MiR-4270 acts as a tumor suppressor by directly targeting Bcl-xL in human osteosarcoma cells.

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.

A cryptic microdeletion del(12)(p11.21p11.23) within an unbalanced translocation t(7;12)(q21.13;q23.1) implicates new candidate loci for intellectual disability and Kallmann syndrome.

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.

OTX2 duplications: a recurrent cause of oculo-auriculo-vertebral spectrum.

BACKGROUND: Oculo-auriculo-vertebral spectrum (OAVS) is the second most common cause of head and neck malformations in children after orofacial clefts. OAVS is clinically heterogeneous and characterised by a broad range of clinical features including ear anomalies with or without hearing loss, hemifacial microsomia, orofacial clefts, ocular defects and vertebral abnormalities. Various genetic causes were associated with OAVS and copy number variations represent a recurrent cause of OAVS, but the responsible gene often remains elusive. METHODS: We described an international cohort of 17 patients, including 10 probands and 7 affected relatives, presenting with OAVS and carrying a 14q22.3 microduplication detected using chromosomal microarray analysis. For each patient, clinical data were collected using a detailed questionnaire addressed to the referring clinicians. We subsequently studied the effects of OTX2 overexpression in a zebrafish model. RESULTS: We defined a 272 kb minimal common region that only overlaps with the OTX2 gene. Head and face defects with a predominance of ear malformations were present in 100% of patients. The variability in expressivity was significant, ranging from simple chondromas to severe microtia, even between intrafamilial cases. Heterologous overexpression of OTX2 in zebrafish embryos showed significant effects on early development with alterations in craniofacial development. CONCLUSIONS: Our results indicate that proper OTX2 dosage seems to be critical for the normal development of the first and second branchial arches. Overall, we demonstrated that OTX2 genomic duplications are a recurrent cause of OAVS marked by auricular malformations of variable severity.

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 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.

Enhanced chondrogenesis of bone marrow-derived stem cells by using a combinatory cell therapy strategy with BMP-2/TGF-ß1, hypoxia, and COL1A1/HtrA1 siRNAs.

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-ß1. The best chondrogenic induction, with the least hypertrophic induction, was obtained with the combination of BMP-2 and TGF-ß1 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-ß1 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.

Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering.

Umbilical cord blood (UCB) is a promising alternative source of mesenchymal stem cells (MSCs), because UCB-MSCs are abundant and harvesting them is a painless non-invasive procedure. Potential clinical applications of UCB-MSCs have been identified, but their ability for chondrogenic differentiation has not yet been fully evaluated. The aim of our work was to characterize and determine the chondrogenic differentiation potential of human UCB-MSCs (hUCB-MSCs) for cartilage tissue engineering using an approach combining 3D culture in type I/III collagen sponges and chondrogenic factors. Our results showed that UCB-MSCs have a high proliferative capacity. These cells differentiated easily into an osteoblast lineage but not into an adipocyte lineage. Furthermore, BMP-2 and TGF-ß1 potentiated chondrogenic differentiation, as revealed by a strong increase in mature chondrocyte-specific mRNA (COL2A1, COL2B, ACAN) and protein (type II collagen) markers. Although growth factors increased the transcription of hypertrophic chondrocyte markers such as COL10A1 and MMP13, the cells present in the neo-tissue maintained their phenotype and did not progress to terminal differentiation and mineralization of the extracellular matrix after subcutaneous implantation in nude mice. Our study demonstrates that our culture model has efficient chondrogenic differentiation, and that hUCB-MSCs can be a reliable source for cartilage tissue engineering.

Pregnancy outcomes in prenatally diagnosed 47, XXX and 47, XYY syndromes: a 30-year French, retrospective, multicentre study.

OBJECTIVE: Sex chromosome aneuploidies are frequently detected fortuitously in a prenatal diagnosis. Most cases of 47, XXX and 47, XYY syndromes are diagnosed in this context, and parents are thus faced with an unexpected situation. The objective of the present study was to characterize a French cohort of prenatally diagnosed cases of 47, XXX and 47, XYY and to evaluate the termination of pregnancy (TOP) rate before and after France's implementation of multidisciplinary centres for prenatal diagnosis in 1997. METHODS: This retrospective study identified respectively 291 and 175 cases of prenatally diagnosed 47, XXX and 47, XYY between 1976 and 2012. For each case, the indication, maternal age, karyotype and outcome were recorded. RESULTS: Most diagnoses of the two conditions were fortuitous. The occurrence of 47, XXX was associated with advanced maternal age. The overall TOP rate was higher for 47, XXX (22.9%) than for 47, XYY (14.6%), although this difference was not statistically significant. However, the TOP rates fell significantly after 1997 (from 41.1% to 11.8% for 47, XXX and from 25.8% to 6.7% for 47, XYY). CONCLUSION: The TOP rates after prenatal diagnoses of 47, XXX and 47, XYY fell significantly after 1997, following France's implementation of multidisciplinary centres for prenatal diagnosis. © 2016 John Wiley & Sons, Ltd.

