Position effects at the FGF8 locus are associated with femoral hypoplasia

Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The ∼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals’ cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.M.S. and A.S.-S. were supported by the Polish National Science Centre (UMO-2016/23/N/NZ2/02362 to M.S. and UMO-2016/21/D/NZ5/00064 to A.S.-S.). A.S.-S. was also supported by the Polish National Science Centre scholarship for PhD students (UMO-2013/08/T/NZ2/00027). C.L. is supported by postdoctoral Beatriu de Pinós from Secretaria d’Universitats I Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya and by the Marie Sklodowska-Curie COFUND program from H2020 (2018-BP-00055). A.J. was supported by the Polish National Science Centre (UMO-2016/22/E/NZ5/00270) as well as the Polish National Centre for Research and Development (LIDER/008/431/L-4/12/NCBR/2013). M.S. is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (SP1532/3-1, SP1532/4-1, and SP1532/5-1), the Max Planck Foundation, and the Deutsches Zentrum für Luft- und Raumfahrt (DLR 01GM1925)

Genetic landscape of a large cohort of Primary Ovarian Insufficiency : New genes and pathways and implications for personalized medicine

Background Primary Ovarian Insufficiency (POI), a public health problem, affects 1-3.7% of women under 40 yield-ing infertility and a shorter lifespan. Most causes are unknown. Recently, genetic causes were identified, mostly in single families. We studied an unprecedented large cohort of POI to unravel its molecular pathophysiology.Methods 375 patients with 70 families were studied using targeted (88 genes) or whole exome sequencing with pathogenic/likely-pathogenic variant selection. Mitomycin-induced chromosome breakages were studied in patients' lymphocytes if necessary. Findings A high-yield of 29.3% supports a clinical genetic diagnosis of POI. In addition, we found strong evidence of pathogenicity for nine genes not previously related to a Mendelian phenotype or POI: ELAVL2, NLRP11, CENPE, SPATA33, CCDC150, CCDC185, including DNA repair genes: C17orf53(HROB), HELQ, SWI5 yielding high chromo-somal fragility. We confirmed the causal role of BRCA2, FANCM, BNC1, ERCC6, MSH4, BMPR1A, BMPR1B, BMPR2, ESR2, CAV1, SPIDR, RCBTB1 and ATG7 previously reported in isolated patients/families. In 8.5% of cases, POI is the only symptom of a multi-organ genetic disease. New pathways were identified: NF-kB, post-translational regulation, and mitophagy (mitochondrial autophagy), providing future therapeutic targets. Three new genes have been shown to affect the age of natural menopause supporting a genetic link.Interpretation We have developed high-performance genetic diagnostic of POI, dissecting the molecular pathogene-sis of POI and enabling personalized medicine to i) prevent/cure comorbidities for tumour/cancer susceptibility genes that could affect life-expectancy (37.4% of cases), or for genetically-revealed syndromic POI (8.5% of cases), ii) predict residual ovarian reserve (60.5% of cases). Genetic diagnosis could help to identify patients who may benefit from the promising in vitro activation-IVA technique in the near future, greatly improving its success in treating infertility.Funding Universite? Paris Saclay, Agence Nationale de Biome?decine.Copyright (c) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)Peer reviewe

Marqueurs chromosomiques surnuméraires sur caryotype foetal (étude d'une série de 20 cas)

CAEN-BU Médecine pharmacie (141182102) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Implication de la voie de signalisation Sonic Hedgehog (Shh) dans 2 syndromes (la trisomie 18 et le syndrome de Smith-Lemli-Opitz (SLO))

