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.

Terminal 6q deletions cause brain malformations, a phenotype mimicking heterozygous DLL1 pathogenic variants: A multicenter retrospective case series.

OBJECTIVE: Terminal 6q deletion is a rare genetic condition associated with a neurodevelopmental disorder characterized by intellectual disability and structural brain anomalies. Interestingly, a similar phenotype is observed in patients harboring pathogenic variants in the DLL1 gene. Our study aimed to further characterize the prenatal phenotype of this syndrome as well as to attempt to establish phenotype-genotype correlations. METHOD: We collected ultrasound findings from 22 fetuses diagnosed with a pure 6qter deletion. We reviewed the literature and compared our 22 cases with 14 fetuses previously reported as well as with patients with heterozygous DLL1 pathogenic variants. RESULTS: Brain structural alterations were observed in all fetuses. The most common findings (>70%) were cerebellar hypoplasia, ventriculomegaly, and corpus callosum abnormalities. Gyration abnormalities were observed in 46% of cases. Occasional findings included cerebral heterotopia, aqueductal stenosis, vertebral malformations, dysmorphic features, and kidney abnormalities. CONCLUSION: This is the first series of fetuses diagnosed with pure terminal 6q deletion. Based on our findings, we emphasize the prenatal sonographic anomalies, which may suggest the syndrome. Furthermore, this study highlights the importance of chromosomal microarray analysis to search for submicroscopic deletions of the 6q27 region involving the DLL1 gene in fetuses with these malformations.

A +3 variant at a donor splice site leads to a skipping of the MYH11 exon 32, a recurrent RNA defect causing Heritable Thoracic Aortic Aneurysm and Dissection and/or Patent Ductus Arteriosus.

BACKGROUND: Pathogenic variants in MYH11 are associated with either heritable thoracic aortic aneurysm and dissection (HTAAD), patent ductus arteriosus (PDA) syndrome, or megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS). METHODS AND RESULTS: We report a family referred for molecular diagnosis with HTAAD/PDA phenotype in which we found a variant at a non-conserved position of the 5' donor splice site of intron 32 of MYH11 potentially altering splicing (NM_002474.3:c.4578+3A>C). Although its cosegregation with disease was observed, it remained of unknown significance. Later, aortic surgery in the proband gave us the opportunity to perform a transcript analysis. This showed a skipping of the exon 32, an RNA defect previously reported to be translated to an in-frame loss of 71 amino acids and a dominant-negative effect in the smooth muscle myosin rod. This RNA defect is also reported in 3 other HTAAD/PDA pedigrees. CONCLUSION: This report confirms that among rare variants in MYH11, skipping of exon 32 is recurrent. This finding is of particular interest to establish complex genotype-phenotype correlations where some alleles are associated with autosomal dominant HTAAD/PDA, while others result in recessive or dominant visceral myopathies.

Parental mosaicism in Marfan and Ehlers-Danlos syndromes and related disorders.

Marfan syndrome (MFS) is a heritable connective tissue disorder (HCTD) caused by pathogenic variants in FBN1 that frequently occur de novo. Although individuals with somatogonadal mosaicisms have been reported with respect to MFS and other HCTD, the overall frequency of parental mosaicism in this pathology is unknown. In an attempt to estimate this frequency, we reviewed all the 333 patients with a disease-causing variant in FBN1. We then used direct sequencing, combined with High Resolution Melting Analysis, to detect mosaicism in their parents, complemented by NGS when a mosaicism was objectivized. We found that (1) the number of apparently de novo events is much higher than the classically admitted number (around 50% of patients and not 25% as expected for FBN1) and (2) around 5% of the FBN1 disease-causing variants were not actually de novo as anticipated, but inherited in a context of somatogonadal mosaicisms revealed in parents from three families. High Resolution Melting Analysis and NGS were more efficient at detecting and evaluating the level of mosaicism compared to direct Sanger sequencing. We also investigated individuals with a causal variant in another gene identified through our "aortic diseases genes" NGS panel and report, for the first time, on an individual with a somatogonadal mosaicism in COL5A1. Our study shows that parental mosaicism is not that rare in Marfan syndrome and should be investigated with appropriate methods given its implications in patient's management.

Neuropsychological study in 19 French patients with White-Sutton syndrome and POGZ mutations.

