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Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease caused by mutations in sarcomeric genes. However, extensive genetic screening failed to identify a mutation in about a third of cases. One possible explanation is that other diseases, caused by other genes, may mimic HCM. Objective: To investigate the possible involvement of Danon's disease, an X linked lysosomal disease, in a large population of patients with HCM. Methods: A population of 197 index cases was considered; 124 were subsequently excluded because of a mutation in sarcomeric genes and 23 because of autosomal dominant inheritance. Fifty index cases were therefore included in molecular analysis (direct sequencing) of the lysosome associated membrane protein 2 (LAMP2) gene responsible for Danon's disease. Results: Two new mutations leading to premature stop codons were identified in patients who evolved towards severe heart failure (< 25 years old): 657C>T and 173_179del. The prevalence was therefore 1 % of the total population (two of 197) or 4% of enrolled index cases (two of 50). Interestingly, Danon's disease was responsible for half of the cases (Iwo of four) with HCM and clinical skeletal myopathy but was not involved in isolated HCM (none of 41). Conclusions: Danon's disease may be involved in patients with previously diagnosed as HCM. A diagnosis strategy is proposed. To distinguish HCM from Danon's disease is important because the clinical evolution, prognosis, mode of inheritance, and therefore genetic counselling are very different. [ABSTRACT FROM AUTHOR]

The family history of cancer in children treated for a solid malignant tumour in the Paediatric Oncology Department at Institute Gustave-Roussy, has been investigated. In order to determine the role of germline p53 mutations in genetic predisposition to childhood cancer, germline p53 mutations were sought in individuals with at least one relative (first- or second-degree relative or first cousin) affected by any cancer before 46 years of age, or affected by multiple cancers. Screening for germline p53 mutation was possible in 268 index cases among individuals fulfilling selection criteria. Seventeen (6.3%) mutations were identified, of which 13 were inherited and four were de novo. Using maximum likelihood methods that incorporate retrospective family data and correct for ascertainment bias, the lifetime risk of cancer for mutation carriers was estimated to be 73% for males and nearly 100% for females with a high risk of breast cancer accounting for the difference. The risk of cancer associated with such mutations is very high and no evidence of low penetrance mutation was found. These mutations are frequently inherited but de novo mutations are not rare. [ABSTRACT FROM AUTHOR]

Tau proteins belong to the microtubule associated protein family and are mainly expressed in neurons. Tau accumulates in patients' brain in several neurodegenerative diseases, including Fronto-temporal dementia and Alzheimer's disease. Recently, we described a 17q21.31 duplication in patients presenting different cognitive or motor symptoms and characterized by the accumulation of different Tau isoforms. This duplication involves four genes, including the MAPT gene that encodes the Tau protein. The main pathophysiological consequence associated with this duplication was a 1.6-1.9-fold increase in the MAPT messenger RNA as measured in blood samples of duplication carriers. However, the pathophysiological consequences of this duplication in a cell type relevant for neurodegenerative diseases have never been explored so far. In this study, we developed the first model of primary tauopathy linked to a 17q21.31 duplication in iPSC-induced neurons from 2 unrelated carriers. As in patients' blood, we demonstrated that this duplication was associated with an increase in MAPT mRNA resulting in elevated Tau protein levels in iPSC-derived cortical neurons. We believe that these iPSC lines will be a pertinent tool to elucidate how a same genetic cause could lead to distinct types of tauopathies and the pathophysiological mechanisms associated with Tau-mediated neurodegeneration in the 17q21.31 duplication context., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer's disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aβ peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 70 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variants (R332W, S577P, and R654W) and two maturation-competent variants (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of Aβ secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variants on SorLA protein suggested that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions., (© 2021. The Author(s).)

