Further delineation of the MECP2 duplication syndrome phenotype in 59 French male patients, with a particular focus on morphological and neurological features.

The Xq28 duplication involving the MECP2 gene (MECP2 duplication) has been mainly described in male patients with severe developmental delay (DD) associated with spasticity, stereotypic movements and recurrent infections. Nevertheless, only a few series have been published. We aimed to better describe the phenotype of this condition, with a focus on morphological and neurological features. Through a national collaborative study, we report a large French series of 59 affected males with interstitial MECP2 duplication. Most of the patients (93%) shared similar facial features, which evolved with age (midface hypoplasia, narrow and prominent nasal bridge, thick lower lip, large prominent ears), thick hair, livedo of the limbs, tapered fingers, small feet and vasomotor troubles. Early hypotonia and global DD were constant, with 21% of patients unable to walk. In patients able to stand, lower limbs weakness and spasticity led to a singular standing habitus: flexion of the knees, broad-based stance with pseudo-ataxic gait. Scoliosis was frequent (53%), such as divergent strabismus (76%) and hypermetropia (54%), stereotypic movements (89%), without obvious social withdrawal and decreased pain sensitivity (78%). Most of the patients did not develop expressive language, 35% saying few words. Epilepsy was frequent (59%), with a mean onset around 7.4 years of age, and often (62%) drug-resistant. Other medical issues were frequent: constipation (78%), and recurrent infections (89%), mainly lung. We delineate the clinical phenotype of MECP2 duplication syndrome in a large series of 59 males. Pulmonary hypertension appeared as a cause of early death in these patients, advocating its screening early in life.

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.

Large national series of patients with Xq28 duplication involving MECP2: Delineation of brain MRI abnormalities in 30 affected patients.

Xq28 duplications encompassing MECP2 have been described in male patients with a severe neurodevelopmental disorder associated with hypotonia and spasticity, severe learning disability, stereotyped movements, and recurrent pulmonary infections. We report on standardized brain magnetic resonance imaging (MRI) data of 30 affected patients carrying an Xq28 duplication involving MECP2 of various sizes (228 kb to 11.7 Mb). The aim of this study was to seek recurrent malformations and attempt to determine whether variations in imaging features could be explained by differences in the size of the duplications. We showed that 93% of patients had brain MRI abnormalities such as corpus callosum abnormalities (n = 20), reduced volume of the white matter (WM) (n = 12), ventricular dilatation (n = 9), abnormal increased hyperintensities on T2-weighted images involving posterior periventricular WM (n = 6), and vermis hypoplasia (n = 5). The occipitofrontal circumference varied considerably between >+2SD in five patients and <-2SD in four patients. Among the nine patients with dilatation of the lateral ventricles, six had a duplication involving L1CAM. The only patient harboring bilateral posterior subependymal nodular heterotopia also carried an FLNA gene duplication. We could not demonstrate a correlation between periventricular WM hyperintensities/delayed myelination and duplication of the IKBKG gene. We thus conclude that patients with an Xq28 duplication involving MECP2 share some similar but non-specific brain abnormalities. These imaging features, therefore, could not constitute a diagnostic clue. The genotype-phenotype correlation failed to demonstrate a relationship between the presence of nodular heterotopia, ventricular dilatation, WM abnormalities, and the presence of FLNA, L1CAM, or IKBKG, respectively, in the duplicated segment.

Variants in CUL4B are associated with cerebral malformations.

Variants in cullin 4B (CUL4B) are a known cause of syndromic X-linked intellectual disability. Here, we describe an additional 25 patients from 11 families with variants in CUL4B. We identified nine different novel variants in these families and confirmed the pathogenicity of all nontruncating variants. Neuroimaging data, available for 15 patients, showed the presence of cerebral malformations in ten patients. The cerebral anomalies comprised malformations of cortical development (MCD), ventriculomegaly, and diminished white matter volume. The phenotypic heterogeneity of the cerebral malformations might result from the involvement of CUL-4B in various cellular pathways essential for normal brain development. Accordingly, we show that CUL-4B interacts with WDR62, a protein in which variants were previously identified in patients with microcephaly and a wide range of MCD. This interaction might contribute to the development of cerebral malformations in patients with variants in CUL4B.

Y-craniosynostosis by premature fusion of the metopic and coronal sutures: a new nosological entity or a variety of Saethre-Chotzen syndrome?

