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Abstract In a patient diagnosed with both Kallmann syndrome (KS) and intellectual disability (ID), who carried an apparently balanced translocation t(7;12)(q22;q24)dn, array comparative genomic hybridization (aCGH) disclosed a cryptic heterozygous 4.7 Mb deletion del(12)(p11.21p11.23), unrelated to the translocation breakpoint. This novel discovery prompted us to consider the possibility that the combination of KS and neurological disorder in this patient could be attributed to gene(s) within this specific deletion at 12p11.21-12p11.23, rather than disrupted or dysregulated genes at the translocation breakpoints. To further support this hypothesis, we expanded our study by screening five candidate genes at both breakpoints of the chromosomal translocation in a cohort of 48 KS patients. However, no mutations were found, thus reinforcing our supposition. In order to delve deeper into the characterization of the 12p11.21-12p11.23 region, we enlisted six additional patients with small copy number variations (CNVs) and analyzed eight individuals carrying small CNVs in this region from the DECIPHER database. Our investigation utilized a combination of complementary approaches. Firstly, we conducted a comprehensive phenotypic-genotypic comparison of reported CNV cases. Additionally, we reviewed knockout animal models that exhibit phenotypic similarities to human conditions. Moreover, we analyzed reported variants in candidate genes and explored their association with corresponding phenotypes. Lastly, we examined the interacting genes associated with these phenotypes to gain further insights. As a result, we identified a dozen candidate genes: TSPAN11 as a potential KS candidate gene, TM7SF3, STK38L, ARNTL2, ERGIC2, TMTC1, DENND5B, and ETFBKMT as candidate genes for the neurodevelopmental disorder, and INTS13, REP15, PPFIBP1, and FAR2 as candidate genes for KS with ID. Notably, the high-level expression pattern of these genes in relevant human tissues further supported their candidacy. Based on our findings, we propose that dosage alterations of these candidate genes may contribute to sexual and/or cognitive impairments observed in patients with KS and/or ID. However, the confirmation of their causal roles necessitates further identification of point mutations in these candidate genes through next-generation sequencing.

Chondrosarcomas and osteosarcomas are malignant bone tumors with a poor prognosis when unresectable or metastasized. Moreover, radiotherapy and chemotherapy could be ineffective. MiRNAs represent an alternative therapeutic approach. Based on high-throughput functional screening, we identified four miRNAs with a potential antiproliferative effect on SW1353 chondrosarcoma cells. Individual functional validations were then performed in SW1353 cells, as well as in three osteosarcoma cell lines. The antiproliferative and cytotoxic effects of miRNAs were evaluated in comparison with a positive control, miR-342-5p. The cytotoxic effect of four selected miRNAs was not confirmed on SW1353 cells, but we unambiguously revealed that miR-4270 had a potent cytotoxic effect on HOS and MG-63 osteosarcoma cell lines, but not on SaOS-2 cell line. Furthermore, like miR-342-5p, miR-4270 induced apoptosis in these two cell lines. In addition, we provided the first report of Bcl-xL as a direct target of miR-4270. MiR-4270 also decreased the expression of the anti-apoptotic protein Mcl-1, and increased the expression of the pro-apoptotic protein Bak. Our findings demonstrated that miR-4270 has tumor suppressive activity in osteosarcoma cells, particularly through Bcl-xL downregulation.

Introduction: Prenatal ultrasound (US) anomalies are detected in around 5%–10% of pregnancies. In prenatal diagnosis, exome sequencing (ES) diagnostic yield ranges from 6% to 80% depending on the inclusion criteria. We describe the first French national multicenter pilot study aiming to implement ES in prenatal diagnosis following the detection of anomalies on US.Patients and methods: We prospectively performed prenatal trio-ES in 150 fetuses with at least two US anomalies or one US anomaly known to be frequently linked to a genetic disorder. Trio-ES was only performed if the results could influence pregnancy management. Chromosomal microarray (CMA) was performed before or in parallel.Results: A causal diagnosis was identified in 52/150 fetuses (34%) with a median time to diagnosis of 28 days, which rose to 56/150 fetuses (37%) after additional investigation. Sporadic occurrences were identified in 34/56 (60%) fetuses and unfavorable vital and/or neurodevelopmental prognosis was made in 13/56 (24%) fetuses. The overall diagnostic yield was 41% (37/89) with first-line trio-ES versus 31% (19/61) after normal CMA. Trio-ES and CMA were systematically concordant for identification of pathogenic CNV.Conclusion: Trio-ES provided a substantial prenatal diagnostic yield, similar to postnatal diagnosis with a median turnaround of approximately 1 month, supporting its routine implementation during the detection of prenatal US anomalies.

Summary: Background: Primary Ovarian Insufficiency (POI), a public health problem, affects 1-3.7% of women under 40 yielding 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 chromosomal 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 pathogenesis 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: Université Paris Saclay, Agence Nationale de Biomédecine.

