Publications by authors named "Thierry Frebourg"

245 Publications

MYT1L-associated neurodevelopmental disorder: description of 40 new cases and literature review of clinical and molecular aspects.

Hum Genet 2021 Nov 8. Epub 2021 Nov 8.

Department of Genetics, IHU Necker-Enfants Malades, University Paris Descartes, Paris, France.

Pathogenic variants of the myelin transcription factor-1 like (MYT1L) gene include heterozygous missense, truncating variants and 2p25.3 microdeletions and cause a syndromic neurodevelopmental disorder (OMIM#616,521). Despite enrichment in de novo mutations in several developmental disorders and autism studies, the data on clinical characteristics and genotype-phenotype correlations are scarce, with only 22 patients with single nucleotide pathogenic variants reported. We aimed to further characterize this disorder at both the clinical and molecular levels by gathering a large series of patients with MYT1L-associated neurodevelopmental disorder. We collected genetic information on 40 unreported patients with likely pathogenic/pathogenic MYT1L variants and performed a comprehensive review of published data (total = 62 patients). We confirm that the main phenotypic features of the MYT1L-related disorder are developmental delay with language delay (95%), intellectual disability (ID, 70%), overweight or obesity (58%), behavioral disorders (98%) and epilepsy (23%). We highlight novel clinical characteristics, such as learning disabilities without ID (30%) and feeding difficulties during infancy (18%). We further describe the varied dysmorphic features (67%) and present the changes in weight over time of 27 patients. We show that patients harboring highly clustered missense variants in the 2-3-ZNF domains are not clinically distinguishable from patients with truncating variants. We provide an updated overview of clinical and genetic data of the MYT1L-associated neurodevelopmental disorder, hence improving diagnosis and clinical management of these patients.
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http://dx.doi.org/10.1007/s00439-021-02383-zDOI Listing
November 2021

Circulating DNA changes are predictive of disease progression after transarterial chemoembolization.

Int J Cancer 2021 Oct 8. Epub 2021 Oct 8.

UNIROUEN, Inserm 1245, IRON group, Normandie Univ, Rouen, France.

Transarterial chemoembolization (TACE) is used to treat patients with unresectable hepatocellular carcinoma (HCC). We evaluated the clinical impact of a-fetoprotein (AFP) and circulating cell-free and tumor DNA (cfDNA and ctDNA) changes around the TACE procedure. Our prospective monocentric study enrolled consecutive patients treated with TACE, with samples collected at baseline (D - 1), Day 2 (D + 2) and 1 month (M + 1) after TACE. cfDNA was quantified by the fluorometric method, and ctDNA was quantified by digital polymerase chain reaction designed for two hotspot TERT mutations. Computerized tomography scans or magnetic resonance imaging were performed at M + 1 every 3 months following TACE and independently reviewed. The objective was to identify thresholds of cfDNA, ctDNA and AFP changes associated with progressive disease (PD) using receiver operating characteristic curves. Thirty-eight patients were included from March 2018 to March 2019. All markers significantly increased from D - 1 to D + 2 (P < .005), and cfDNA and ctDNA significantly decreased from D + 2 to M + 1 (P < .0001). The analysis of changes from D - 1 to M + 1 identified thresholds at +31.4% for cfDNA and 0% for ctDNA that were significantly associated with PD at M + 1 (44.4% [>+31.4%] vs 3.8% [≤+31.4%] and 50.0% [>0%] vs 5.0% [≤0%], respectively). No significant threshold was identified for AFP. Using a score combining cfDNA and ctDNA, the patients were classified into high- or low-risk PD groups at M + 1, with PD rates of 80.0% vs 4.3% (P = .001) and median progression-free survival times of 1.3 vs 10.3 months (P = .002). Our study suggests that cfDNA and ctDNA increases around the TACE procedure and are associated with therapeutic failure.
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http://dx.doi.org/10.1002/ijc.33829DOI Listing
October 2021

Diagnostic chest X-rays and breast cancer risk among women with a hereditary predisposition to breast cancer unexplained by a BRCA1 or BRCA2 mutation.

Breast Cancer Res 2021 08 3;23(1):79. Epub 2021 Aug 3.

Institut Claudius Regaud - IUCT-Oncopole, Service d'Oncologie Médicale, Toulouse, France.

Background: Diagnostic ionizing radiation is a risk factor for breast cancer (BC). BC risk increases with increased dose to the chest and decreases with increased age at exposure, with possible effect modification related to familial or genetic predisposition. While chest X-rays increase the BC risk of BRCA1/2 mutation carriers compared to non-carriers, little is known for women with a hereditary predisposition to BC but who tested negative for a BRCA1 or BRCA2 (BRCA1/2) mutation.

Methods: We evaluated the effect of chest X-rays from diagnostic medical procedures in a dataset composed of 1552 BC cases identified through French family cancer clinics and 1363 unrelated controls. Participants reported their history of X-ray exposures in a detailed questionnaire and were tested for 113 DNA repair genes. Logistic regression and multinomial logistic regression models were used to assess the association with BC.

