16p13.11 microduplication in 45 new patients: refined clinical significance and genotype-phenotype correlations.

BACKGROUND: The clinical significance of 16p13.11 duplications remains controversial while frequently detected in patients with developmental delay (DD), intellectual deficiency (ID) or autism spectrum disorder (ASD). Previously reported patients were not or poorly characterised. The absence of consensual recommendations leads to interpretation discrepancy and makes genetic counselling challenging. This study aims to decipher the genotype-phenotype correlations to improve genetic counselling and patients' medical care. METHODS: We retrospectively analysed data from 16 013 patients referred to 12 genetic centers for DD, ID or ASD, and who had a chromosomal microarray analysis. The referring geneticists of patients for whom a 16p13.11 duplication was detected were asked to complete a questionnaire for detailed clinical and genetic data for the patients and their parents. RESULTS: Clinical features are mainly speech delay and learning disabilities followed by ASD. A significant risk of cardiovascular disease was noted. About 90% of the patients inherited the duplication from a parent. At least one out of four parents carrying the duplication displayed a similar phenotype to the propositus. Genotype-phenotype correlations show no impact of the size of the duplicated segment on the severity of the phenotype. However, NDE1 and miR-484 seem to have an essential role in the neurocognitive phenotype. CONCLUSION: Our study shows that 16p13.11 microduplications are likely pathogenic when detected in the context of DD/ID/ASD and supports an essential role of NDE1 and miR-484 in the neurocognitive phenotype. Moreover, it suggests the need for cardiac evaluation and follow-up and a large study to evaluate the aortic disease risk.

Growth charts in Kabuki syndrome 1.

Kabuki syndrome (KS, KS1: OMIM 147920 and KS2: OMIM 300867) is caused by pathogenic variations in KMT2D or KDM6A. KS is characterized by multiple congenital anomalies and neurodevelopmental disorders. Growth restriction is frequently reported. Here we aimed to create specific growth charts for individuals with KS1, identify parameters used for size prognosis and investigate the impact of growth hormone therapy on adult height. Growth parameters and parental size were obtained for 95 KS1 individuals (41 females). Growth charts for height, weight, body mass index (BMI) and occipitofrontal circumference were generated in standard deviation values for the first time in KS1. Statural growth of KS1 individuals was compared to parental target size. According to the charts, height, weight, BMI, and occipitofrontal circumference were lower for KS1 individuals than the normative French population. For males and females, the mean growth of KS1 individuals was -2 and -1.8 SD of their parental target size, respectively. Growth hormone therapy did not increase size beyond the predicted size. This study, from the largest cohort available, proposes growth charts for widespread use in the management of KS1, especially for size prognosis and screening of other diseases responsible for growth impairment beyond a calculated specific target size.

Whole genome paired-end sequencing elucidates functional and phenotypic consequences of balanced chromosomal rearrangement in patients with developmental disorders.

BACKGROUND: Balanced chromosomal rearrangements associated with abnormal phenotype are rare events, but may be challenging for genetic counselling, since molecular characterisation of breakpoints is not performed routinely. We used next-generation sequencing to characterise breakpoints of balanced chromosomal rearrangements at the molecular level in patients with intellectual disability and/or congenital anomalies. METHODS: Breakpoints were characterised by a paired-end low depth whole genome sequencing (WGS) strategy and validated by Sanger sequencing. Expression study of disrupted and neighbouring genes was performed by RT-qPCR from blood or lymphoblastoid cell line RNA. RESULTS: Among the 55 patients included (41 reciprocal translocations, 4 inversions, 2 insertions and 8 complex chromosomal rearrangements), we were able to detect 89% of chromosomal rearrangements (49/55). Molecular signatures at the breakpoints suggested that DNA breaks arose randomly and that there was no major influence of repeated elements. Non-homologous end-joining appeared as the main mechanism of repair (55% of rearrangements). A diagnosis could be established in 22/49 patients (44.8%), 15 by gene disruption (KANSL1, FOXP1, SPRED1, TLK2, MBD5, DMD, AUTS2, MEIS2, MEF2C, NRXN1, NFIX, SYNGAP1, GHR, ZMIZ1) and 7 by position effect (DLX5, MEF2C, BCL11B, SATB2, ZMIZ1). In addition, 16 new candidate genes were identified. Systematic gene expression studies further supported these results. We also showed the contribution of topologically associated domain maps to WGS data interpretation. CONCLUSION: Paired-end WGS is a valid strategy and may be used for structural variation characterisation in a clinical setting.