Background: The interpretation of germline TP53 variants is critical to ensure appropriate medical management of patients with cancer and follow-up of variant carriers. This interpretation remains complex and is becoming a growing challenge considering the exponential increase in TP53 tests. We developed a functional assay directly performed on patients' blood., Methods: Peripheral blood mononuclear cells were cultured, activated, exposed to doxorubicin and the p53-mediated transcriptional response was quantified using reverse transcription-multiplex ligation probe amplification and RT-QMPSF assays, including 10 p53 targets selected from transcriptome analysis, and two amplicons to measure p53 mRNA levels. We applied this blood functional assay to 77 patients addressed for TP53 analysis., Results: In 51 wild-type TP53 individuals, the mean p53 functionality score was 12.7 (range 7.5-22.8). Among eight individuals harbouring likely pathogenic or pathogenic variants, the scores were reduced (mean 4.8, range 3.1-7.1), and p53 mRNA levels were reduced in patients harbouring truncating variants. We tested 14 rare unclassified variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro191Arg), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108&#95;Phe109delinsVal), p.(Asn131del), p.(Leu265del), c.-117G>T) and 12 yielded functionally abnormal scores. Remarkably, the assay revealed that the c.*1175A>C polymorphic variant within TP53 poly-adenylation site can impact p53 function with the same magnitude as a null variant, when present on both alleles, and may act as a modifying factor in pathogenic variant carriers., Conclusion: This blood p53 assay should therefore be a useful tool for the rapid clinical classification of germline TP53 variants and detection of non-coding functional variants., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Infantile hypercalcemia is an autosomal recessive disorder caused either by mutations in the CYP24A1 gene (20q13.2) or in the SLC34A1 gene (5q35.3). This disease is characterized by hypercalcemia, hypercalciuria and nephrocalcinosis in paediatric patients. Maternal uniparental disomy of chromosome 20 [UPD(20)mat], resulting in aberrant expression of imprinted transcripts at the GNAS locus, is a poorly characterized condition. UPD(20)mat patients manifest a phenotype similar to that of Silver-Russell syndrome and small for gestational age-short stature., Case Presentation: We report here the genetic and clinical characterization of a male child with a phenotype of infantile hypercalcemia, postnatal growth retardation, and minor dysmorphic features. Genetic analysis using a next generation sequencing panel revealed a homozygous pathogenic variant of CYP24A1. The absence of the variant in the father led to microsatellite segregation analysis, suggestive of UPD. SNP-array revealed a large terminal copy neutral loss of heterozygosity leading to CYP24A1 homozygosity. SNP-array data of parent-child trio confirmed a UPD(20)mat responsible for both infantile hypercalcemia and Silver-Russell syndrome-like traits., Conclusion: This is the first report of uniparental disomy of chromosome 20 revealed by infantile hypercalcemia related to CYP24A1 biallelic homozygous variants, underlying the importance of controlling allelic segregation in cases of homozygosity.