Results: Chest X-ray exposure doubled BC risk. A 3% increased BC risk per additional exposure was observed. Being 20 years old or younger at first exposure or being exposed before first full-term pregnancy did not seem to modify this risk. Birth after 1960 or carrying a rare likely deleterious coding variant in a DNA repair gene other than BRCA1/2 modified the effect of chest X-ray exposure.

Conclusion: Ever/never chest X-ray exposure increases BC risk 2-fold regardless of age at first exposure and, by up to 5-fold when carrying 3 or more rare variants in a DNA repair gene. Further studies are needed to evaluate other DNA repair genes or variants to identify those which could modify radiation sensitivity. Identification of subpopulations that are more or less susceptible to ionizing radiation is important and potentially clinically relevant.
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http://dx.doi.org/10.1186/s13058-021-01456-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8336294PMC
August 2021

[A, not so robertsonian, translocation!]

Ann Biol Clin (Paris) 2021 06;79(3):261-264

Department of Genetics, Rouen University Hospital and Inserm U1245, UNIROUEN, Normandie Univ, Normandy Center for Genomic and Personalized Medicine, France.

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http://dx.doi.org/10.1684/abc.2021.1646DOI Listing
June 2021

CEA, CA19-9, circulating DNA and circulating tumour cell kinetics in patients treated for metastatic colorectal cancer (mCRC).

Br J Cancer 2021 Aug 10;125(5):725-733. Epub 2021 Jun 10.

Department of Hepatogastroenterology and Department of Medical Oncology, Henri Becquerel Centre, Normandie Univ, UNIROUEN, Inserm U1245, IRON Group, Rouen University Hospital, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.

Background: We previously reported that CEA kinetics are a marker of progressive disease (PD) in metastatic colorectal cancer (mCRC). This study was specifically designed to confirm CEA kinetics for predicting PD and to evaluate CA19-9, cell-free DNA (cfDNA), circulating tumour DNA (ctDNA) and circulating tumour cell (CTC) kinetics.

Methods: Patients starting a chemotherapy (CT) with pre-treatment CEA > 5 ng/mL and/or CA19.9 > 30 UI/mL were prospectively included. Samples were collected from baseline to cycle 4 for CEA and CA19-9 and at baseline and the sixth week for other markers. CEA kinetics were calculated from the first to the third or fourth CT cycle.

Results: A total of 192 mCRC patients were included. CEA kinetics based on the previously identified >0.05 threshold was significantly associated with PD (p < 0.0001). By dichotomising by the median value, cfDNA, ctDNA and CA19-9 were associated with PD, PFS and OS in multivariate analysis. A circulating scoring system (CSS) combining CEA kinetics and baseline CA19-9 and cfDNA values classified patients based on high (n = 58) and low risk (n = 113) of PD and was independently associated with PD (ORa = 4.6, p < 0.0001), PFS (HRa = 2.07, p < 0.0001) and OS (HRa = 2.55, p < 0.0001).

Conclusions: CEA kinetics alone or combined with baseline CA19-9 and cfDNA are clinically relevant for predicting outcomes in mCRC.

Trial Registration Number: NCT01212510.
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http://dx.doi.org/10.1038/s41416-021-01431-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405627PMC
August 2021

Recurrence of an early postzygotic rescue of an inherited unbalanced translocation resulting in mosaic segmental uniparental isodisomy of chromosome 11q in siblings.

Am J Med Genet A 2021 10 27;185(10):3057-3061. Epub 2021 May 27.

Normandie Univ, UNIROUEN, Inserm U1245, CHU Rouen, Department of Genetics and reference center for developmental disorders, FHU G4 Génomique, F-76000, Rouen, France.

Balanced translocations are associated with a risk of transmission of unbalanced chromosomal rearrangements in the offspring. Such inherited chromosomal abnormalities are typically non-mosaic as they are present in the germline. We report the recurrence in two siblings of a mosaicism for a chromosomal rearrangement inherited from their asymptomatic father who carried a balanced t(2;11)(q35;q25) translocation. Both siblings exhibited a similar phenotype including intellectual disability, dysmorphic features, kyphoscoliosis, and cervical spinal stenosis. Karyotyping, fluorescence in situ hybridization and SNP array analysis of blood lymphocytes of both siblings identified two cell lines: one carrying a 2q35q37.3 duplication and a 11q25qter deletion (~90% cells), and one carrying an 11q uniparental isodisomy of maternal origin (~10% cells). We hypothesize that these mosaics were related to a postzygotic rescue mechanism which unexpectedly recurred in both siblings.
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http://dx.doi.org/10.1002/ajmg.a.62361DOI Listing
October 2021

When a maternal heterozygous mutation of the CYP24A1 gene leads to infantile hypercalcemia through a maternal uniparental disomy of chromosome 20.

Mol Cytogenet 2021 May 5;14(1):23. Epub 2021 May 5.

Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics and Reference Center for Developmental Disorders, Normandy Center for Genomic and Personalized Medicine, 76000, Rouen, France.

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.
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http://dx.doi.org/10.1186/s13039-021-00543-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101107PMC
May 2021

Hypersociability associated with developmental delay, macrocephaly and facial dysmorphism points to CHD3 mutations.

Eur J Med Genet 2021 Apr 9;64(4):104166. Epub 2021 Feb 9.

Normandie Univ, UNIROUEN, Inserm U1245, CHU Rouen, Department of Genetics and reference center for developmental disorders, FHU G4 Génomique, F-76000 Rouen, France. Electronic address:

CHD3-related syndrome, also known as Snijders Blok-Campeau syndrome, is a rare developmental disorder described in 2018, caused by de novo pathogenic variants in the CHD3 gene. This syndrome is characterized by global developmental delay, speech delay, intellectual disability, hypotonia and behavioral disorders including autism spectrum disorder (ASD). Typical dysmorphic features include macrocephaly, hypertelorism, enophthalmia, sparse eyebrows, bulging forehead, midface hypoplasia, prominent nose and pointed chin. To our knowledge, there have been no other clinical descriptions of patients since the initial publication. We report the clinical description of a 21-year-old patient harboring a pathogenic de novo variant in CHD3. We reviewed the clinical features of the 35 previously reported patients. Main features were severe intellectual disability, dysmorphic facies, macrocephaly, cryptorchidism, pectus carinatum, severe ophthalmologic abnormalities and behavioral disorders including ASD, and a frank happy demeanor. Hypersociability, which was a noticeable clinical feature in our case, despite ASD, is an uncommon behavioral feature in syndromic intellectual disabilities. Our report supports hypersociability as a suggestive feature of CHD3-related syndrome along with developmental delay, macrocephaly and a dysmorphic facies.
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http://dx.doi.org/10.1016/j.ejmg.2021.104166DOI Listing
April 2021

Further delineation of the NTHL1 associated syndrome: A report from the French Oncogenetic Consortium.

Clin Genet 2021 05 12;99(5):662-672. Epub 2021 Feb 12.

Department of Genetics, Normandy Center for Genomic and Personalized Medicine, Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Rouen, France.

Biallelic pathogenic variants in the NTHL1 (Nth like DNA glycosylase 1) gene cause a recently identified autosomal recessive hereditary cancer syndrome predisposing to adenomatous polyposis and colorectal cancer. Half of biallelic carriers also display multiple colonic or extra-colonic primary tumors, mainly breast, endometrium, urothelium, and brain tumors. Published data designate NTHL1 as an important contributor to hereditary cancers but also underline the scarcity of available informations. Thanks to the French oncogenetic consortium (Groupe Génétique et Cancer), we collected NTHL1 variants from 7765 patients attending for hereditary colorectal cancer or polyposis (n = 3936) or other hereditary cancers (n = 3829). Here, we describe 10 patients with pathogenic biallelic NTHL1 germline variants, that is, the second largest NTHL1 series. All carriers were from the "colorectal cancer or polyposis" series. All nine biallelic carriers who underwent colonoscopy presented adenomatous polyps. For digestive cancers, average age at diagnosis was 56.2 and we reported colorectal, duodenal, caecal, and pancreatic cancers. Extra-digestive malignancies included sarcoma, basal cell carcinoma, breast cancer, urothelial carcinoma, and melanoma. Although tumor risks remain to be precisely defined, these novel data support NTHL1 inclusion in diagnostic panel testing. Colonic surveillance should be conducted based on MUTYH recommendations while extra-colonic surveillance has to be defined.
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http://dx.doi.org/10.1111/cge.13925DOI Listing
May 2021

Functional characterization of ABCC8 variants of unknown significance based on bioinformatics predictions, splicing assays, and protein analyses: Benefits for the accurate diagnosis of congenital hyperinsulinism.

Hum Mutat 2021 Apr 28;42(4):408-420. Epub 2021 Jan 28.

Inserm U1245, UFR de Médecine et Pharmacie, UNIROUEN, Normandie University, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.

ABCC8 encodes the SUR1 subunit of the β-cell ATP-sensitive potassium channel whose loss of function causes congenital hyperinsulinism (CHI). Molecular diagnosis is critical for optimal management of CHI patients. Unfortunately, assessing the impact of ABCC8 variants on RNA splicing remains very challenging as this gene is poorly expressed in leukocytes. Here, we performed bioinformatics analysis and cell-based minigene assays to assess the impact on splicing of 13 ABCC8 variants identified in 20 CHI patients. Next, channel properties of SUR1 proteins expected to originate from minigene-detected in-frame splicing defects were analyzed after ectopic expression in COSm6 cells. Out of the analyzed variants, seven induced out-of-frame splicing defects and were therefore classified as recessive pathogenic, whereas two led to skipping of in-frame exons. Channel functional analysis of the latter demonstrated their pathogenicity. Interestingly, the common rs757110 SNP increased exon skipping in our system suggesting that it may act as a disease modifier factor. Our strategy allowed determining the pathogenicity of all selected ABCC8 variants, and CHI-inheritance pattern for 16 out of the 20 patients. This study highlights the value of combining RNA and protein functional approaches in variant interpretation and reveals the minigene splicing assay as a new tool for CHI molecular diagnostics.
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http://dx.doi.org/10.1002/humu.24164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049974PMC
April 2021

Gene- and pathway-level analyses of iCOGS variants highlight novel signaling pathways underlying familial breast cancer susceptibility.

