3q29 duplications: A cohort of 46 patients and a literature review.

Duplications of the 3q29 cytoband are rare chromosomal copy number variations (CNVs) (overlapping or recurrent ~1.6 Mb 3q29 duplications). They have been associated with highly variable neurodevelopmental disorders (NDDs) with various associated features or reported as a susceptibility factor to the development of learning disabilities and neuropsychiatric disorders. The smallest region of overlap and the phenotype of 3q29 duplications remain uncertain. We here report a French cohort of 31 families with a 3q29 duplication identified by chromosomal microarray analysis (CMA), including 14 recurrent 1.6 Mb duplications, eight overlapping duplications (>1 Mb), and nine small duplications (<1 Mb). Additional genetic findings that may be involved in the phenotype were identified in 11 patients. Focusing on apparently isolated 3q29 duplications, patients present mainly mild NDD as suggested by a high rate of learning disabilities in contrast to a low proportion of patients with intellectual disabilities. Although some are de novo, most of the 3q29 duplications are inherited from a parent with a similar mild phenotype. Besides, the study of small 3q29 duplications does not provide evidence for any critical region. Our data suggest that the overlapping and recurrent 3q29 duplications seem to lead to mild NDD and that a severe or syndromic clinical presentation should warrant further genetic analyses.

Prenatal Diagnosis of Primrose Syndrome.

Primrose syndrome is a very rare congenital malformation. Symptoms of this disorder may appear during childhood, but the diagnosis is identified in adulthood in the majority of cases. The prenatal diagnosis of Primrose syndrome is not developed in the literature. We present herein a case series of 3 cases with characteristic sonographic features. A dysmorphic metopic suture, downslanting palpebral fissures, a wide forehead, and agenesis of corpus callosum are the main signs. A missense mutation in ZBTB20 identified in whole exome sequencing can confirm the prenatal diagnosis of Primrose syndrome.

iPSCs derived from infertile men carrying complex genetic abnormalities can generate primordial germ-like cells.

Despite increasing insight into the genetics of infertility, the developmental disease processes remain unclear due to the lack of adequate experimental models. The advent of induced pluripotent stem cell (iPSC) technology has provided a unique tool for in vitro disease modeling enabling major advances in our understanding of developmental disease processes. We report the full characterization of complex genetic abnormalities in two infertile patients with either azoospermia or XX male syndrome and we identify genes of potential interest implicated in their infertility. Using the erythroblasts of both patients, we generated primed iPSCs and converted them into a naive-like pluripotent state. Naive-iPSCs were then differentiated into primordial germ-like cells (PGC-LCs). The expression of early PGC marker genes SOX17, CD-38, NANOS3, c-KIT, TFAP2C, and D2-40, confirmed progression towards the early germline stage. Our results demonstrate that iPSCs from two infertile patients with significant genetic abnormalities are capable of efficient production of PGCs. Such in vitro model of infertility will certainly help identifying causative factors leading to early germ cells development failure and provide a valuable tool to explore novel therapeutic strategies.

Case Report: BMPR2-Targeted MinION Sequencing as a Tool for Genetic Analysis in Patients With Pulmonary Arterial Hypertension.

Background: Mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2) represent a major genetic cause of pulmonary arterial hypertension (PAH). Identification of BMPR2 mutations is crucial for the genetic diagnosis of PAH. MinION nanopore sequencer is a portable third-generation technology that enables long-read sequencing at a low-cost. This nanopore technology-based device has not been used previously for PAH diagnosis. This study aimed to determine the feasibility of using MinION nanopore sequencing for the genetic analysis of PAH patients, focused on BMPR2. Methods: We developed a protocol for the custom bioinformatics pipeline analysis of long reads generated by long-PCR. To evaluate the potential of using MinION sequencing in PAH, we analyzed five samples, including those of two idiopathic PAH patients and a family of three members with one affected patient. Sanger sequencing analysis was performed to validate the variants. Results: The median read length was around 3.4 kb and a good mean quality score of approximately 19 was obtained. The total number of reads generated was uniform among the cases and ranged from 2,268,263 to 3,126,719. The coverage was consistent across flow cells in which the average number of reads per base ranged from 80,375 to 135,603. We identified two polymorphic variants and three mutations in four out of five patients. Certain indel variant calling-related errors were observed, mostly outside coding sequences. Conclusion: We have shown the ability of this portable nanopore sequencer to detect BMPR2 mutations in patients with PAH. The MinION nanopore sequencer is a promising tool for screening BMPR2 mutations, especially in small laboratories and research groups.