In utero alcohol exposure can induce severe neurodevelopmental disabilities leading to long-term behavioral deficits. Because alcohol induces brain defects, many studies have focused on nervous cells. However, recent reports have shown that alcohol markedly affects cortical angiogenesis in both animal models and infants with fetal alcohol spectrum disorder (FASD). In addition, the vascular system is known to contribute to controlling gamma-aminobutyric acid (GABA)ergic interneuron migration in the developing neocortex. Thus, alcohol-induced vascular dysfunction may contribute to the neurodevelopmental defects in FASD. The present study aimed at investigating the effects of alcohol on endothelial activity of pial microvessels. Ex vivo experiments on cortical slices from mouse neonates revealed that in endothelial cells from pial microvessels acute alcohol exposure inhibits both glutamate-induced calcium mobilization and activities of matrix metalloproteinase-9 (MMP-9) and tissue plasminogen activator (tPA). The inhibitory effect of alcohol on glutamate-induced MMP-9 activity was abrogated in tPA-knockout and Grin1 flox /VeCad cre mice suggesting that alcohol interacts through the endothelial NMDAR/tPA/MMP-9 vascular pathway. Contrasting with the effects from acute alcohol exposure, in mouse neonates exposed to alcohol in utero during the last gestational week, glutamate exacerbated both calcium mobilization and endothelial protease activities from pial microvessels. This alcohol-induced vascular dysfunction was associated with strong overexpression of the N-methyl-d-aspartate receptor subunit GluN1 and mispositioning of the Gad67-GFP interneurons that normally populate the superficial cortical layers. By comparing several human control fetuses with a fetus chronically exposed to alcohol revealed that alcohol exposure led to mispositioning of the calretinin-positive interneurons, whose density was decreased in the superficial cortical layers II-III and increased in deepest layers. This study provides the first mechanistic and functional evidence that alcohol impairs glutamate-regulated activity of pial microvessels. Endothelial dysfunction is characterized by altered metalloproteinase activity and interneuron mispositioning, which was also observed in a fetus with fetal alcohol syndrome. These data suggest that alcohol-induced endothelial dysfunction may contribute in ectopic cortical GABAergic interneurons, that has previously been described in infants with FASD., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Actionable somatic molecular alterations are found in 15% to 20% of NSCLC in Europe. NSCLC is a tumor observed in patients with germline TP53 variants causing Li-Fraumeni syndrome (LFS), but its somatic molecular profile is unknown., Methods: Retrospective study of clinical and molecular profiles of patients with NSCLC and germline TP53 variants., Results: Among 22 patients with NSCLC and LFS (n = 23 lung tumors), 64% were women, median age was 51 years, 84% were nonsmokers, 73% had adenocarcinoma histological subtype, and 84% were diagnosed with advanced-stage disease. These patients harbored 16 distinct germline TP53 variants; the most common was p.R158H (5/22; three in the same family). Personal and family histories of cancer were reported in 71% and 90% of patients, respectively. In most cases (87%, 13/15), lung cancer was diagnosed with a late onset. Of the 21 tumors analyzed, somatic oncogenic driver mutations were found in 19 of 21 (90%), EGFR mutations in 18 (exon 19 deletion in 12 cases, L858R in three cases, and G719A, exon 20 insertion, and missing mutation subtype, each with one case), and ROS1 fusion in one case. A PI3KCA mutation was concurrently detected at diagnosis in three EGFR exon 19-deleted tumors (3/12). The median overall survival was 37.3 months in 14 patients treated with EGFR inhibitors; seven developed resistance, five (71%) acquired EGFR-T790M mutation, and one had SCLC transformation., Conclusions: Driver oncogenic alterations were observed in 90% of the LFS tumors, mainly EGFR mutations; one ROS1 fusion was also observed. The germline TP53 variants and lung cancer carcinogenesis driven by oncogenic processes need further evaluation., (Copyright © 2020 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Background: Heterozygous germline PMS2 variants are responsible for about 5% of Lynch syndrome (LS) but their prevalence is most likely underestimated because of complicated routine screening caused by highly homologous pseudogenes. Consequently, there is limited knowledge on the implication of the PMS2 gene in LS., Methods: We report 200 PMS2 heterozygous variants identified in 195 French patients, including 112 unique variants classified as class-3/4/5., Results: Genomic rearrangements account for 18% of alterations. The c.137G>T variant was observed in 18% of the patients, but a founder effect could not be clearly identified by haplotype analysis. Among class-4/5 variant carriers, the median age at first tumour onset was 49 years with a predominance of colorectal (80%) and endometrial (8.1%) cancers. Seven patients developed colorectal cancers before the age of 30 with the youngest at the age of 21. Only 6.2% of class-4/5 carriers had a family history fulfilling Amsterdam I/II criteria among patients with available data. Tumours from PMS2 variant carriers exhibited microsatellite instability (96%) and loss of PMS2 expression (76%), confirming the high predictive value of somatic analysis., Conclusion: Our results provide further insight into the role of the PMS2 gene in LS. While PMS2 variants are mostly detected in families not fulfilling Amsterdam criteria, which supports their lower penetrance, they can nevertheless cause early-onset cancers, highlighting the variability of their penetrance., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Constitutional epimutations are an alternative to genetic mutations in the etiology of genetic diseases. Some of these epimutations, termed secondary, correspond to the epigenetic effects of cis-acting genetic defects transmitted to the offspring following a Mendelian inheritance pattern. In Lynch syndrome, a few families with such apparently heritable MLH1 epimutations have been reported so far., Methods: We designed a long-range polymerase chain reaction next-generation sequencing strategy to screen MLH1 entire gene and applied it to 4 French families with heritable epimutations and 10 additional patients with no proven transmission of their epimutations., Results: This strategy successfully detected the insertion of an Alu element in MLH1 coding sequence in one family. Two previously unreported MLH1 variants were also identified in other epimutation carriers: a nucleotide substitution within intron 1 and a single-nucleotide deletion in the 5'-UTR. Detection of a partial MLH1 duplication in another family required multiplex ligation-dependent probe amplification technology. We demonstrated the segregation of these variants with MLH1 methylation and studied the functional consequences of these defects on transcription., Conclusion: This is the largest cohort of patients with MLH1 secondary epimutations associated with a broad spectrum of genetic defects. This study provides further insight into the complexity of molecular mechanisms leading to secondary epimutations.