BACKGROUND: New forms and varieties of craniosynostoses are continuously identified due to the current increased interest of clinicians and genetists especially since the introduction of microarray-based comparative genomic hybridization (Array-CGH) techniques in the diagnostic setting of patients with craniofacial anomalies. METHODS: In this report, we describe the case of an infant who associated the early fusion of the metopic and both the coronal sutures. The interaction of the early fusion of the anterior group of the main cranial sutures gave the infant a particular clinical phenotypes with a Y configuration of the frontal bone and a globally reduced size of the skull. Such a deformity was observed in utero and was subsequently confirmed by the postnatal imaging of the head. RESULTS: This phenotype was never described previously in antenatal period to our knowledge. The array-CGH showed a heterozygous 9.0 Mb deletion in the chromosomal region 7p21.1p21.3 encompassing approximately 25 other genes, spanning from THSD7A to TWIST1/FERD3L. CONCLUSION: This case further illustrates the variability of the clinical spectrum of craniofacial disorders associated with TWIST1 abnormalities. It is important to note that the Saethre-Chotzen syndrome caused by microdeletion is generally characterized by a mental disability. However, of interest, the postoperative psychomotor development of the child considered hereby was within the normal limits.

Intragenic CAMTA1 rearrangements cause non-progressive congenital ataxia with or without intellectual disability.

BACKGROUND: Non-progressive congenital ataxias (NPCA) with or without intellectual disability (ID) are clinically and genetically heterogeneous conditions. As a consequence, the identification of the genes responsible for these phenotypes remained limited. OBJECTIVE: Identification of a new gene responsible for NPCA and ID. Methods Following the discovery of three familial or sporadic cases with an intragenic calmodulin-binding transcription activator 1 (CAMTA1) rearrangement identified by an array-CGH and recruited from a national collaboration, the authors defined the clinical and molecular characteristics of such rearrangements, and searched for patients with point mutations by direct sequencing. RESULTS: Intragenic copy number variations of CAMTA1 were all located in the CG-1 domain of the gene. It segregated with autosomal dominant ID with non-progressive congenital cerebellar ataxia (NPCA) in two unrelated families, and was de novo deletion located in the same domain in a child presenting with NPCA. In the patients with ID, the deletion led to a frameshift, producing a truncated protein, while this was not the case for the patient with isolated childhood ataxia. Brain MRI of the patients revealed a pattern of progressive atrophy of cerebellum medium lobes and superior vermis, parietal lobes and hippocampi. DNA sequencing of the CG-1 domain in 197 patients with sporadic or familial non-syndromic intellectual deficiency, extended to full DNA sequencing in 50 patients with ID and 47 additional patients with childhood ataxia, identified no pathogenic mutation. CONCLUSION: The authors have evidence that loss-of-function of CAMTA1, a brain-specific calcium responsive transcription factor, is responsible for NPCA with or without ID. Accession numbers CAMTA1 reference sequence used was ENST00000303635. Protein sequence was ENSP00000306522.

Microtriplication of 11q24.1: a highly recognisable phenotype with short stature, distinctive facial features, keratoconus, overweight, and intellectual disability.

BACKGROUND: Partial tetrasomy is mainly described as a cytogenetically visible rearrangement due to a supernumerary chromosome (i(12p), i(18p), inv dup(15)). Except for chromosome 15q11q13, intrachromosomal triplications are rare and so far not associated with a recognisable phenotype. METHODS AND RESULTS: This report describes two unrelated patients with a de novo non-recurrent submicroscopic interstitial triplication 11q24.1 detected with array comparative genomic hybridisation and confirmed by fluorescence in situ hybridisation, molecular combing, and quantitative PCR. Microsatellite analysis suggested that a common mechanism of rearrangement might have been involved. These patients share remarkably similar clinical features including distinctive facial dysmorphisms, short stature with small extremities, keratoconus, overweight, and intellectual disability. The overlapping region of 1.8 Mb contains 11 RefSeq genes and three microRNA related genes. Interestingly, the overexpression of ASAM, a gene encoding an adipocyte specific adhesion molecule, may contribute to patients' obesity. Upregulation of BILD, known to mediate apoptosis in a caspase dependent manner, could deserve further investigation into the pathological mechanism of keratoconus. CONCLUSION: Isolated duplications of distal 11q region have been previously reported and associated with intellectual disability but without a consistent set of clinical features. These findings support the proposal that microtriplication 11q24.1 is a well recognisable clinical entity.

Search for the best indicators for the presence of a VPS13B gene mutation and confirmation of diagnostic criteria in a series of 34 patients genotyped for suspected Cohen syndrome.