Two new cases of interstitial 7q35q36.1 deletion including CNTNAP2 and KMT2C.

BACKGROUND: Terminal deletions of the long arm of chromosome 7 are well known and frequently associated with syndromic holoprosencephaly due to the involvement of the SHH (aliases HHG1, SMMCI, TPT, TPTPS, and MCOPCB5) gene region. However, interstitial deletions including CNTNAP2 (aliases Caspr2, KIAA0868, and NRXN4) and excluding the SHH region are less common. METHODS: We report the clinical and molecular characterization associated with pure 7q35 and 7q35q36.1 deletion in two unrelated patients as detected by oligonucleotide-based array-CGH analysis. RESULTS: The common clinical features were abnormal maternal serum screening during first-trimester pregnancy, low occipitofrontal circumference at birth, hypotonia, abnormal feet, developmental delay, impaired language development, generalized seizures, hyperactive behavior, friendly personality, and cranio-facial dysmorphism. Both deletions occurred de novo and sequencing of CNTNAP2, a candidate gene for epilepsy and autism showed absence of mutation on the contralateral allele. CONCLUSION: Combined haploinsufficiency of GALNTL5 (alias GalNAc-T5L), CUL1, SSPO (aliases SCO-spondin, KIAA0543, and FLJ36112), AOC1 (alias DAO), RHEB, and especially KMT2C (alias KIAA1506 and HALR) with monoallelic disruption of CNTNAP2 may explain neurologic abnormalities, hypotonia, and exostoses. Haploinsufficiency of PRKAG2 (aliases AAKG, AAKG2, H91620p, WPWS, and CMH6) and KCNH2 (aliases Kv11.1, HERG, and erg1) genes may be responsible of long QT syndrome observed for one patient.

Lack of consensus in the choice of termination of pregnancy for Turner syndrome in France.

BACKGROUND: The observed rate of termination of pregnancy (TOP) for Turner syndrome varies worldwide and even within countries. In this vignette study we quantified agreement among ten multidisciplinary prenatal diagnosis centers in Paris. METHODS: We submitted online three cases of Turner syndrome (increased nuchal translucency, normal ultrasound, aortic coarctation) to fetal medicine experts: one obstetrician, one pediatrician and one geneticist in each of the ten Parisian centers. Each case was presented in the form of a progressive clinical history with conditional links dependent upon responses. The background to each case was provided, along with the medical history of the parents and the counseling they got from medical staff. The experts indicated online whether or not they would accept the parents' request for TOP. We assessed the percentage of agreement for acceptance or refusal of TOP. We also used a multilevel logistic regression model to evaluate differences among obstetrician-gynecologists, pediatricians and cytogeneticists. RESULTS: Overall agreement among the experts to accept or refuse TOP was, respectively, 25 and 28%. The percentage of disagreement was 47%. The percentage of agreement to accept TOP was 33, 8 and 33% for obstetrician-gynecologists, pediatricians and cytogeneticists, respectively. The respective percentages of agreement to refuse TOP were 19, 47 and 26%. CONCLUSION: Our results show the lack of consensus with regard to decisions related to termination of pregnancy for Turner Syndrome. This lack of consensus in turn underscores the importance of multidisciplinary management of these pregnancies in specialized fetal medicine centers.

Chromosomal translocations and semen quality: A study on 144 male translocation carriers.