Purpose: Integration of gene panels in the diagnosis of hereditary breast and ovarian cancer (HBOC) requires a careful evaluation of the risk associated with pathogenic or likely pathogenic variants (PVs) detected in each gene. Here we analyzed 34 genes in 5131 suspected HBOC index cases by next-generation sequencing., Methods: Using the Exome Aggregation Consortium data sets plus 571 individuals from the French Exome Project, we simulated the probability that an individual from the Exome Aggregation Consortium carries a PV and compared it to the estimated frequency within the HBOC population., Results: Odds ratio conferred by PVs within BRCA1, BRCA2, PALB2, RAD51C, RAD51D, ATM, BRIP1, CHEK2, and MSH6 were estimated at 13.22 [10.01-17.22], 8.61 [6.78-10.82], 8.22 [4.91-13.05], 4.54 [2.55-7.48], 5.23 [1.46-13.17], 3.20 [2.14-4.53], 2.49 [1.42-3.97], 1.67 [1.18-2.27], and 2.50 [1.12-4.67], respectively. PVs within RAD51C, RAD51D, and BRIP1 were associated with ovarian cancer family history (OR = 11.36 [5.78-19.59], 12.44 [2.94-33.30] and 3.82 [1.66-7.11]). PALB2 PVs were associated with bilateral breast cancer (OR = 16.17 [5.48-34.10]) and BARD1 PVs with triple-negative breast cancer (OR = 11.27 [3.37-25.01]). Burden tests performed in both patients and the French Exome Project population confirmed the association of PVs of BRCA1, BRCA2, PALB2, and RAD51C with HBOC., Conclusion: Our results validate the integration of PALB2, RAD51C, and RAD51D in the diagnosis of HBOC and suggest that the other genes are involved in an oligogenic determinism.

Extreme microcephaly and rhombencephalosynapsis represent unusual pathological conditions, each of which occurs in isolation or in association with various other cerebral and or extracerebral anomalies. Unlike microcephaly for which several disease-causing genes have been identified with different modes of inheritance, the molecular bases of rhombencephalosynapsis remain unknown and rhombencephalosynapsis presents mainly as a sporadic condition consistent with de novo dominant variations. We report for the first time the association of extreme microcephaly with almost no sulcation and rhombencephalosynapsis in a fœtus for which comparative patient-parent exome sequencing strategy revealed a heterozygous de novo missense variant in the ADGRL2 gene. ADGRL2 encodes latrophilin 2, an adhesion G-protein-coupled receptor whose exogenous ligand is α-latrotoxin. Adgrl2 immunohistochemistry and in situ hybridization revealed expression in the telencephalon, mesencephalon and rhombencephalon of mouse and chicken embryos. In human brain embryos and fœtuses, Adgrl2 immunoreactivity was observed in the hemispheric and cerebellar germinal zones, the cortical plate, basal ganglia, pons and cerebellar cortex. Microfluorimetry experiments evaluating intracellular calcium release in response to α-latrotoxin binding showed significantly reduced cytosolic calcium release in the fœtus amniocytes vs amniocytes from age-matched control fœtuses and in HeLa cells transfected with mutant ADGRL2 cDNA vs wild-type construct. Embryonic lethality was also observed in constitutive Adgrl2 -/- mice. In Adgrl2 +/- mice, MRI studies revealed microcephaly and vermis hypoplasia. Cell adhesion and wound healing assays demonstrated that the variation increased cell adhesion properties and reduced cell motility. Furthermore, HeLa cells overexpressing mutant ADGRL2 displayed a highly developed cytoplasmic F-actin network related to cytoskeletal dynamic modulation. ADGRL2 is the first gene identified as being responsible for extreme microcephaly with rhombencephalosynapsis. Increased cell adhesion, reduced cell motility and cytoskeletal dynamic alterations induced by the variant therefore represent a new mechanism responsible for microcephaly.