Exome sequencing in clinical settings: preferences and experiences of parents of children with rare diseases (SEQUAPRE study).

Exome sequencing (ES) has revolutionized diagnostic procedures in medical genetics, particularly for developmental diseases. The variety and complexity of the information produced has raised issues regarding its use in a clinical setting. Of particular interest are patients' expectations regarding the information disclosed, the accompaniment provided, and the value patients place on these. To explore these issues in parents of children with developmental disorders and no diagnosis with known etiology, a multidisciplinary group of researchers from social and behavioral sciences and patient organizations conducted a mixed-methodology study (quantitative and qualitative) in two centers of expertise for rare diseases in France. The quantitative study aimed to determine the preferences of 513 parents regarding the disclosure of ES results. It showed that parents wished to have exhaustive information, including variants of unknown significance possibly linked to their child's disorder and secondary findings. This desire for information could be a strategy to maximize the chances of obtaining a diagnosis. The qualitative study aimed to understand the expectations and reactions of 57 parents interviewed just after the return of ES results. In-depth analysis showed that parents had ambivalent feelings about the findings whatever the results returned. The contrasting results from these studies raise questions about the value of the information provided and parents' high expectations regarding the results. The nature of parental expectations has emerged as an important topic in efforts to optimize accompaniment and support for families during the informed decision-making process and after disclosure of the results in an overall context of uncertainty.

Recessive Spondylocarpotarsal Synostosis Syndrome Due to Compound Heterozygosity for Variants in MYH3.

Spondylocarpotarsal synostosis syndrome (SCTS) is characterized by intervertebral fusions and fusion of the carpal and tarsal bones. Biallelic mutations in FLNB cause this condition in some families, whereas monoallelic variants in MYH3, encoding embryonic heavy chain myosin 3, have been implicated in dominantly inherited forms of the disorder. Here, five individuals without FLNB mutations from three families were hypothesized to be affected by recessive SCTS on account of sibling recurrence of the phenotype. Initial whole-exome sequencing (WES) showed that all five were heterozygous for one of two independent splice-site variants in MYH3. Despite evidence indicating that three of the five individuals shared two allelic haplotypes encompassing MYH3, no second variant could be located in the WES datasets. Subsequent genome sequencing of these three individuals demonstrated a variant altering a 5' UTR splice donor site (rs557849165 in MYH3) not represented by exome-capture platforms. When the cohort was expanded to 16 SCTS-affected individuals without FLNB mutations, nine had truncating mutations transmitted by unaffected parents, and six inherited the rs557849165 variant in trans, an observation at odds with the population allele frequency for this variant. The rs557849165 variant disrupts splicing in the 5' UTR but is still permissive of MYH3 translational initiation, albeit with reduced efficiency. Although some MYH3 variants cause dominant SCTS, these data indicate that others (notably truncating variants) do not, except in the context of compound heterozygosity for a second hypomorphic allele. These observations make genetic diagnosis challenging in the context of simplex presentations of the disorder.

Outcome of isolated agenesis of the corpus callosum: A population-based prospective study.

OBJECTIVES: Neurodevelopmental outcome of apparently isolated agenesis of the corpus callosum (ACC) remains a major concern with uncertain prognosis. Despite "normal" IQ reported in a majority of patients, the rates of learning disabilities and severe outcome (ranging from 0% to 20%) are not clearly established. METHODS: A large population-based series was investigated based on a longitudinal follow-up until school age, using Wechsler Intelligence scales at 3, 5, and 7 years. RESULTS: Fifty women were referred to a tertiary referral unit for an "apparently" isolated ACC confirmed by ultrasound, foetal MRI, and karyotyping or array CGH. Twelve pregnancies were terminated, one foetus died in utero, one pregnancy outcome was unknown, and 36 babies were born. Two were lost to follow-up. Thirty-four children could be classified into three groups. Group 1 comprised two children (6%) with severe intellectual disability (one Mowat-Wilson syndrome and one ASD). Group 2 comprised 10 children (29%) who had learning disabilities and borderline intellectual functioning (VIQ and/or PIQ scores >70 and <85); three patients had hypopituitarism with additional MRI anomalies revealed after birth. Group 3 comprised 22 children (65%) who had both VIQ and PIQ >85 (-1 SD) with a normal school level. Longitudinal follow-up revealed weaker PIQ in younger children which improved with age. CONCLUSION: Our data indicate that intellectual ability is normal (IQ > 85) in approximately two thirds and borderline in just over a quarter of patients. However, a low risk of severe cognitive impairment exists, and this information should be shared with couples during prenatal counselling.