White-Sutton syndrome is a rare developmental disorder characterized by global developmental delay, intellectual disabilities (ID), and neurobehavioral abnormalities secondary to pathogenic pogo transposable element-derived protein with zinc finger domain (POGZ) variants. The purpose of our study was to describe the neurocognitive phenotype of an unbiased national cohort of patients with identified POGZ pathogenic variants. This study is based on a French collaboration through the AnDDI-Rares network, and includes 19 patients from 18 families with POGZ pathogenic variants. All clinical data and neuropsychological tests were collected from medical files. Among the 19 patients, 14 patients exhibited ID (six mild, five moderate and three severe). The five remaining patients had learning disabilities and shared a similar neurocognitive profile, including language difficulties, dysexecutive syndrome, attention disorders, slowness, and social difficulties. One patient evaluated for autism was found to have moderate autism spectrum disorder. This study reveals that the cognitive phenotype of patients with POGZ pathogenic variants can range from learning disabilities to severe ID. It highlights that pathogenic variations in the same genes can be reported in a large spectrum of neurocognitive profiles, and that children with learning disabilities could benefit from next generation sequencing techniques.

Next-generation sequencing in a series of 80 fetuses with complex cardiac malformations and/or heterotaxy.

Herein, we report the screening of a large panel of genes in a series of 80 fetuses with congenital heart defects (CHDs) and/or heterotaxy and no cytogenetic anomalies. There were 49 males (61%/39%), with a family history in 28 cases (35%) and no parental consanguinity in 77 cases (96%). All fetuses had complex CHD except one who had heterotaxy and midline anomalies while 52 cases (65%) had heterotaxy in addition to CHD. Altogether, 29 cases (36%) had extracardiac and extra-heterotaxy anomalies. A pathogenic variant was found in 10/80 (12.5%) cases with a higher percentage in the heterotaxy group (8/52 cases, 15%) compared with the non-heterotaxy group (2/28 cases, 7%), and in 3 cases with extracardiac and extra-heterotaxy anomalies (3/29, 10%). The inheritance was recessive in six genes (DNAI1, GDF1, MMP21, MYH6, NEK8, and ZIC3) and dominant in two genes (SHH and TAB2). A homozygous pathogenic variant was found in three cases including only one case with known consanguinity. In conclusion, after removing fetuses with cytogenetic anomalies, next-generation sequencing discovered a causal variant in 12.5% of fetal cases with CHD and/or heterotaxy. Genetic counseling for future pregnancies was greatly improved. Surprisingly, unexpected consanguinity accounts for 20% of cases with identified pathogenic variants.

NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients.

The relationships between impaired cortical development and consequent malformations in neurodevelopmental disorders, as well as the genes implicated in these processes, are not fully elucidated to date. In this study, we report six novel cases of patients affected by BBSOAS (Boonstra-Bosch-Schaff optic atrophy syndrome), a newly emerging rare neurodevelopmental disorder, caused by loss-of-function mutations of the transcriptional regulator NR2F1. Young patients with NR2F1 haploinsufficiency display mild to moderate intellectual disability and show reproducible polymicrogyria-like brain malformations in the parietal and occipital cortex. Using a recently established BBSOAS mouse model, we found that Nr2f1 regionally controls long-term self-renewal of neural progenitor cells via modulation of cell cycle genes and key cortical development master genes, such as Pax6. In the human fetal cortex, distinct NR2F1 expression levels encompass gyri and sulci and correlate with local degrees of neurogenic activity. In addition, reduced NR2F1 levels in cerebral organoids affect neurogenesis and PAX6 expression. We propose NR2F1 as an area-specific regulator of mouse and human brain morphology and a novel causative gene of abnormal gyrification.

Single Circulating Fetal Trophoblastic Cells Eligible for Non Invasive Prenatal Diagnosis: the Exception Rather than the Rule.

Non-Invasive Prenatal Diagnosis (NIPD), based on the analysis of circulating cell-free fetal DNA (cff-DNA), is successfully implemented for an increasing number of monogenic diseases. However, technical issues related to cff-DNA characteristics remain, and not all mutations can be screened with this method, particularly triplet expansion mutations that frequently concern prenatal diagnosis requests. The objective of this study was to develop an approach to isolate and analyze Circulating Trophoblastic Fetal Cells (CFTCs) for NIPD of monogenic diseases caused by triplet repeat expansion or point mutations. We developed a method for CFTC isolation based on DEPArray sorting and used Huntington's disease as the clinical model for CFTC-based NIPD. Then, we investigated whether CFTC isolation and Whole Genome Amplification (WGA) could be used for NIPD in couples at risk of transmitting different monogenic diseases. Our data show that the allele drop-out rate was 3-fold higher in CFTCs than in maternal cells processed in the same way. Moreover, we give new insights into CFTCs by compiling data obtained by extensive molecular testing by microsatellite multiplex PCR genotyping and by WGA followed by mini-exome sequencing. CFTCs appear to be often characterized by a random state of genomic degradation.