Int J Cancer 2021 04 9;148(8):1895-1909. Epub 2021 Jan 9.

Département d'Anticipation et de Suivi des Cancers, Oncogénétique Clinique, Institut Paoli-Calmettes, Marseille, France.

Single-nucleotide polymorphisms (SNPs) in over 180 loci have been associated with breast cancer (BC) through genome-wide association studies involving mostly unselected population-based case-control series. Some of them modify BC risk of women carrying a BRCA1 or BRCA2 (BRCA1/2) mutation and may also explain BC risk variability in BC-prone families with no BRCA1/2 mutation. Here, we assessed the contribution of SNPs of the iCOGS array in GENESIS consisting of BC cases with no BRCA1/2 mutation and a sister with BC, and population controls. Genotyping data were available for 1281 index cases, 731 sisters with BC, 457 unaffected sisters and 1272 controls. In addition to the standard SNP-level analysis using index cases and controls, we performed pedigree-based association tests to capture transmission information in the sibships. We also performed gene- and pathway-level analyses to maximize the power to detect associations with lower-frequency SNPs or those with modest effect sizes. While SNP-level analyses identified 18 loci, gene-level analyses identified 112 genes. Furthermore, 31 Kyoto Encyclopedia of Genes and Genomes and 7 Atlas of Cancer Signaling Network pathways were highlighted (false discovery rate of 5%). Using results from the "index case-control" analysis, we built pathway-derived polygenic risk scores (PRS) and assessed their performance in the population-based CECILE study and in a data set composed of GENESIS-affected sisters and CECILE controls. Although these PRS had poor predictive value in the general population, they performed better than a PRS built using our SNP-level findings, and we found that the joint effect of family history and PRS needs to be considered in risk prediction models.
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http://dx.doi.org/10.1002/ijc.33457DOI Listing
April 2021

Assessment of Multiplex Digital Droplet RT-PCR as a Diagnostic Tool for SARS-CoV-2 Detection in Nasopharyngeal Swabs and Saliva Samples.

Clin Chem 2021 04;67(5):736-741

Department of Genetics, Rouen University Hospital and Inserm U1245, UNIROUEN, Normandie University, Normandy Center for Genomic and Personalized Medicine, Rouen, France.

Background: Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity.

Methods: We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting 6 SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection.

Results: For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs.

Conclusion: Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.
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http://dx.doi.org/10.1093/clinchem/hvaa323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799276PMC
April 2021

Germline Testing in Breast Cancers: Why, When and How?

Cancers (Basel) 2020 Dec 14;12(12). Epub 2020 Dec 14.

Department of Genetics, Rouen University Hospital, Normandy Centre for Genomic and Personalized Medicine, 76000 Rouen, France.

Germline variants represent a main genetic cause of breast cancers before 31 years of age. Development of cancer multi-gene panels has resulted in an exponential increase of germline testing in breast cancer patients. Interpretation of variants, which are mostly missense, is complex and requires excluding clonal haematopoiesis and circulating tumour DNA. In breast cancer patients harbouring germline disease-causing variants, radiotherapy contributing to the development of subsequent tumours should be, if possible, avoided and, within families, annual follow-up including whole-body MRI should be offered to carriers. We consider that, in breast cancer patients, germline testing should be performed before treatment and offered systematically only to patients with: (i) invasive breast carcinoma or in situ (DCIS) before 31; or (ii) bilateral or multifocal or HER2+ invasive breast carcinoma/DCIS or phyllode tumour before 36; or (iii) invasive breast carcinoma before 46 and another core tumour (breast cancer, soft-tissue sarcoma, osteosarcoma, central nervous system tumour, adrenocortical carcinoma); or (iv) invasive breast carcinoma before 46 and one first- or second-degree relative with a core tumour before 56. In contrast, women presenting with breast cancer after 46, without suggestive personal or familial history, should not be tested for
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http://dx.doi.org/10.3390/cancers12123762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764913PMC
December 2020

Blood functional assay for rapid clinical interpretation of germline variants.

J Med Genet 2021 12 13;58(12):796-805. Epub 2020 Oct 13.

Normandie University, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics, F76000, Normandy Centre for Genomic and Personalized Medicine, University of Rouen Faculty of Medicine and Pharmacy, Rouen, France.

Background: The interpretation of germline 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 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 analysis.