In utero ultrasound diagnosis of corpus callosum agenesis leading to the identification of orofaciodigital type 1 syndrome in female fetuses.

BACKGROUND: OFD1 syndrome is a rare ciliopathy inherited on a dominant X-linked mode, typically lethal in males in the first or second trimester of pregnancy. It is characterized by oral cavity and digital anomalies possibly associated with cerebral and renal signs. Its prevalence is between 1/250,000 and 1/50,000 births. It is due to heterozygous mutations of OFD1 and mutations are often de novo (75%). Familial forms show highly variable phenotypic expression. OFD1 encodes a protein involved in centriole growth, distal appendix formation, and ciliogenesis. CASES: We report the investigation of three female fetuses in which corpus callosum agenesis was detected by ultrasound during the second trimester of pregnancy. In all three fetuses, fetopathological examination allowed the diagnosis of OFD1 syndrome, which was confirmed by molecular analysis. CONCLUSIONS: To our knowledge, these are the first case reports of antenatal diagnosis of OFD1 syndrome in the absence of familial history, revealed following detection of agenesis of the corpus callosum. They highlight the impact of fetal examination following termination of pregnancy for brain malformations. They also highlight the contribution of ciliary genes to corpus callosum development.

Mutations in GREB1L Cause Bilateral Kidney Agenesis in Humans and Mice.

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute a major cause of chronic kidney disease in children and 20% of prenatally detected anomalies. CAKUT encompass a spectrum of developmental kidney defects, including renal agenesis, hypoplasia, and cystic and non-cystic dysplasia. More than 50 genes have been reported as mutated in CAKUT-affected case subjects. However, the pathophysiological mechanisms leading to bilateral kidney agenesis (BKA) remain largely elusive. Whole-exome or targeted exome sequencing of 183 unrelated familial and/or severe CAKUT-affected case subjects, including 54 fetuses with BKA, led to the identification of 16 heterozygous variants in GREB1L (growth regulation by estrogen in breast cancer 1-like), a gene reported as a target of retinoic acid signaling. Four loss-of-function and 12 damaging missense variants, 14 being absent from GnomAD, were identified. Twelve of them were present in familial or simplex BKA-affected case subjects. Female BKA-affected fetuses also displayed uterus agenesis. We demonstrated a significant association between GREB1L variants and BKA. By in situ hybridization, we showed expression of Greb1l in the nephrogenic zone in developing mouse kidney. We generated a Greb1l knock-out mouse model by CRISPR-Cas9. Analysis at E13.5 revealed lack of kidneys and genital tract anomalies in male and female Greb1l-/- embryos and a slight decrease in ureteric bud branching in Greb1l+/- embryos. We showed that Greb1l invalidation in mIMCD3 cells affected tubulomorphogenesis in 3D-collagen culture, a phenotype rescued by expression of the wild-type human protein. This demonstrates that GREB1L plays a major role in early metanephros and genital development in mice and humans.

Blepharocheilodontic syndrome is a CDH1 pathway-related disorder due to mutations in CDH1 and CTNND1.

PURPOSE: Blepharocheilodontic (BCD) syndrome is a rare autosomal dominant condition characterized by eyelid malformations, cleft lip/palate, and ectodermal dysplasia. The molecular basis of BCD syndrome remains unknown. METHODS: We recruited 11 patients from 8 families and performed exome sequencing for 5 families with de novo BCD syndrome cases and targeted Sanger sequencing in the 3 remaining families. RESULTS: We identified five CDH1 heterozygous missense mutations and three CTNND1 heterozygous truncating mutations leading to loss-of-function or haploinsufficiency. Establishment of detailed genotype-phenotype correlations was not possible because of the size of the cohort; however, the phenotype seems to appear more severe in case of CDH1 mutations. Functional analysis of CDH1 mutations confirmed their deleterious impact and suggested accelerated E-cadherin degradation. CONCLUSION: Mutations in CDH1 encoding the E-cadherin were previously reported in hereditary diffuse gastric cancer as well as in nonsyndromic cleft lip/palate. Mutations in CTNND1 have never been reported before. The encoded protein, p120ctn, prevents E-cadherin endocytosis and stabilizes its localization at the cell surface. Conditional deletion of Cdh1 and Ctnnd1 in various animal models induces features reminiscent of BCD syndrome and underlines critical role of the E-cadherin-p120ctn interaction in eyelid, craniofacial, and tooth development. Our data assert BCD syndrome as a CDH1 pathway-related disorder due to mutations in CDH1 and CTNND1 and widen the phenotypic spectrum of E-cadherin anomalies.Genet Med advance online publication 09 March 2017.