RESEARCH QUESTION: Chromosomal translocations are known genetic causes of male infertility. Are certain translocations or chromosomal regions more directly associated with sperm defects? Is there a threshold of sperm impairment that can be relevant for detection of translocations? DESIGN: This is a monocentric retrospective observational study covering a 10-year period. Eighty-one patients carrying a reciprocal translocation (RCT) and 63 carrying a Robertsonian translocation (ROBT) were compared with 105 fertile patients. Semen quality before and after sperm migration was compared. The aims were to define whether a threshold based on sperm analysis could be proposed for detection of translocations and to identify whether some redundant chromosomal regions might be associated with sperm quality defects. RESULTS: The number of progressive spermatozoa retrieved after sperm preparation (NPS-ASP) was altered in both RCT and ROBT carriers compared with controls, with a stronger alteration in ROBT. Based on the NPS-ASP results in this large group of translocation carriers, a relatively robust threshold, fixed at less than 5 million, may be proposed for detection of translocations. The alteration of NPS-ASP was independent of the chromosome involved in ROBT, while in RCT, four redundant chromosomal regions (1q21, 6p21, 16q21, 17q11.2) were associated with poor or very poor NPS-ASP. CONCLUSIONS: The NPS-ASP appears to be a good parameter to assess sperm function and would be a useful tool to detect chromosomal translocations. Four redundant regions have been identified on four chromosomes, suggesting that they may contain genes of interest to study sperm functions.

15q24.1 BP4-BP1 microdeletion unmasking paternally inherited functional polymorphisms combined with distal 15q24.2q24.3 duplication in a patient with epilepsy, psychomotor delay, overweight, ventricular arrhythmia.

15q24 microdeletion and microduplication syndromes are genetic disorders caused by non-allelic homologous recombination between low-copy repeats (LCRs) in the 15q24 chromosome region. Individuals with 15q24 microdeletion and microduplication syndromes share a common 1.2 Mb critical interval, spanning from LCR15q24B to LCR15q24C. Patients with 15q24 microdeletion syndrome exhibit distinct dysmorphic features, microcephaly, variable developmental delay, multiples congenital anomalies while individuals with reciprocal 15q24 microduplication syndrome show mild developmental delay, facial dysmorphism associated with skeletal and genital abnormalities. We report the first case of a 10 year-old girl presenting mild developmental delay, psychomotor retardation, epilepsy, ventricular arrhythmia, overweight and idiopathic central precocious puberty. 180K array-CGH analysis identified a 1.38 Mb heterozygous interstitial 15q24.1 BP4-BP1 microdeletion including HCN4 combined with a concomitant 2.6 Mb heterozygous distal 15q24.2q24.3 microduplication. FISH analysis showed that both deletion and duplication occurred de novo in the proband. Of note, both copy number imbalances did not involve the 1.2 Mb minimal deletion/duplication critical interval of the 15q24.1q24.2 chromosome region (74.3-75.5 Mb). Sequencing of candidate genes for epilepsy and obesity showed that the proband was hemizygous for paternal A-at risk allele of BBS4 rs7178130 and NPTN rs7171755 predisposing to obesity, epilepsy and intellectual deficits. Our study highlights the complex interaction of functional polymorphisms and/or genetic variants leading to variable clinical manifestations in patients with submicroscopic chromosomal aberrations.

First prenatal case of proximal 19p13.12 microdeletion syndrome: New insights and new delineation of the syndrome.

Proximal 19p13.12 microdeletion has been rarely reported. Only five postnatal cases with intellectual disability, facial dysmorphism, branchial arch defects and overlapping deletions involving proximal 19p13.12 have been documented. Two critical intervals were previously defined: a 700 kb for branchial arch defects and a 350 kb for hypertrichosis-synophrys-protruding front teeth. We describe the first prenatal case, a fetal death in utero at 39 weeks of gestation. Agilent 180K array-CGH analysis identified a heterozygous interstitial 745 kb deletion at 19p13.12 chromosome region, encompassing both previously reported critical intervals, including at least 6 functionally relevant genes: NOTCH3, SYDE1, AKAP8, AKAP8L, WIZ and BRD4. Quantitative PCR showed that the deletion occurred de novo with a median size of 753 kb. NOTCH3 and SYDE1 were candidate genes for placental pathology whilst AKAP8, AKAP8L, WIZ and BRD4 were highly expressed in the branchial arches. Molecular characterization and sequencing of candidate genes for placental pathology and branchial arch defects were carried out in order to correlate the genotype-phenotype relationship and unravel the underlying mechanism of proximal 19p13.12 microdeletion syndrome. This case also contributes to define the novel critical interval and expand the clinical phenotype spectrum of proximal 19p13.12 microdeletion syndrome.