Introduction: Pseudohypoparathyroidism type 1A (PHP1A) and pseudopseudohypoparathyroidism (PPHP) (Inactivating PTH/PTHrP Signaling Disorders type 2, IPPSD2) are two rare autosomal disorders caused by loss-of-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.

Abstract Mesenchymal stem cells (MSCs) hold promise for cartilage engineering. Here, we aimed to determine the best culture conditions to induce chondrogenesis of MSCs isolated from bone marrow (BM) of aged osteoarthritis (OA) patients. We showed that these BM-MSCs proliferate slowly, are not uniformly positive for stem cell markers, and maintain their multilineage potential throughout multiple passages. The chondrogenic lineage of BM-MSCs was induced in collagen scaffolds, under normoxia or hypoxia, by BMP-2 and/or TGF-β1. The best chondrogenic induction, with the least hypertrophic induction, was obtained with the combination of BMP-2 and TGF-β1 under hypoxia. Differentiated BM-MSCs were then transfected with siRNAs targeting two markers overexpressed in OA chondrocytes, type I collagen and/or HtrA1 protease. siRNAs significantly decreased mRNA and protein levels of type I collagen and HtrA1, resulting in a more typical chondrocyte phenotype, but with frequent calcification of the subcutaneously implanted constructs in a nude mouse model. Our 3D culture model with BMP-2/TGF-β1 and COL1A1/HtrA1 siRNAs was not effective in producing a cartilage-like matrix in vivo. Further optimization is needed to stabilize the chondrocyte phenotype of differentiated BM-MSCs. Nevertheless, this study offers the opportunity to develop a combinatory cellular therapy strategy for cartilage tissue engineering.

Cartilage is a non-innervated and non-vascularized tissue. It is composed of one main cell type, the chondrocyte, which governs homeostasis within the cartilage tissue, but has low metabolic activity. Articular cartilage undergoes substantial stresses that lead to chondral defects, and inevitably osteoarthritis (OA) due to the low intrinsic repair capacity of cartilage. OA remains an incurable degenerative disease. In this context, several dietary supplements have shown promising results, notably in the relief of OA symptoms. In this study, we investigated the effects of collagen hydrolysates derived from fish skin (Promerim®30 and Promerim®60) and fish cartilage (Promerim®40) on the phenotype and metabolism of human articular chondrocytes (HACs). First, we demonstrated the safety of Promerim® hydrolysates on HACs cultured in monolayers. Then we showed that, Promerim® hydrolysates can increase the HAC viability and proliferation, while decreasing HAC SA-β-galactosidase activity. To evaluate the effect of Promerim® on a more relevant model of culture, HAC were cultured as organoids in the presence of Promerim® hydrolysates with or without IL-1β to mimic an OA environment. In such conditions, Promerim® hydrolysates led to a decrease in the transcript levels of some proteases that play a major role in the development of OA, such as Htra1 and metalloproteinase-1. Promerim® hydrolysates downregulated HtrA1 protein expression. In contrast, the treatment of cartilage organoids with Promerim® hydrolysates increased the neosynthesis of type I collagen (Promerim®30, 40 and 60) and type II collagen isoforms (Promerim®30 and 40), the latter being the major characteristic component of the cartilage extracellular matrix. Altogether, our results demonstrate that the use of Promerim® hydrolysates hold promise as complementary dietary supplements in combination with the current classical treatments or as a preventive therapy to delay the occurrence of OA in humans.

Articular cartilage experiences mechanical constraints leading to chondral defects that inevitably evolve into osteoarthritis (OA), because cartilage has poor intrinsic repair capacity. Although OA is an incurable degenerative disease, several dietary supplements may help improve OA outcomes. In this study, we investigated the effects of Dielen® hydrolyzed fish collagens from skin (Promerim®30 and Promerim®60) and cartilage (Promerim®40) to analyze the phenotype and metabolism of equine articular chondrocytes (eACs) cultured as organoids. Here, our findings demonstrated the absence of cytotoxicity and the beneficial effect of Promerim® hydrolysates on eAC metabolic activity under physioxia; further, Promerim®30 also delayed eAC senescence. To assess the effect of Promerim® in a cartilage-like tissue, eACs were cultured as organoids under hypoxia with or without BMP-2 and/or IL-1β. In some instances, alone or in the presence of IL-1β, Promerim®30 and Promerim®40 increased protein synthesis of collagen types I and II, while decreasing transcript levels of proteases involved in OA pathogenesis, namely Htra1, and the metalloproteinases Mmp1-3, Adamts5, and Cox2. Both Promerim® hydrolysates also decreased Htra1 protein amounts, particularly in inflammatory conditions. The effect of Promerim® was enhanced under inflammatory conditions, possibly due to a decrease in the synthesis of inflammation-associated molecules. Finally, Promerim® favored in vitro repair in a scratch wound assay through an increase in cell proliferation or migration. Altogether, these data show that Promerim®30 and 40 hold promise as dietary supplements to relieve OA symptoms in patients and to delay OA progression.