Relevance of C5b9 immunostaining in the diagnosis of neonatal hemochromatosis.

BACKGROUND: Neonatal hemochromatosis caused by a gestational alloimmune mechanism or gestational alloimmune liver disease (GALD) is a rare perinatal disorder characterized by intra- and extrahepatic iron overload. It is believed to result from complement-mediated liver injury, in which the classical complement pathway is activated by maternal antibody/fetal antigen complexes, leading to hepatocyte lysis by the membrane attack complex C5b9. According to some authors, C5b9 expression in more than 75% of liver parenchyma is specific for GALD. METHODS: We conducted a retrospective multicentric immunohistochemical study with anti-C5b9 in GALD cases (n = 25) and non-GALD cases with iron overload (n = 36) and without iron overload (n = 18). RESULTS: C5b9 was expressed in 100% of GALD cases but involved more than 75% of the liver parenchyma in only 26% of the cases. C5b9 was detected in 26.75% of the non-GALD cases with more than 75% of positive parenchyma in maternal erythrocytic alloimmunization, herpes and enterovirus hepatitis, bile acid synthetic defect, DGUOK mutation, Gaucher disease, cystic fibrosis, and giant-cell hepatitis with autoimmune hemolytic anemia. CONCLUSION: Diagnosis and therapeutic management of GALD cannot only be based on C5b9 expression in liver samples as it is not specific of this disease.

Antenatal manifestations of inborn errors of metabolism: prenatal imaging findings.

Prenatal manifestations of inborn errors of metabolism (IEM) are related to severe disorders involving metabolic pathways active in the fetal period and not compensated by maternal or placental metabolism. Some prenatal imaging findings can be suggestive of such conditions-especially in cases of consanguinity and/or recurrence of symptoms-after exclusion of the most frequent nonmetabolic etiologies. Most of these prenatal imaging findings are nonspecific. They include mainly ascites and hydrops fetalis, intrauterine growth restriction (IUGR), central nervous system (CNS) anomalies, echogenic kidneys, epiphyseal stippling, craniosynostosis, and a wide spectrum of dysostoses. These anomalies can be isolated, but in most cases, an IEM is suggested by an association of features. It must be stressed that the diagnosis of an IEM in the prenatal period is based on a close collaboration between specialists in fetal imaging, medicine, genetics, biology, and pathology.

Otopalatodigital spectrum disorders: refinement of the phenotypic and mutational spectrum.

Otopalatodigital spectrum disorders (OPDSD) constitute a group of dominant X-linked osteochondrodysplasias including four syndromes: otopalatodigital syndromes type 1 and type 2 (OPD1 and OPD2), frontometaphyseal dysplasia, and Melnick-Needles syndrome. These syndromes variably associate specific facial and extremities features, hearing loss, cleft palate, skeletal dysplasia and several malformations, and show important clinical overlap over the different entities. FLNA gain-of-function mutations were identified in these conditions. FLNA encodes filamin A, a scaffolding actin-binding protein. Here, we report phenotypic descriptions and molecular results of FLNA analysis in a large series of 27 probands hypothesized to be affected by OPDSD. We identified 11 different missense mutations in 15 unrelated probands (n=15/27, 56%), of which seven were novel, including one of unknown significance. Segregation analyses within families made possible investigating 20 additional relatives carrying a mutation. This series allows refining the phenotypic and mutational spectrum of FLNA mutations causing OPDSD, and providing suggestions to avoid the overdiagnosis of OPD1.

Antenatal manifestations of inborn errors of metabolism: autopsy findings suggestive of a metabolic disorder.

This review highlights the importance of performing an autopsy when faced with fetal abortion or termination of pregnancy with suspicion of an inborn error of metabolism. Radiological, macroscopic and microscopic features found at autopsy as well as placental anomalies that can suggest such a diagnosis are detailed. The following metabolic disorders encountered in fetuses are discussed: lysosomal storage diseases, peroxisomal disorders, cholesterol synthesis disorders, congenital disorders of glycosylation, glycogenosis type IV, mitochondrial respiratory chain disorders, transaldolase deficiency, generalized arterial calcification of infancy, hypophosphatasia, arylsulfatase E deficiency, inborn errors of serine metabolism, asparagine synthetase deficiency, hyperphenylalaninemia, glutaric aciduria type I, non-ketotic hyperglycinemia, pyruvate dehydrogenase deficiency, pyruvate carboxylase deficiency, glutamine synthase deficiency, sulfite oxidase and molybdenum cofactor deficiency.