Results: In 51 wild-type 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_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 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 variants and detection of non-coding functional variants.
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http://dx.doi.org/10.1136/jmedgenet-2020-107059DOI Listing
December 2021

In utero alcohol exposure exacerbates endothelial protease activity from pial microvessels and impairs GABA interneuron positioning.

Neurobiol Dis 2020 11 3;145:105074. Epub 2020 Sep 3.

Normandie Univ, UNIROUEN, INSERM U1245 and Rouen University Hospital, Department of Neonatal Paediatrics and Intensive Care, F 76000, Normandy Centre for Genomic and Personalized Medicine, Rouen, France. Electronic address:

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/VeCad 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.
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http://dx.doi.org/10.1016/j.nbd.2020.105074DOI Listing
November 2020

Reply to Kratz et al.

Eur J Hum Genet 2020 11 13;28(11):1483-1485. Epub 2020 Aug 13.

Division of Evolution and Genomic Sciences, Manchester Centre for Genomic Medicine, University of Manchester, MAHSC, St Mary's Hospital, Manchester University Hospitals NHS Foundation Trust, Manchester, UK.

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http://dx.doi.org/10.1038/s41431-020-00710-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576774PMC
November 2020

Large-scale comparative evaluation of user-friendly tools for predicting variant-induced alterations of splicing regulatory elements.

Hum Mutat 2020 Oct 16;41(10):1811-1829. Epub 2020 Aug 16.

Inserm U1245, UNIROUEN, Normandie University, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.

Discriminating which nucleotide variants cause disease or contribute to phenotypic traits remains a major challenge in human genetics. In theory, any intragenic variant can potentially affect RNA splicing by altering splicing regulatory elements (SREs). However, these alterations are often ignored mainly because pioneer SRE predictors have proved inefficient. Here, we report the first large-scale comparative evaluation of four user-friendly SRE-dedicated algorithms (QUEPASA, HEXplorer, SPANR, and HAL) tested both as standalone tools and in multiple combined ways based on two independent benchmark datasets adding up to >1,300 exonic variants studied at the messenger RNA level and mapping to 89 different disease-causing genes. These methods display good predictive power, based on decision thresholds derived from the receiver operating characteristics curve analyses, with QUEPASA and HAL having the best accuracies either as standalone or in combination. Still, overall there was a tight race between the four predictors, suggesting that all methods may be of use. Additionally, QUEPASA and HEXplorer may be beneficial as well for predicting variant-induced creation of pseudoexons deep within introns. Our study highlights the potential of SRE predictors as filtering tools for identifying disease-causing candidates among the plethora of variants detected by high-throughput DNA sequencing and provides guidance for their use in genomic medicine settings.
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http://dx.doi.org/10.1002/humu.24091DOI Listing
October 2020

Clinical and molecular description of 19 patients with GATAD2B-Associated Neurodevelopmental Disorder (GAND).

Eur J Med Genet 2020 Oct 17;63(10):104004. Epub 2020 Jul 17.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

De novo pathogenic variants in the GATAD2B gene have been associated with a syndromic neurodevelopmental disorder (GAND) characterized by severe intellectual disability (ID), impaired speech, childhood hypotonia, and dysmorphic features. Since its first description in 2013, nine patients have been reported in case reports and a series of 50 patients was recently published, which is consistent with the relative frequency of GATAD2B pathogenic variants in public databases. We report the detailed phenotype of 19 patients from various ethnic backgrounds with confirmed pathogenic GATAD2B variants including intragenic deletions. All individuals presented developmental delay with a median age of 2.5 years for independent walking and of 3 years for first spoken words. GATAD2B variant carriers showed very little subsequent speech progress, two patients over 30 years of age remaining non-verbal. ID was mostly moderate to severe, with one profound and one mild case, which shows a wider spectrum of disease severity than previously reported. We confirm macrocephaly as a major feature in GAND (53%). Most common dysmorphic features included broad forehead, deeply set eyes, hypertelorism, wide nasal base, and pointed chin. Conversely, prenatal abnormalities, non-cerebral malformations, epilepsy, and autistic behavior were uncommon. Other features included feeding difficulties, behavioral abnormalities, and unspecific abnormalities on brain MRI. Improving our knowledge of the clinical phenotype is essential for correct interpretation of the molecular results and accurate patient management.
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http://dx.doi.org/10.1016/j.ejmg.2020.104004DOI Listing
October 2020

Rhabdomyosarcoma associated with germline TP53 alteration in children and adolescents: The French experience.

Pediatr Blood Cancer 2020 09 13;67(9):e28486. Epub 2020 Jul 13.

Gustave Roussy Cancer Center, Department of Children and Adolescents Oncology, Paris-Saclay University, Villejuif, France.

Objective: To describe the clinical characteristics and outcome of patients with Li-Fraumeni-associated rhabdomyosarcoma (RMS).

Method: Retrospective analysis of data from 31 French patients with RMS diagnosed before the age of 20 years associated with a TP53 pathogenic germline variant. Cases were identified through the French Li-Fraumeni database. Central histologic review was performed in 16 cases.