12q21 Microdeletion in a fetus with Meckel syndrome involving CEP290/MKS4.

We report on a fetus with Meckel syndrome diagnosed during the 21st gestational week, hydrocephalus and bilateral hyperechogenic kidneys were then detected on ultrasonography. Fetal pathological examination showed facial dysmorphism, occipital meningoencephalocele, characteristic renal cysts, mild hepatic ductal dysplasia, hydrocephalus in association with extreme cerebellar vermis hypoplasia and brainstem anomalies. Molecular and cytogenetic analysis identified a paternally inherited CEP290/MKS4 (MIM611134) (12q21) nonsense mutation and a maternal 12q21 microdeletion. Two cases with such a mechanism have previously been described in the literature, one of them involves an inherited microdeletion. The observation of such cases highlights the existence of a pathogenic mechanism which involves deletion and point mutation, and illustrates how homozygosity can hide hemizygosity when usual sequencing methods are used. The identification of hemizygosity enables to determine precisely the molecular mechanism and to understand some phenotypic variations. As they act as complete loss of function allele, deletions might give indication on the severity of the associated point mutation. This clinical report highlights the importance of fetal pathology following termination of pregnancies in order to guide molecular analysis and the potential role of cytogenetic cryptic disorders in autosomal recessive disease. The use of polymorphic marker analysis in association with FISH or arrayCGH provided an accurate identification of molecular mechanisms, accurate genetic counseling and optimized strategy for next pregnancies or preimplantation diagnosis.

The 2q37-deletion syndrome: an update of the clinical spectrum including overweight, brachydactyly and behavioural features in 14 new patients.

The 2q37 locus is one of the most commonly deleted subtelomeric regions. Such a deletion has been identified in >100 patients by telomeric fluorescence in situ hybridization (FISH) analysis and, less frequently, by array-based comparative genomic hybridization (array-CGH). A recognizable '2q37-deletion syndrome' or Albright's hereditary osteodystrophy-like syndrome has been previously described. To better map the deletion and further refine this deletional syndrome, we formed a collaboration with the Association of French Language Cytogeneticists to collect 14 new intellectually deficient patients with a distal or interstitial 2q37 deletion characterized by FISH and array-CGH. Patients exhibited facial dysmorphism (13/14) and brachydactyly (10/14), associated with behavioural problems, autism or autism spectrum disorders of varying severity and overweight or obesity. The deletions in these 14 new patients measured from 2.6 to 8.8 Mb. Although the major role of HDAC4 has been demonstrated, the phenotypic involvement of several other genes in the deleted regions is unknown. We further refined the genotype-phenotype correlation for the 2q37 deletion. To do this, we examined the smallest overlapping deleted region for candidate genes for skeletal malformations (facial dysmorphism and brachydactyly), overweight, behavioural problems and seizures, using clinical data, a review of the literature, and the Manteia database. Among the candidate genes identified, we focus on the roles of PRLH, PER2, TWIST2, CAPN10, KIF1A, FARP2, D2HGDH and PDCD1.

Identification by array-Comparative Genomic Hybridization (array-CGH) of a large deletion of luteinizing hormone receptor gene combined with a missense mutation in a patient diagnosed with a 46,XY disorder of sex development and application to prenatal diagnosis.

This paper reports the case of an infant presenting with sexual ambiguity at birth. The child presented with labia majora synechia, thready genital tubercle and perineal hypospadias. The karyotype was 46,XY. Low testosterone levels with no response to hCG administration, associated with high LH level for her age, high FSH level, high inhibin B levels and normal AMH indicated a lack of LH receptivity and prompted us to screen the LHCGR gene for mutations. A previously described missense mutation (p.Cys131Arg) was identified at homozygous state in the propositus and at heterozygous state in the mother. This variation, however, was not found in the father. Our attention was drawn by the presence of several single nucleotide polymorphisms (SNPs), identified at homozygous state without any paternal contribution from exon 1 to exon 10 of LHCGR, suggesting a paternal deletion. Array DNA analysis was performed revealing a large deletion extending from 61,493 to 135,344 bp and including the LHCGR gene. Adequate genetic counselling was provided. This paper describes the first application of prenatal diagnosis in LHCGR deficiency for 46,XY disorders of sex development with the subsequent delivery of a normal baby.