Novel NEK8 Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation.

Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. NEK8/NPHP9 mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, situs inversus, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of NEK8 mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to NEK8 loss of function, highlighting their likely gain-of-function effect. We also showed that NEK8 missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of Nek8-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of Jck mice. Moreover, co-injection of nek8 MO with WT or mutated NEK8-GFP RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human YAP RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of Nek8-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that NEK8 human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.

[Innovations in diagnosis and treatment about a case of primary failure eruption linked to a PTHR1 gene mutation]. / Innovations dans le diagnostic et le traitement d'un cas de défaut primaire d'éruption lié à une mutation du gène PTHR1.

Primary failure of eruption is a rare condition marked by non-eruption of the posterior teeth due to mutation of a gene responsible for tooth eruption. Today, this anomaly can be detected early using innovative 3D-imaging techniques. Genetic and histologic testing will confirm the diagnosis and unfavorable prognosis. Alveolar growth must be followed in other areas too in order to avoid structural and functional asymmetry. An analysis of the diagnostic and therapeutic options using bone-borne anchorage is presented via the long-term monitoring of a female patient presenting primary failure of eruption linked to mutation of the PTHR1 gene.

Radiographic features of the skeleton in disorders of post-squalene cholesterol biosynthesis.

Disorders of post-squalene cholesterol biosynthesis are inborn errors of metabolism characterised by multiple congenital abnormalities, including significant skeletal involvement. The most frequent and best-characterised example is the Smith-Lemli-Opitz syndrome. Nine other disorders are known, namely autosomal-recessive Antley-Bixler syndrome, Greenberg dysplasia, X-linked dominant chondrodysplasia punctata, X-linked recessive male emopamil-binding protein deficiency, CHILD syndrome, CK syndrome, sterol C4 methyloxidase-like deficiency, desmosterolosis and lathosterolosis. This study provides an overview of the radiologic features observed in these diseases. A common pattern of limb abnormalities is recognisable, including polydactyly, which is typically post-axial and rarely interdigital and can involve all four limbs, and syndactyly of the toes. Chondrodysplasia punctata is specifically associated with a subgroup of disorders of cholesterol biosynthesis (Greenberg dysplasia, CHILD syndrome, X-linked dominant chondrodysplasia punctata, male emopamil-binding protein deficiency). The possible occurrence of epiphyseal stippling in the Smith-Lemli-Opitz syndrome, initially reported, does not appear to be confirmed. Stippling is also associated with other congenital disorders such as chromosomal abnormalities, brachytelephalangic chondrodysplasia punctata (X-linked recessive chondrodysplasia punctata, disruptions of vitamin K metabolism, maternal autoimmune diseases), rhizomelic chondrodysplasia punctata (peroxisomal disorders) and lysosomal storage disorders. In the differential diagnosis of epiphyseal stippling, a moth-eaten appearance of bones, asymmetry, or presence of a common pattern of limb abnormalities indicate inborn errors of cholesterol biosynthesis. We highlight the specific differentiating radiologic features of disorders of post-squalene cholesterol biosynthesis.

Clinical and molecular characterization of the 20q11.2 microdeletion syndrome: six new patients.

Interstitial microdeletions of 20q chromosome are rare, only 17 patients have been reported in the literature to date. Among them, only six carried a proximal 20q11.21-q11.23 deletion, with a size ranging from 2.6 to 6.8 Mb. The existence of a 20q11.2 microdeletion syndrome has been proposed, based on five previously reported cases that displayed anomalies of the extremities, intellectual disability, feeding difficulties, craniofacial dysmorphism and variable malformations. To further characterize this syndrome, we report on six new patients with 20q11.2 microdeletions diagnosed by whole-genome array-based comparative genomic hybridization. These patient reports more precisely refined the phenotype and narrowed the minimal critical region involved in this syndrome. Careful clinical assessment confirms the distinctive clinical phenotype. The craniofacial dysmorphism consists of high forehead, frontal bossing, enophthalmos, and midface hypoplasia. We have identified a 1.62 megabase minimal critical region involved in this syndrome encompassing three genes­GDF5, EPB41L1, andSAMHD1­which are strong candidates for different aspects of the phenotype. These results support that 20q11.2 microdeletion syndrome is a new contiguous gene deletion syndrome with a recognizable phenotype.