Background: Development of tumours such as adrenocortical carcinomas (ACC), choroid plexus tumours (CPT) or female breast cancers before age 31 or multiple primary cancers belonging to the Li-Fraumeni (LFS) spectrum is, independently of the familial history, highly suggestive of a germline TP53 mutation. The aim of this study was to determine the contribution of de novo and mosaic mutations to LFS., Methods and Results: Among 328 unrelated patients harbouring a germline TP53 mutation identified by Sanger sequencing and/or QMPSF, we could show that the mutations had occurred de novo in 40 cases, without detectable parental age effect. Sanger sequencing revealed two mosaic mutations in a child with ACC and in an unaffected father of a child with medulloblastoma. Re-analysis of blood DNA by next-generation sequencing, performed at a depth above 500X, from 108 patients suggestive of LFS without detectable TP53 mutations, allowed us to identify 6 additional cases of mosaic TP53 mutations, in 2/49 children with ACC, 2/21 children with CPT, in 1/31 women with breast cancer before age 31 and in a patient who developed an osteosarcoma at age 12, a breast carcinoma and a breast sarcoma at age 35., Conclusions: This study performed on a large series of TP53 mutation carriers allows estimating the contribution to LFS of de novo mutations to at least 14% (48/336) and suggests that approximately one-fifth of these de novo mutations occur during embryonic development. Considering the medical impact of TP53 mutation identification, medical laboratories in charge of TP53 testing should ensure the detection of mosaic mutations., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have recently been involved in breast and ovarian cancer predisposition: RAD51B, RAD51C, and RAD51D in ovarian cancer, RAD51B and XRCC2 in breast cancer. The aim of this study was to estimate the contribution of deleterious variants in the five RAD51 paralogs to breast and ovarian cancers. The five RAD51 paralog genes were analyzed by next-generation sequencing technologies in germline DNA from 2649 consecutive patients diagnosed with breast and/or ovarian cancer. Twenty-one different deleterious variants were identified in the RAD51 paralogs in 30 patients: RAD51B (n = 4), RAD51C (n = 12), RAD51D (n = 7), XRCC2 (n = 2), and XRCC3 (n = 5). The overall deleterious variant rate was 1.13% (95% confidence interval (CI): 0.72-1.55%) (30/2649), including 15 variants in breast cancer only cases (15/2063; 0.73% (95% CI: 0.34-1.11%)) and 15 variants in cases with at least one ovarian cancer (15/570; 2.63% (95% CI: 1.24-4.02%)). This study is the first evaluation of the five RAD51 paralogs in breast and ovarian cancer predisposition and it demonstrates that deleterious variants can be present in breast cancer only cases. Moreover, this is the first time that XRCC3 deleterious variants have been identified in breast and ovarian cancer cases.

Background: The direct comparison of CA19.9, circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA) using endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has never been performed for the diagnosis of solid pancreatic tumours (SPTs)., Methods: We included 68 patients with a SPT referred for EUS-FNA. CTCs were analysed using size-based platform and ctDNA using digital PCR. The sensitivity, specificity, negative and positive predictive values were evaluated for each marker and their combination., Results: SPTs corresponded to 58 malignant tumours (52 pancreatic adenocarcinoma (PA) and 6 others) and 10 benign lesions. The sensitivity and specificity for PA diagnosis were 73% and 88% for EUS-FNA, 67% and 80% for CTC, 65% and 75% for ctDNA and 79% and 93% for CA19.9, respectively. The positivity of at least 2 markers was associated with a sensitivity and specificity of 78% and 91%, respectively. CtDNA was the only marker associated with overall survival (median 5.2 months for ctDNA+ vs 11.0 months for ctDNA-, P=0.01)., Conclusions: CA19.9 alone and in combination with ctDNA and/or CTC analysis may represent an efficient method for diagnosing PA in patients with SPTs. Further studies including a larger cohort of patients with both malignant and benign lesions will be necessary to confirm these promising results.