Results: The median age at diagnosis was 2.3 years, and the median follow-up was 9.1 years (0.3-34.8). The main tumor sites were head and neck (n = 13), extremities (n = 8), and trunk (n = 8). The local pathology report classified the 31 tumors in embryonal (n = 26), alveolar (n = 1), pleomorphic (n = 1), and spindle-cell (n = 1) RMS (missing = 2). After histological review, anaplasia (diffuse or focal) was reported in 12/16 patients. Twenty-five patients had localized disease, three had lymph node involvement, and three distant metastases. First-line therapy combined surgery (n = 27), chemotherapy (n = 30), and radiotherapy (n = 14) and led to RMS control in all, but one patient. Eleven patients relapsed, and 18 patients had second malignancies. The 10-year event-free, progression-free, and overall survival rates were 36% (95% CI: 20-56), 62% (95% CI: 43-77) and 76% (95% CI: 56-88), respectively. The 10-year cumulative risk of second malignancies was 40% (95% CI: 22-60).

Conclusion: The high incidence of multiple primary tumors strongly influences the long-term prognosis of RMS associated with TP53 pathogenic germline variants. Anaplastic RMS in childhood, independently of the familial history, should lead to TP53 analysis at treatment initiation to reduce, whenever possible, the burden of genotoxic drugs and radiotherapy in carriers and to ensure the early detection of second malignancies.
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http://dx.doi.org/10.1002/pbc.28486DOI Listing
September 2020

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using Exon 3 as a Model System.

Cancer Res 2020 09 8;80(17):3593-3605. Epub 2020 Jul 8.

Department of Genetics, Institut Curie, Paris, France.

is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored exon 3 (e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for haploinsufficiency. predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL transcripts. Of 100 e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of leaky splicing variants, some of which may not increase cancer risk.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484206PMC
September 2020

Detection of copy-number variations from NGS data using read depth information: a diagnostic performance evaluation.

Eur J Hum Genet 2021 01 26;29(1):99-109. Epub 2020 Jun 26.

Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics, Normandy Center for Genomic and Personalized Medicine, Rouen, France.

The detection of copy-number variations (CNVs) from NGS data is underexploited as chip-based or targeted techniques are still commonly used. We assessed the performances of a workflow centered on CANOES, a bioinformatics tool based on read depth information. We applied our workflow to gene panel (GP) and whole-exome sequencing (WES) data, and compared CNV calls to quantitative multiplex PCR of short fluorescent fragments (QMSPF) or array comparative genomic hybridization (aCGH) results. From GP data of 3776 samples, we reached an overall positive predictive value (PPV) of 87.8%. This dataset included a complete comprehensive QMPSF comparison of four genes (60 exons) on which we obtained 100% sensitivity and specificity. From WES data, we first compared 137 samples with aCGH and filtered comparable events (exonic CNVs encompassing enough aCGH probes) and obtained an 87.25% sensitivity. The overall PPV was 86.4% following the targeted confirmation of candidate CNVs from 1056 additional WES. In addition, our CANOES-centered workflow on WES data allowed the detection of CNVs with a resolution of single exons, allowing the detection of CNVs that were missed by aCGH. Overall, switching to an NGS-only approach should be cost-effective as it allows a reduction in overall costs together with likely stable diagnostic yields. Our bioinformatics pipeline is available at: https://gitlab.bioinfo-diag.fr/nc4gpm/canoes-centered-workflow .
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http://dx.doi.org/10.1038/s41431-020-0672-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852510PMC
January 2021

Haploinsufficiency of the Primary Familial Brain Calcification Gene SLC20A2 Mediated by Disruption of a Regulatory Element.

Mov Disord 2020 08 7;35(8):1336-1345. Epub 2020 Jun 7.

Department of Genetics and CNR-MAJ, Normandy Center for Genomic and Personalized Medicine, Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Rouen, France.

Objective: Primary familial brain calcification (PFBC) is a rare cerebral microvascular calcifying disorder with diverse neuropsychiatric expression. Five genes were reported as PFBC causative when carrying pathogenic variants. Haploinsufficiency of SLC20A2, which encodes an inorganic phosphate importer, is a major cause of autosomal-dominant PFBC. However, PFBC remains genetically unexplained in a proportion of patients, suggesting the existence of additional genes or cryptic mutations. We analyzed exome sequencing data of 71 unrelated, genetically unexplained PFBC patients with the aim to detect copy number variations that may disrupt the expression of core PFBC-causing genes.

Methods: After the identification of a deletion upstream of SLC20A2, we assessed its consequences on gene function by reverse transcriptase droplet digital polymerase chain reaction (RT-ddPCR), an ex vivo inorganic phosphate uptake assay, and introduced the deletion of a putative SLC20A2 enhancer mapping to this region in human embryonic kidney 293 (HEK293) cells by clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 9 (Cas9).