Clinical and genetic data of 22 new patients with SMAD3 pathogenic variants and review of the literature.

BACKGROUND: Pathogenic SMAD3 variants are responsible for a cardiovascular phenotype, mainly thoracic aortic aneurysms and dissections. Precocious identification of the vascular risk such as aortic dilatation in mutated patients has a major impact in terms of management, particularly to avoid dissection and sudden death. These vascular damages are classically associated with premature osteoarthritis and skeletal abnormalities. However, variable expressivity and incomplete penetrance are common with SMAD3 variants. METHODS: To investigate the clinical variability observed within SMAD3 patients, we reviewed the phenotypic and genetic data of 22 new patients from our Centre and of 133 patients reported in the literature. From this cohort of 155 mutated individuals, we first aimed to delineate an estimated frequency of the main clinical signs associated with SMAD3 pathogenic variants and, then, to look for genotype-phenotype correlations, mainly to see if the aortic phenotype (AP) could be predicted by the SMAD3 variant type. RESULTS: We showed, herein, the absence of correlation between the SMAD3 variant type and the occurrence of an AP in patients. CONCLUSION: Therefore, this report brings additional data for the genotype-phenotype correlations of SMAD3 variants and the need to explore in more detail the effects of genetic modifiers that could influence the phenotype.

Immunopathological manifestations in Kabuki syndrome: a registry study of 177 individuals.

PURPOSE: Kabuki syndrome (KS) (OMIM 147920 and 300867) is a rare genetic disorder characterized by specific facial features, intellectual disability, and various malformations. Immunopathological manifestations seem prevalent and increase the morbimortality. To assess the frequency and severity of the manifestations, we measured the prevalence of immunopathological manifestations as well as genotype-phenotype correlations in KS individuals from a registry. METHODS: Data were for 177 KS individuals with KDM6A or KMT2D pathogenic variants. Questionnaires to clinicians were used to assess the presence of immunodeficiency and autoimmune diseases both on a clinical and biological basis. RESULTS: Overall, 44.1% (78/177) and 58.2% (46/79) of KS individuals exhibited infection susceptibility and hypogammaglobulinemia, respectively; 13.6% (24/177) had autoimmune disease (AID; 25.6% [11/43] in adults), 5.6% (10/177) with ≥2 AID manifestations. The most frequent AID manifestations were immune thrombocytopenic purpura (7.3% [13/177]) and autoimmune hemolytic anemia (4.0% [7/177]). Among nonhematological manifestations, vitiligo was frequent. Immune thrombocytopenic purpura was frequent with missense versus other types of variants (p = 0.027). CONCLUSION: The high prevalence of immunopathological manifestations in KS demonstrates the importance of systematic screening and efficient preventive management of these treatable and sometimes life-threatening conditions.

Multiplex targeted high-throughput sequencing in a series of 352 patients with congenital limb malformations.

Congenital limb malformations (CLM) comprise many conditions affecting limbs and more than 150 associated genes have been reported. Due to this large heterogeneity, a high proportion of patients remains without a molecular diagnosis. In the last two decades, advances in high throughput sequencing have allowed new methodological strategies in clinical practice. Herein, we report the screening of 52 genes/regulatory sequences by multiplex high-throughput targeted sequencing, in a series of 352 patients affected with various CLM, over a 3-year period of time. Patients underwent a clinical triage by expert geneticists in CLM. A definitive diagnosis was achieved in 35.2% of patients, the yield varying considerably, depending on the phenotype. We identified 112 single nucleotide variants and 26 copy-number variations, of which 52 are novel pathogenic or likely pathogenic variants. In 6% of patients, variants of uncertain significance have been found in good candidate genes. We showed that multiplex targeted high-throughput sequencing works as an efficient and cost-effective tool in clinical practice for molecular diagnosis of congenital limb malformations. Careful clinical evaluation of patients may maximize the yield of CLM panel testing.