Prenatal diagnosis of isochromosome 20q in a fetus with vertebral anomaly and rocker-bottom feet.

OBJECTIVE: Isochromosome of the long arm of chromosome 20 (i(20q)) is a rare structural abnormality in prenatal diagnosis. Thirty prenatal cases of mosaic i(20q) have been reported, among which only four are associated with fetal malformations. We describe a new prenatal case of i(20q) with fetal malformations. MATERIALS AND METHODS: We also observed a discrepancy between uncultured and cultured amniotic fluid cells by using conventional cytogenetic, fluorescence in situ hybridization and array-SNP analysis. RESULTS: The short arm deletion of chromosome 20 arising from the isochromosome encompassed two candidate genes PAX1 and JAG1 involved in cranio-facial and vertebral development. CONCLUSION: The data would allow establishing a phenotype-genotype correlation. Thus, we proposed to define a recognizable syndrome combining cranio-facial dysmorphism, vertebral bodies' anomalies, feet and cerebral malformations.

Prenatal Diagnosis of a 2.5 Mb De Novo 17q24.1q24.2 Deletion Encompassing KPNA2 and PSMD12 Genes in a Fetus with Craniofacial Dysmorphism, Equinovarus Feet, and Syndactyly.

Interstitial 17q24.1 or 17q24.2 deletions were reported after conventional cytogenetic analysis or chromosomal microarray analysis in patients presenting intellectual disability, facial dysmorphism, and/or malformations. We report on a fetus with craniofacial dysmorphism, talipes equinovarus, and syndactyly associated with a de novo 2.5 Mb 17q24.1q24.2 deletion. Among the deleted genes, KPNA2 and PSMD12 are discussed for the correlation with the fetal phenotype. This is the first case of prenatal diagnosis of 17q24.1q24.2 deletion.

Induced pluripotent stem cell generation from a man carrying a complex chromosomal rearrangement as a genetic model for infertility studies.

Despite progress in human reproductive biology, the cause of male infertility often remains unknown, due to the lack of appropriate and convenient in vitro models of meiosis. Induced pluripotent stem cells (iPSCs) derived from the cells of infertile patients could provide a gold standard model for generating primordial germ cells and studying their development and the process of spermatogenesis. We report the characterization of a complex chromosomal rearrangement (CCR) in an azoospermic patient, and the successful generation of specific-iPSCs from PBMC-derived erythroblasts. The CCR was characterized by karyotype, fluorescence in situ hybridization and oligonucleotide-based array-comparative genomic hybridization. The CCR included five breakpoints and was caused by the inverted insertion of a chromosome 12 segment into the short arm of one chromosome 7 and a pericentric inversion of the structurally rearranged chromosome 12. Gene mapping of the breakpoints led to the identification of a candidate gene, SYCP3. Erythroblasts from the patient were reprogrammed with Sendai virus vectors to generate iPSCs. We assessed iPSC pluripotency by RT-PCR, immunofluorescence staining and teratoma induction. The generation of specific-iPSCs from patients with a CCR provides a valuable in vitro genetic model for studying the mechanisms by which chromosomal abnormalities alter meiosis and germ cell development.

Teaching invasive prenatal procedures: effectiveness of two simple simulators in training.