La trisomie 18 (T18) et le syndrome de Smith-Lemli-Opitz (SLO) sont 2 syndromes polymalformatifs pour lesquels un déficit en cholestérol est observé. En prénatal, ils s associent à un taux effondré d œstriol non conjugué maternel (uE3), marqueur du dépistage de la trisomie 21, reflétant le taux de cholestérol fœtal. L ajout de cholestérol à Sonic Hedgehog (Shh) lui assure une activité maximale. Certaines malformations surviennent dans des tissus où Shh joue un rôle primordial. L objectif de cette étude rétrospective portant sur des cas de T18 et de SLO fœtaux était de déterminer si le déficit en cholestérol était à l origine de l altération de la voie Shh et de malformations. Le nombre de celles-ci a été corrélé au taux d uE3 maternel. Nous avons dosé le cholestérol dans le milieu de culture d amniocytes de T18, SLO et de témoins par LC/MSMS, puis testé l expression de gènes de la voie Shh par RT-PCR quantitative : les facteurs GLI, et des gènes cibles de Shh : BMP2, BMP4, TGFb1, COL1A1 et COL1A2. L expression protéique de Shh, BMP2 et COL1 a été testée. Enfin, l expression de ces gènes a été évaluée dans des amniocytes témoins traités par inhibiteur de synthèse du cholestérol. L activité de liaison des GLI a été étudiée par gel retard. On observe une corrélation inverse entre la sévérité phénotypique et le taux d uE3 dans le SLO et la T18. Le taux de cholestérol dosé dans le milieu de culture d amniocytes est corrélé au taux d uE3 et significativement plus bas en cas de T18 et de SLO. On rapporte une diminution d expression des gènes testés et une différence d activité de liaison des GLI en cas de T18. Cette perturbation n est pas significative dans les amniocytes traités.Trisomy 18 (T18) and Smith Lemli Opitz (SLO) syndrome are two polymalformative conditions in which cholesterol defect was noticed. Prenatally, they are associated with a decreased maternal unconjugated estriol (uE3) level, one of the second trimester maternal serum screening test that reflects fetal cholesterol level. Addition of cholesterol allows Shh protein maturation. Many malformations in these 2 syndromes occur in Shh dependent tissus. We thus sought to assess whether cholesterol defect could affect Shh pathway and explain malformations. We selected trisomy 18 and SLO cases in which maternal uE3 level was decreased and reported fetal malformations. We correlated the number of malformations with maternal uE3 level. We carried out cholesterol dosage in culture medium of trisomy 18, SLO and control amniocytes through LC/MSMS. We then analyzed Shh pathway in testing gene expression of several Shh components : GLI transcription factors, and Shh targets : BMP2, BMP4, TGFb1, COL1A1 and COL1A2. Protein expression of Shh, BMP2 and COL1 was tested. Gene expression was also tested in control amniocytes treated with an anti-cholesterol. Finally, GLI binding activity was tested using gel retardation assay. We observed an inverse correlation between phenotypic severity and maternal uE3 level in SLO and trisomy 18. Cholesterol level in culture medium of amniocytes is correlated with maternal uE3 level and significantly lower in T18 and SLO amniocytes. There is an alteration in Shh pathway as expression of several genes is decreased in T18 and SLO amniocytes. We also observed a different GLI binding activity in T18 amniocytes. This alteration is not significant in treated amniocytes.CAEN-BU Médecine pharmacie (141182102) / SudocSudocFranceF

The impact of functional characterization of variants in calcium sensing receptor gene (CASR) on the clinical diagnosis

International audienceThe human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that signals via intracellular calcium mobilization and MAPK pathway to regulate extracellular calcium homeostasis. Loss-or gain-of-function heterozygous CASR variants lead to familial hypocalciuric hypercalcemia type 1 (FHH1) or autosomal-dominant hypocalcemia (ADH), respectively

Two novel deletions in the 5'untranslated region of GNAS gene as cause of pseudohypoparathyroidism type 1A

International audienc

Involvement and alteration of the Sonic Hedgehog pathway is associated with decreased cholesterol level in trisomy 18 and SLO amniocytes.

International audienceBACKGROUND: Trisomy 18 and Smith-Lemli-Opitz syndrome are two polymalformative conditions in which a cholesterol defect has been noted. When they occur prenatally, they are associated with a decreased maternal unconjugated estriol (uE(3)) level. Cholesterol plays an essential role in the Sonic Hedgehog pathway, allowing Shh protein maturation leading to its maximal activity. Many malformations in these two syndromes occur in Shh dependent tissues. We thus sought to assess whether a cholesterol defect could affect the Shh pathway and explain some of the observed malformations. MATERIALS AND METHODS: We selected 14 cases of trisomy 18 and 3 cases of SLO in which the maternal uE(3) level was decreased and reported malformations were observed after fetopathological examination. We correlated the number of malformations with maternal uE(3) level. We then carried out cholesterol concentrations in separate culture media consisting of trisomy 18, SLO and control amniocytes. Finally, we analyzed the Shh pathway by testing the gene expression of several Shh components: GLI transcription factors, BMP2, BMP4, TGFβ1, COL1A1 and COL1A2. RESULTS AND DISCUSSION: There was an inverse correlation between phenotypic severity and maternal uE(3) levels in SLO and trisomy 18. The cholesterol levels in the amniocyte culture media were correlated with maternal uE3 levels and were significantly lower in T18 and SLO amniocytes, reflecting cholesterol defects. There was an alteration in the Shh pathway since expression of several genes was decreased in T18 and SLO amniocytes. However, these cholesterol defects were not solely responsible for the altered Shh pathway and the malformations observed

A novel synonymous variant in exon 1 of GNAS gene results in a cryptic splice site and causes pseudohypoparathyroidism type 1A and pseudo-pseudohypoparathyroidism in a French family

International audiencePseudohypoparathyroidism type 1A (PHP1A) and pseudopseudohypoparathyroidism (PPHP) (Inactivating PTH/PTHrP Signaling Disorders type 2, IPPSD2) are two rare autosomal disorders caused by lossof-function mutations on either maternal or paternal allele, respectively, in the imprinted GNAS gene, which encodes the α subunit of the ubiquitously-expressed stimulatory G protein (Gαs). Case presentation: We investigated a synonymous GNAS variant NM_001077488.2: c.108C>A / p.(Val36=) identified in a family presenting with IPPSD2 phenotype. In silico splicing prediction algorithms were in favor of a deleterious effect of this variant, by creating a new donor splicing site. The GNAS expression studies in blood suggested haploinsufficiency and showed an alternate splice product demonstrating the unmasking of a cryptic site, leading to a 34 base pairs deletion and the creation of a probable unstable RNA. We present the first familial case of IPPSD2 caused by a pathogenic synonymous variant in GNAS gene