Bardet-Biedl syndrome: Antenatal presentation of forty-five fetuses with biallelic pathogenic variants in known Bardet-Biedl syndrome genes.

Bardet-Biedl syndrome (BBS) is an emblematic ciliopathy associated with retinal dystrophy, obesity, postaxial polydactyly, learning disabilities, hypogonadism and renal dysfunction. Before birth, enlarged/cystic kidneys as well as polydactyly are the hallmark signs of BBS to consider in absence of familial history. However, these findings are not specific to BBS, raising the problem of differential diagnoses and prognosis. Molecular diagnosis during pregnancies remains a timely challenge for this heterogeneous disease (22 known genes). We report here the largest cohort of BBS fetuses to better characterize the antenatal presentation. Prenatal ultrasound (US) and/or autopsy data from 74 fetuses with putative BBS diagnosis were collected out of which molecular diagnosis was established in 51 cases, mainly in BBS genes (45 cases) following the classical gene distribution, but also in other ciliopathy genes (6 cases). Based on this, an updated diagnostic decision tree is proposed. No genotype/phenotype correlation could be established but postaxial polydactyly (82%) and renal cysts (78%) were the most prevalent symptoms. However, autopsy revealed polydactyly that was missed by prenatal US in 55% of the cases. Polydactyly must be carefully looked for in pregnancies with apparently isolated renal anomalies in fetuses.

A Broad Test Based on Fluorescent-Multiplex PCR for Noninvasive Prenatal Diagnosis of Cystic Fibrosis.

BACKGROUND: Analysis of cell-free fetal DNA in maternal plasma is very promising for early diagnosis of monogenic diseases. However, it has been limited by the need to set up patient- or disease-specific custom-made approaches. Here we propose a universal test based on fluorescent multiplex PCR and size fragment analysis for an indirect diagnosis of cystic fibrosis (CF). METHODS: The test, based on haplotyping, includes nine intra- and extragenic short tandem repeats of the CFTR locus, the coamplification of p.Phe508del (the most frequent mutation in CF patients worldwide), and a specific SRY sequence. The assay is able to determine the inherited paternal allele. RESULTS: Our simple approach was successfully applied to 30 couples and provided clear results from the maternal plasma. The mean rate of informative markers was sufficient to propose it for use in indirect diagnosis. CONCLUSIONS: This noninvasive prenatal diagnosis test, focused on indirect diagnosis of CF, offers many advantages over current methods: it is simple, rapid, and cost-effective. It allows for the testing of a large number of couples with high risk of CF, whatever the familial mutation of the CFTR gene. It provides an alternative method to reduce the number of invasive tests.

ATP6V0A2-related cutis laxa in 10 novel patients: Focus on clinical variability and expansion of the phenotype.

In ATP6V0A2-related cutis laxa, the skin phenotype varies from a wrinkly skin to prominent cutis laxa and typically associates with skeletal and neurological manifestations. The phenotype remains incompletely characterized, especially in adult patients. Glycosylation defects and reduced acidification of secretory vesicles contribute to the pathogenesis, but the consequences at the clinical level remain to be determined. Moreover, the morphology of the elastic fibres has not been studied in ATP6V0A2-related cutis laxa, nor its relation with potential clinical risks. We report on the extreme variability in ATP6V0A2-related cutis laxa in 10 novel patients, expand the phenotype with emphysema and von Willebrand disease and hypothesize on the pathogenesis that might link both with deficiency of glycosylation and with elastic fibre anomalies. Our data will affect clinical management of patients with ATP6V0A2-related cutis laxa.

Inflammatory facioscapulohumeral muscular dystrophy type 2 in 18p deletion syndrome.

Facioscapulohumeral muscular dystrophy (FSHD) has been shown to be related to genetic and epigenetic derepression of DUX4 (mapping to chromosome 4), a gene located within a repeat array of D4Z4 sequences of polymorphic length. FSHD type 1 (FSHD1) is associated with pathogenic D4Z4 repeat array contraction, while FSHD type 2 (FSHD2) is associated with SMCHD1 variants (a chromatin modifier gene that maps to the short arm of chromosome 18). Both FSHD types require permissive polyadenylation signal (4qA) downstream of the D4Z4 array.

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.

Bi-allelic variants in COL3A1 encoding the ligand to GPR56 are associated with cobblestone-like cortical malformation, white matter changes and cerebellar cysts.