Background: Cystinuria is an autosomal recessive disorder of dibasic amino acid transport in the kidney and the intestine leading to increased urinary cystine excretion and nephrolithiasis. Two genes, SLC3A1 and SLC7A9 , coding respectively for rBAT and b0,+AT, account for the genetic basis of cystinuria., Methods: This study reports the clinical and molecular characterization of a French cohort including 112 cystinuria patients and 25 relatives from 99 families. Molecular screening was performed using sequencing and Quantitative Multiplex PCR of Short Fluorescent Fragments analyses. Functional minigene-based assays have been used to characterize splicing variants., Results: Eighty-eight pathogenic nucleotide changes were identified in SLC3A1 (63) and SLC7A9 (25) genes, of which 42 were novel. Interestingly, 17% (15/88) and 11% (10/88) of the total number of variants correspond, respectively, to large-scale rearrangements and splicing mutations. Functional minigene-based assays were performed for six variants located outside the most conserved sequences of the splice sites; three variants affect splice sites, while three others modify exonic splicing regulatory elements (ESR), in good agreement with a new in silico prediction based on ΔtESRseq values., Conclusion: This report expands the spectrum of SLC3A1 and SLC7A9 variants and supports that digenic inheritance is unlikely. Furthermore, it highlights the relevance of assessing large-scale rearrangements and splicing mutations to fully characterize cystinuria patients at the molecular level.

Congenital hydrocephalus is considered as either acquired due to haemorrhage, infection or neoplasia or as of developmental nature and is divided into two subgroups, communicating and obstructive. Congenital hydrocephalus is either syndromic or non-syndromic, and in the latter no cause is found in more than half of the patients. In patients with isolated hydrocephalus, L1CAM mutations represent the most common aetiology. More recently, a founder mutation has also been reported in the MPDZ gene in foetuses presenting massive hydrocephalus, but the neuropathology remains unknown. We describe here three novel homozygous null mutations in the MPDZ gene in foetuses whose post-mortem examination has revealed a homogeneous phenotype characterized by multiple ependymal malformations along the aqueduct of Sylvius, the third and fourth ventricles as well as the central canal of the medulla, consisting in multifocal rosettes with immature cell accumulation in the vicinity of ependymal lining early detached from the ventricular zone. MPDZ also named MUPP1 is an essential component of tight junctions which are expressed from early brain development in the choroid plexuses and ependyma. Alterations in the formation of tight junctions within the ependyma very likely account for the lesions observed and highlight for the first time that primary multifocal ependymal malformations of the ventricular system is genetically determined in humans. Therefore, MPDZ sequencing should be performed when neuropathological examination reveals multifocal ependymal rosette formation within the aqueduct of Sylvius, of the third and fourth ventricles and of the central canal of the medulla.

Purpose: To assess the prognostic and predictive value of circulating ESR1 mutation and its kinetics before and after progression on aromatase inhibitor (AI) treatment., Patients and Methods: ESR1 circulating D538G and Y537S/N/C mutations were retrospectively analyzed by digital droplet PCR after first-line AI failure in patients treated consecutively from 2010 to 2012 for hormone receptor-positive metastatic breast cancer. Progression-free survival (PFS) and overall survival (OS) were analyzed according to circulating mutational status and subsequent lines of treatment. The kinetics of ESR1 mutation before (3 and 6 months) and after (3 months) AI progression were determined in the available archive plasmas., Results: Circulating ESR1 mutations were found at AI progression in 44/144 patients included (30.6%). Median follow-up from AI initiation was 40 months (range 4-94). The median OS was decreased in patients with circulating ESR1 mutation than in patients without mutation (15.5 versus 23.8 months, P=0.0006). The median PFS was also significantly decreased in patients with ESR1 mutation than in patients without mutation (5.9 vs 7 months, P=0.002). After AI failure, there was no difference in outcome for patients receiving chemotherapy (n = 58) versus non-AI endocrine therapy (n=51) in patients with and without ESR1 mutation. ESR1 circulating mutations were detectable in 75% of all cases before AI progression, whereas the kinetics 3 months after progression did not correlate with outcome., Conclusion: ESR1 circulating mutations are independent risk factors for poor outcome after AI failure, and are frequently detectable before clinical progression. Interventional studies based on ESR1 circulating status are warranted.

The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient's RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.