Results: The 8p11.21 deletion, segregating with PFBC in a family, mapped 35 kb upstream of SLC20A2. The deletion carriers/normal controls ratio of relative SLC20A2 mRNA levels was 60.2% (P < 0.001). This was comparable with that of patients carrying an SLC20A2 premature stop codon (63.4%; P < 0.001). The proband exhibited a 39.3% decrease of inorganic phosphate uptake in blood (P = 0.015). In HEK293 cells, we observed a 39.8% decrease in relative SLC20A2 mRNA levels after normalization on DNA copy number (P < 0.001).

Discussion: We identified a deletion of an enhancer of SLC20A2 expression, with carriers showing haploinsufficiency in similar ranges to loss-of-function alleles, and we observed reduced mRNA levels after deleting this element in a cellular model. We propose a 3-step strategy to identify and easily assess the effect of such events. © 2020 International Parkinson and Movement Disorder Society.
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http://dx.doi.org/10.1002/mds.28090DOI Listing
August 2020

Guidelines for the Li-Fraumeni and heritable TP53-related cancer syndromes.

Eur J Hum Genet 2020 10 26;28(10):1379-1386. Epub 2020 May 26.

Manchester Centre for Genomic Medicine, Division of Evolution and Genomic Sciences, University of Manchester, MAHSC, St Mary's Hospital, Manchester University Hospitals NHS Foundation Trust, Manchester, UK.

Fifty years after the recognition of the Li-Fraumeni syndrome (LFS), our perception of cancers related to germline alterations of TP53 has drastically changed: (i) germline TP53 alterations are often identified among children with cancers, in particular soft-tissue sarcomas, adrenocortical carcinomas, central nervous system tumours, or among adult females with early breast cancers, without familial history. This justifies the expansion of the LFS concept to a wider cancer predisposition syndrome designated heritable TP53-related cancer (hTP53rc) syndrome; (ii) the interpretation of germline TP53 variants remains challenging and should integrate epidemiological, phenotypical, bioinformatics prediction, and functional data; (iii) the penetrance of germline disease-causing TP53 variants is variable, depending both on the type of variant (dominant-negative variants being associated with a higher cancer risk) and on modifying factors; (iv) whole-body MRI (WBMRI) allows early detection of tumours in variant carriers and (v) in cancer patients with germline disease-causing TP53 variants, radiotherapy, and conventional genotoxic chemotherapy contribute to the development of subsequent primary tumours. It is critical to perform TP53 testing before the initiation of treatment in order to avoid in carriers, if possible, radiotherapy and genotoxic chemotherapies. In children, the recommendations are to perform clinical examination and abdominal ultrasound every 6 months, annual WBMRI and brain MRI from the first year of life, if the TP53 variant is known to be associated with childhood cancers. In adults, the surveillance should include every year clinical examination, WBMRI, breast MRI in females from 20 until 65 years and brain MRI until 50 years.
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http://dx.doi.org/10.1038/s41431-020-0638-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609280PMC
October 2020

High Prevalence of Somatic Oncogenic Driver Alterations in Patients With NSCLC and Li-Fraumeni Syndrome.

J Thorac Oncol 2020 07 14;15(7):1232-1239. Epub 2020 Mar 14.

Clinical Genetic Unit, Cancer Medicine Department, Gustave Roussy, Villejuif, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jtho.2020.03.005DOI Listing
July 2020

A Connected Network of Interacting Proteins Is Involved in Human-Tau Toxicity in .

Front Neurosci 2020 11;14:68. Epub 2020 Feb 11.

UNIROUEN, Inserm U1245, CNR-MAJ, F 76000, Department of Genetics, Normandy Center for Genomic and Personalized Medicine, Rouen University Hospital, Normandie Université, Rouen, France.

Tauopathies are neurodegenerative diseases characterized by the presence of aggregates of abnormally phosphorylated Tau. Deciphering the pathophysiological mechanisms that lead from the alteration of Tau biology to neuronal death depends on the identification of Tau cellular partners. Combining genetic and transcriptomic analyses in , we identified 77 new modulators of human Tau-induced toxicity, bringing to 301 the number of Tau genetic interactors identified so far in flies. Network analysis showed that 229 of these genetic modulators constitute a connected network. The addition of 77 new genes strengthened the network structure, increased the intergenic connectivity and brought up key hubs with high connectivities, namely , and Interestingly, we established for the first time a genetic link between Tau-induced toxicity and , a recognized Alzheimer Disease protective factor. In addition, our data support the importance of the presynaptic compartment in mediating Tau toxicity.
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http://dx.doi.org/10.3389/fnins.2020.00068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026268PMC
February 2020

Rare genetic susceptibility variants assessment in autism spectrum disorder: detection rate and practical use.

Transl Psychiatry 2020 02 24;10(1):77. Epub 2020 Feb 24.

Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics and Reference Center for Developmental Disorders, F 76000, Normandy Center for Genomic and Personalized Medicine, Rouen, France.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a strong genetic component whose knowledge evolves quickly. Next-generation sequencing is the only effective technology to deal with the high genetic heterogeneity of ASD in a clinical setting. However, rigorous criteria to classify rare genetic variants conferring ASD susceptibility are currently lacking. We have performed whole-exome sequencing to identify both nucleotide variants and copy number variants (CNVs) in 253 ASD patients, including 68 patients with intellectual disability (ID) and 90 diagnosed as Asperger syndrome. Using explicit criteria to classify both susceptibility genes and susceptibility variants we prioritized 217 genes belonging to the following categories: syndromic genes, genes with an excess of de novo protein truncating variants and genes targeted by rare CNVs. We obtained a susceptibility variant detection rate of 19.7% (95% CI: [15-25.2%]). The rate for CNVs was 7.1% (95% CI: [4.3-11%]) and 12.6% (95% CI: [8.8-17.4%]) for nucleotide variants. The highest rate (30.1%, 95% CI: [20.2-43.2%]) was obtained in the ASD + ID subgroup. A strong contributor for at risk nucleotide variants was the recently identified set of genes (n = 81) harboring an excess of de novo protein truncating variants. Since there is currently no evidence that the genes targeted here are necessary and sufficient to cause ASD, we recommend to avoid the term "causative of ASD" when delivering the information about a variant to a family and to use instead the term "genetic susceptibility factor contributing to ASD".
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http://dx.doi.org/10.1038/s41398-020-0760-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039996PMC
February 2020

Moderate Overexpression of Tau in Drosophila Exacerbates Amyloid-β-Induced Neuronal Phenotypes and Correlates with Tau Oligomerization.

J Alzheimers Dis 2020 ;74(2):637-647

Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Department of Genetics and CNR-MAJ, Normandy Center for Genomic and Personalized Medicine, Rouen, France.

Alzheimer's disease (AD) is neuropathologically defined by two key hallmarks: extracellular senile plaques composed primarily of amyloid-β (Aβ) peptide and intraneuronal neurofibrillary tangles, containing abnormally hyperphosphorylated tau protein. The tau protein is encoded by the MAPT gene. Recently, the H1 and H2 haplotypes of the MAPT gene were associated with AD risk. The minor MAPT H2 haplotype has been linked with a decreased risk of developing late-onset AD (LOAD). MAPT haplotypes show different levels of MAPT/Tau expression with H1 being ∼1.5-fold more expressed than H2, suggesting that MAPT expression level could be related to LOAD risk. In this study, we investigated whether this moderate difference in MAPT/Tau expression could influence Aβ-induced toxicity in vivo. We show that modest overexpression of tau protein in Drosophila exacerbates neuronal phenotypes in AβPP/BACE1 flies. The exacerbation of neuronal defects correlates with the accumulation of insoluble dTau oligomers, suggesting that the moderate difference in level of tau expression observed between H1 and H2 haplotypes could influence Aβ toxicity through the production of oligomeric tau insoluble species.
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http://dx.doi.org/10.3233/JAD-190906DOI Listing
August 2021

Exome sequencing identifies the first genetic determinants of sirenomelia in humans.

Hum Mutat 2020 05 1;41(5):926-933. Epub 2020 Mar 1.

Department of Genetics and Reference Center for Developmental Disorders, Normandy Center for Genomic and Personalized Medicine, Normandie Univ, UNIROUEN, Inserm U1245 and Rouen University Hospital, Rouen, France.

Sirenomelia is a rare severe malformation sequence of unknown cause characterized by fused legs and severe visceral abnormalities. We present a series of nine families including two rare familial aggregations of sirenomelia investigated by a trio-based exome sequencing strategy. This approach identified CDX2 variants in the two familial aggregations, both fitting an autosomal dominant pattern of inheritance with variable expressivity. CDX2 is a major regulator of caudal development in vertebrate and mouse heterozygotes are a previously described model of sirenomelia. Remarkably, the p.(Arg237His) variant has already been reported in a patient with persistent cloaca. Analysis of the sporadic cases revealed six additional candidate variants including a de novo frameshift variant in the genetically constrained NKD1 gene, encoding a known interactor of CDX2. We provide the first insights for a genetic contribution in human sirenomelia and highlight the role of Cdx and Wnt signaling pathways in the development of this disorder.
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http://dx.doi.org/10.1002/humu.23998DOI Listing
May 2020

Skipping Nonsense to Maintain Function: The Paradigm of Exon 12.

Cancer Res 2020 04 11;80(7):1374-1386. Epub 2020 Feb 11.

Normandie Univ, UNIROUEN, Inserm U1245, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.

Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing ( = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell-based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications. SIGNIFICANCE: This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-2491DOI Listing
April 2020

Characterisation of heterozygous variants in French patients with Lynch syndrome.

J Med Genet 2020 07 28;57(7):487-499. Epub 2020 Jan 28.

Department of Genetics, Rouen University Hospital and UNIROUEN, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.

Background: Heterozygous germline 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 gene in LS.

Methods: We report 200 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 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 gene in LS. While 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.
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http://dx.doi.org/10.1136/jmedgenet-2019-106256DOI Listing
July 2020
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