BACKGROUND: Collagens are one of the major constituents of the pial membrane, which plays a crucial role in neuronal migration and cortical lamination during brain development. Type III procollagen, the chains of which are encoded by COL3A1, is the ligand of the G protein-coupled receptor 56 (GPR56), also known as adhesion G protein-coupled receptor G1. Bi-allelic mutations in GPR56 give rise to cobblestone-like malformation, white matter changes and cerebellar dysplasia. This report shows that bi-allelic mutations in COL3A1 are associated with a similar phenotype. METHODS: Exome analysis was performed in a family consisting of two affected and two non-affected siblings. Brain imaging studies of this family and of two previously reported individuals with bi-allelic mutations in COL3A1 were reviewed. Functional assays were performed on dermal fibroblasts. RESULTS: Exome analysis revealed a novel homozygous variant c.145C>G (p.Pro49Ala) in exon 2 of COL3A1. Brain MRI in the affected siblings as well as in the two previously reported individuals with bi-allelic COL3A1 mutations showed a brain phenotype similar to that associated with mutations in GPR56. CONCLUSION: Homozygous or compound heterozygous mutations in COL3A1 are associated with cobblestone-like malformation in all three families reported to date. The variability of the phenotype across patients suggests that genetic alterations in distinct domains of type III procollagen can lead to different outcomes. The presence of cobblestone-like malformation in patients with bi-allelic COL3A1 mutations emphasises the critical role of the type III collagen-GPR56 axis and the pial membrane in the regulation of brain development and cortical lamination.

Molecular diagnosis of PIK3CA-related overgrowth spectrum (PROS) in 162 patients and recommendations for genetic testing.

PURPOSE: Postzygotic activating mutations of PIK3CA cause a wide range of mosaic disorders collectively referred to as PIK3CA-related overgrowth spectrum (PROS). We describe the diagnostic yield and characteristics of PIK3CA sequencing in PROS. METHODS: We performed ultradeep next-generation sequencing (NGS) of PIK3CA in various tissues from 162 patients referred to our clinical laboratory and assessed diagnostic yield by phenotype and tissue tested. RESULTS: We identified disease-causing mutations in 66.7% (108/162) of patients, with mutant allele levels as low as 1%. The diagnostic rate was higher (74%) in syndromic than in isolated cases (35.5%; P = 9.03 × 10-5). We identified 40 different mutations and found strong oncogenic mutations more frequently in patients without brain overgrowth (50.6%) than in those with brain overgrowth (15.2%; P = 0.00055). Mutant allele levels were higher in skin and overgrown tissues than in blood and buccal samples (P = 3.9 × 10-25), regardless of the phenotype. CONCLUSION: Our data demonstrate the value of ultradeep NGS for molecular diagnosis of PROS, highlight its substantial allelic heterogeneity, and confirm that optimal diagnosis requires fresh skin or surgical samples from affected regions. Our findings may be of value in guiding future recommendations for genetic testing in PROS and other mosaic conditions.Genet Med advance online publication 02 February 2017.

Clinical and molecular findings in 39 patients with KBG syndrome caused by deletion or mutation of ANKRD11.

KBG syndrome, due to ANKRD11 alteration is characterized by developmental delay, short stature, dysmorphic facial features, and skeletal anomalies. We report a clinical and molecular study of 39 patients affected by KBG syndrome. Among them, 19 were diagnosed after the detection of a 16q24.3 deletion encompassing the ANKRD11 gene by array CGH. In the 20 remaining patients, the clinical suspicion was confirmed by the identification of an ANKRD11 mutation by direct sequencing. We present arguments to modulate the previously reported diagnostic criteria. Macrodontia should no longer be considered a mandatory feature. KBG syndrome is compatible with autonomous life in adulthood. Autism is less frequent than previously reported. We also describe new clinical findings with a potential impact on the follow-up of patients, such as precocious puberty and a case of malignancy. Most deletions remove the 5'end or the entire coding region but never extend toward 16q telomere suggesting that distal 16q deletion could be lethal. Although ANKRD11 appears to be a major gene associated with intellectual disability, KBG syndrome remains under-diagnosed. NGS-based approaches for sequencing will improve the detection of point mutations in this gene. Broad knowledge of the clinical phenotype is essential for a correct interpretation of the molecular results. © 2016 Wiley Periodicals, Inc.