the spectrum of mutations involved in erythrocytosis. Notably, we identified patients with only erythrocytosis associated with germline mutations A530S and Y532C previously identified at somatic state in tumors, thereby raising the complexity of the genotype/phenotype correlations. Altogether, this study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2α inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.

A de novo pathogenic variant in the MSH6 gene in a 52 years-old woman.

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.

Mapping of colorectal carcinoma diseases with activation of Wnt/beta-catenin signalling pathway using hierarchical clustering approach.

AIMS: To map the colorectal carcinoma (CRC) diseases with significant Wnt signalling pathway activation for delineating their clinicopathological and molecular profiles. METHODS: Mapping 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. RESULTS: Nuclei 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. CONCLUSIONS: MSS 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.

Classification of 101 BRCA1 and BRCA2 variants of uncertain significance by cosegregation study: A powerful approach.

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.

MEM: An Algorithm for the Reliable Detection of Microsatellite Instability (MSI) on a Small NGS Panel in Colorectal Cancer.

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.

Diagnosis of exon 12-positive polycythemia vera rescued by NGS.

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.

Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

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 VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and 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 regarding the VHL-related hypoxia-signaling pathway.

Mapping clinicopathological entities within colorectal mucinous adenocarcinomas: a hierarchical clustering approach.

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.

Evaluation of the colorectal cancer risk conferred by rare UNC5C alleles.

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.

A de novo germline MLH1 mutation in a Lynch syndrome patient with discordant immunohistochemical and molecular biology test results.

We describe a patient with a Homo sapiens mutL homolog 1 (MLH1)-associated Lynch syndrome with previous diagnoses of two distinct primary cancers: a sigmoid colon cancer at the age of 39 years, and a right colon cancer at the age of 50 years. The mutation identified in his blood and buccal cells, c.1771delG, p.Asp591Ilefs*25, appears to be a de novo event, as it was not transmitted by either of his parents. This type of de novo event is rare in MLH1 as only three cases have been reported in the literature so far. Furthermore, the discordant results observed between replication error phenotyping and immunohistochemistry highlight the importance of the systematic use of both pre-screening tests in the molecular diagnosis of Lynch syndrome.

Delineation of the infrequent mosaicism of KRAS mutational status in metastatic colorectal adenocarcinomas.

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.

Influence of the combined ABO, FUT2, and FUT3 polymorphism on susceptibility to Norwalk virus attachment.

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.