Identification par séquençage du génome entier d’une inversion de 140 Kb dans le locus GNAS impliquant NPELP1 et l’exon 1 du gène XLas dans une famille présentant une pseudohypoparathyroïdie de type Ib autosomique dominante (AD-PHP1b)

International audienceLe locus GNAS code pour la sous-unité alpha de la protéine G stimulatrice (Gsα) et des transcrits supplémentaires soumis à l’empreinte. La pseudohypoparathyroïdie de type 1b (PHP1b) est caractérisée par une résistance rénale à la PTH (et parfois une résistance modérée à la TSH), qui survient généralement en l'absence d’autres caractéristiques de l'ostéodystrophie héréditaire d'Albright. La PHP1b est causée par des modifications épigénétiques au niveau d'une ou plusieurs régions différentiellement méthylées (DMR) dans le locus GNAS. La cause génétique la plus fréquente de PHP1b autosomique dominante (AD-PHP1b) est la délétion du gène STX16 sur l’allèle maternel, qui conduit à une perte isolée de la méthylation (LOM) du DMR A/B. D'autres mécanismes moléculaires plus rares sont décrits, en particulier des anomalies du nombre de copies (délétion/duplication) englobant un ou plusieurs DMR (NEPS55, NESPAS, XLαs).Nous avons étudié une famille avec deux membres atteints, le cas index et sa sœur, présentant un phénotype typique de PHP1b. Un séquençage Sanger et une analyse par MS-MLPA du locus GNAS ont été réalisés pour les deux enfants et leurs parents. Un séquençage du génome entier (WGS) a été réalisée pour le cas index: la préparation de la libraire a utilisé le kit NEBNext, le séquençage a été effectué sur une plateforme Illumina, les séquences ont été alignées au génome humain de référence GRCh38 et les données ont été traitées en utilisant le pipeline GATK. L'inversion a été confirmée par une analyse Sanger et par une analyse PCR de l'ADN génomique en utilisant des amorces conçues pour amplifier spécifiquement les deux points de cassure.Le séquençage Sanger ne retrouvait pas de mutation dans le gène GNAS. L'analyse par MS-MLPA n’a pas été mis en évidence d’anomalies du nombre de copies dans le locus GNAS mais a révélé chez le cas index et sa sœur une LOM complète au DMR A/B, une LOM partielle au DMR XLαs (25%) et une méthylation normale sur les autres DMRs AS et NESP. La méthylation était normale pour les parents. Le WGS a révélé une inversion paracentrique de 140 kb avec comme points de cassure en 5 ’: chr20: 58714222 (intron NPEPL1) et en 3’chr20: 58854618 (exon 1 XLαs). Ces points de cassure ont été confirmés par analyse Sanger pour les deux patients. Des amplicons ont été obtenus pour le cas index, sa sœur et sa mère, alors qu'aucun produit de PCR n'a été généré pour l'ADN du père.Nous avons détecté dans une famille avec AD-PHP1b un nouveau réarrangement par WGS: une inversion en 20q13.3 qui inclut la région située entre NPELP1 et XLαs. À notre connaissance, une seule inversion a été décrite dans AD-PHP1b, qui comprenait l'intégralité du DMR A/B et du gène GNAS. Ces résultats font suspecter la présence d’un nouveau centre d’empreinte dans la région inversée. Des études complémentaires sont nécessaires pour vérifier cette hypothèse

Phenotypic and genotypic characterization of 1q21.1 copy number variants: A report of 34 new individuals and literature review

Recurrent 1q21.1 copy number variants (CNVs) have been associated with a wide spectrum of clinical features, ranging from normal phenotype to moderate intellectual disability, with congenital anomalies and dysmorphic features. They are often inherited from unaffected parents and the pathogenicity is difficult to assess. We describe the phenotypic and genotypic data for 34 probands carrying CNVs in the 1q21.1 chromosome region (24 duplications, 8 deletions and 2 triplications). We also reviewed 89 duplications, 114 deletions and 5 triplications described in the literature, at variable 1q21.1 locations. We aimed to identify the most highly associated clinical features to determine the phenotypic expression in affected individuals. Developmental delay or learning disabilities and neuropsychiatric disorders were common in patients with deletions, duplications and triplications of 1q21.1. Mild dysmorphic features common in these CNVs include a prominent forehead, widely spaced eyes and a broad nose. The CNVs were mostly inherited from apparently unaffected parents. Almost half of the CNVs were distal, overlapping with a common minimal region of 1.2 Mb. We delineated the clinical implications of 1q21.1 CNVs and confirmed that these CNVs are likely pathogenic, although subject to incomplete penetrance and variable expressivity. Long‐term follow‐up should be performed to each newly diagnosed case, and prenatal genetic counseling cautiously discussed, as it remains difficult to predict the phenotype in the event of an antenatal diagnosis