BACKGROUND: Chorionic villus sampling (CVS) and amniocentesis are the major tools of invasive prenatal diagnosis. We studied the effectiveness of two simulators in training in invasive procedures. METHODS: Two affordable and simple simulators were developed, modelling the maternal abdomen and the different tissue layers crossed by the needle. The trainees were evaluated before and after practical and theoretical training. A score evaluating five criteria for technical quality in performing a procedure safely was established. Initial score of 9 or 10 was excluded. The primary endpoint was improvement defined as the change between pre-test and post-test, expressed as a percentage of the pre-test. RESULTS: A total of 54 residents and 63 specialists in obstetrics and gynaecology participated. Residents improved their scores in the practice of amniocentesis (80% [43-167]) and CVS (100% [29-150]), as well as specialists (100% [25-233] and 67% [33-122]). Specialists who earlier performed one CVS or more than five amniocentesis procedures had a lower increase during training than those who had performed fewer than five procedures (p < 0.01). Being inexperienced in CVS was associated with greater improvement (27% vs 56%, p = 0.003). CONCLUSION: A simple simulator improves the ability of physicians to perform invasive procedures in particular when initial experience is low. © 2016 John Wiley & Sons, Ltd.

Confined blood chimerism in a monochorionic dizygotic sex discordant twin pregnancy conceived after induced ovulation.

BACKGROUND: Monochorionic twins are generally considered as a monozygotic twin pregnancy. However, several cases of monochorial dizygotic twin pregnancies have been reported. CASE REPORT: We report on a rare case of monochorionic dizygotic twin pregnancy conceived after induced ovulation in a 32-year-old woman. The diagnosis was made on morphological ultrasound examination at 18+4 weeks of gestation, showing two fetuses with discordant sex. The amniocentesis was declined by the patient. RESULTS: The monochorionic status was confirmed after a histopathalogical study of the placenta. At delivery, both a phenotypically normal boy and a phenotypically normal girl without sexual abnormality were observed. This analysis also revealed the presence of vascular anastomoses between both fetal circulations. Postnatal cytogenetic analyses indicated the presence of a chimerism in peripheral blood lymphocytes. This chimerism was not observed in cells obtained from a buccal swab. Molecular determination of zygosity confirmed the existence of the confined peripheral blood chimerism with the presence of four parental alleles. CONCLUSION: We report on a case of monochorionic dizygotic twin pregnancy. This observation underlies the need to carefully assess twin pregnancies, especially when obtained after assisted reproductive technology.

Complex translocation t(1;12;14)(q42;q14;q32) and HMGA2 deletion in a fetus presenting growth delay and bilateral cataracts.

We report the prenatal detection of a de novo unbalanced complex chromosomal rearrangement (CCR), in a fetus with growth delay and bilateral cataracts. Standard karyotype and FISH analyses on amniotic fluid revealed a complex de novo translocation, resulting in a 46,XY,t(1;12;14)(q42;q14;q32) karyotype. CGH-array showed a significant deletion of 387  kb at 12q14.3, at a distance of only 200-700 kb from the breakpoint at 12q14, which encompassed the HMGA2 gene and occurred de novo. Although 12q14 microdeletions are associated with growth delay in several reports in the literature, we present here the smallest deletion prenatally detected, and we detail the clinical description of the fetus. The correlation between cataracts and this complex genotype is puzzling. Among the genes disrupted by the breakpoint in 12q14, GRIP1 has been associated with abnormal eye development in mice, including lens degeneration. Interestingly, HMGA2 is expressed in the mouse's developing lens, and its expression is decreased in lens of elderly humans, correlated with the severity of lens opacity. In this report, we refine the link between HMGA2 loss of function and growth delay during prenatal development. We also discuss the correlation between cataracts and genotype in this unbalanced CCR case of unexpected complexity.

Small Supernumerary Marker Chromosomes in Human Infertility.

Small supernumerary marker chromosomes (sSMC) are structurally abnormal chromosomes that cannot be unambiguously identified by banding cytogenetics. The objective of this study was to provide an overview of sSMC frequency and characterization in a context of infertility and to review the literature describing sSMC in relation with male and female infertility. Therefore, a systematic literature review on sSMC associated with infertility was conducted by means of a PubMed literature and a sSMC database (http://ssmc-tl.com/sSMC.html) search. A total of 234 patients with infertility were identified as carriers of sSMC. All chromosomes, except chromosomes 10, 19 and the X, were involved in sSMC, and in 72% the sSMC originated from acrocentric chromosomes. Euchromatic imbalances were caused by the presence of sSMC in 30% of the cases. Putative genes have been identified in only 1.2% of sSMC associated with infertility. The implication of sSMC in infertility could be due to a partial trisomy of some genes but also to mechanical effects perturbing meiosis. Further precise molecular and interphase-architecture studies on sSMC are needed in the future to characterize the relationship between this chromosomal anomaly and human infertility.

Inherited 1q21.1q21.2 duplication and 16p11.2 deletion: a two-hit case with more severe clinical manifestations.

We report paternally inherited duplication of 1q12q21.2 of 5.8 Mb associated with maternally inherited deletion of 16p11.2 of 545 Kb, this latter first identified in a fetus exhibiting an absent nasal bone detected during pregnancy. During the neonatal period, the young boy presented developmental delay, epilepsy, congenital anomalies and overweight. The clinical features of the proband with two rearrangements were more severe than in either of the parents carrying only one or the other mutation. Thus our data support a two-hit model in which the concomitant presence of these two copy-number variations exacerbates the neurodevelopmental phenotype.

Double Xp11.22 deletion including SHROOM4 and CLCN5 associated with severe psychomotor retardation and Dent disease.

BACKGROUND: Here we report the clinical and molecular characterization of two Xp11.22 deletions including SHROOM4 and CLCN5 genes. These deletions appeared in the same X chromosome of the same patient. RESULTS: The patient is a six-year-old boy who presented hydrocephalus, severe psychomotor and growth retardation, facial dysmorphism and renal proximal tubulopathy associated with low-molecular-weight proteinuria, hypercalciuria, hyperaminoaciduria, hypophosphatemia and hyperuricemia. Standard and high resolution karyotypes showed a 46,XY formula. Array-CGH revealed two consecutive cryptic deletions in the region Xp11.22, measuring respectively 148 Kb and 2.6 Mb. The two deletions were inherited from the asymptomatic mother. CONCLUSIONS: Array-CGH allowed us to determine candidate genes in the deleted region. The disruption and partial loss of CLCN5 confirmed the diagnostic of Dent disease for this patient. Moreover, the previously described involvement of SHROOM4 in neuronal development is discussed.

Anophthalmia, hearing loss, abnormal pituitary development and response to growth hormone therapy in three children with microdeletions of 14q22q23.

BACKGROUND: Microdeletions of 14q22q23 have been associated with eye abnormalities and pituitary defects. Other phenotypic features in deletion carriers including hearing loss and response to growth hormone therapy are less well recognized. We studied genotype and phenotype of three newly identified children with 14q22q23 deletions, two girls and one boy with bilateral anophthalmia, and compared them with previously published deletion patients and individuals with intragenic defects in genes residing in the region. RESULTS: The three deletions were de novo and ranged in size between 5.8 and 8.9 Mb. All three children lacked one copy of the OTX2 gene and in one of them the deletion involved also the BMP4 gene. All three patients presented partial conductive hearing loss which tended to improve with age. Analysis of endocrine and growth phenotypes showed undetectable anterior pituitary, growth hormone deficiency and progressive growth retardation in all three patients. Growth hormone therapy led to partial catch-up growth in two of the three patients but just prevented further height loss in the third. CONCLUSIONS: The pituitary hypoplasia, growth hormone deficiency and growth retardation associated with 14q22q23 microdeletions are very remarkable, and the latter appears to have an atypical response to growth hormone therapy in some of the cases.

How can the quality of medical data in pharmacovigilance, pharmacoepidemiology and clinical studies be guaranteed?

The development of medicinal products is subject to quality standards aimed at guaranteeing that database contents accurately reflect the source documents. Paradoxically, these standards hardly address the quality of the source data itself. The objective of this work was to propose recommendations to improve data quality in three fields (pharmacovigilance, pharmacoepidemiology and clinical studies). The analysis was focused on the data and on the critical stages presenting critical quality problems, for which the current guidelines are insufficiently detailed, unsuitable and/or poorly applied. Finally, recommendations have been proposed, mainly focused on the origin of the data and its transcription.