BACKGROUND: Cohen syndrome is a rare autosomal recessive inherited disorder that results from mutations of the VPS13B gene. Clinical features consist of a combination of mental retardation, facial dysmorphism, postnatal microcephaly, truncal obesity, slender extremities, joint hyperextensibility, myopia, progressive chorioretinal dystrophy, and intermittent neutropenia. PATIENTS AND METHODS: The aim of the study was to determine which of the above clinical features were the best indicators for the presence of VPS13B gene mutations in a series of 34 patients with suspected Cohen syndrome referred for molecular analysis of VPS13B. RESULTS: 14 VPS13B gene mutations were identified in 12 patients, and no mutation was found in 22 patients. The presence of chorioretinal dystrophy (92% vs 32%, p=0.0023), intermittent neutropenia (92% vs 5%, p<0.001), and postnatal microcephaly (100% vs 48%, p=0.0045) was significantly higher in the group of patients with a VPS13B gene mutation compared to the group of patients without a mutation. All patients with VPS13B mutations had chorioretinal dystrophy and/or intermittent neutropenia. The Kolehmainen diagnostic criteria provided 100% sensibility and 77% specificity when applied to this series. CONCLUSION: From this study and a review of more than 160 genotyped cases from the literature, it is concluded that, given the large size of the gene, VPS13B screening is not indicated in the absence of chorioretinal dystrophy or neutropenia in patients aged over 5 years. The follow-up of young patients could be a satisfactory alternative unless there are some reproductive issues.

A systematic variant screening in familial cases of congenital heart defects demonstrates the usefulness of molecular genetics in this field.

The etiology of congenital heart defect (CHD) combines environmental and genetic factors. So far, there were studies reporting on the screening of a single gene on unselected CHD or on familial cases selected for specific CHD types. Our goal was to systematically screen a proband of familial cases of CHD on a set of genetic tests to evaluate the prevalence of disease-causing variant identification. A systematic screening of GATA4, NKX2-5, ZIC3 and Multiplex ligation-dependent probe amplification (MLPA) P311 Kit was setup on the proband of 154 families with at least two cases of non-syndromic CHD. Additionally, ELN screening was performed on families with supravalvular arterial stenosis. Twenty-two variants were found, but segregation analysis confirmed unambiguously the causality of 16 variants: GATA4 (1 ×), NKX2-5 (6 ×), ZIC3 (3 ×), MLPA (2 ×) and ELN (4 ×). Therefore, this approach was able to identify the causal variant in 10.4% of familial CHD cases. This study demonstrated the existence of a de novo variant even in familial CHD cases and the impact of CHD variants on adult cardiac condition even in the absence of CHD. This study showed that the systematic screening of genetic factors is useful in familial CHD cases with up to 10.4% elucidated cases. When successful, it drastically improved genetic counseling by discovering unaffected variant carriers who are at risk of transmitting their variant and are also exposed to develop cardiac complications during adulthood thus prompting long-term cardiac follow-up. This study provides an important baseline at dawning of the next-generation sequencing era.

The expanding spectrum of COL2A1 gene variants IN 136 patients with a skeletal dysplasia phenotype.

Heterozygous COL2A1 variants cause a wide spectrum of skeletal dysplasia termed type II collagenopathies. We assessed the impact of this gene in our French series. A decision tree was applied to select 136 probands (71 Stickler cases, 21 Spondyloepiphyseal dysplasia congenita cases, 11 Kniest dysplasia cases, and 34 other dysplasia cases) before molecular diagnosis by Sanger sequencing. We identified 66 different variants among the 71 positive patients. Among those patients, 18 belonged to multiplex families and 53 were sporadic. Most variants (38/44, 86%) were located in the triple helical domain of the collagen chain and glycine substitutions were mainly observed in severe phenotypes, whereas arginine to cysteine changes were more often encountered in moderate phenotypes. This series of skeletal dysplasia is one of the largest reported so far, adding 44 novel variants (15%) to published data. We have confirmed that about half of our Stickler patients (46%) carried a COL2A1 variant, and that the molecular spectrum was different across the phenotypes. To further address the question of genotype-phenotype correlation, we plan to screen our patients for other candidate genes using a targeted next-generation sequencing approach.

GJB2 and GJB6 mutations: genotypic and phenotypic correlations in a large cohort of hearing-impaired patients.

OBJECTIVES: To analyze the clinical features of hearing impairment and to search for correlations with the genotype in patients with DFNB1. DESIGN: Case series. SETTING: Collaborative study in referral centers, institutional practice. Patients A total of 256 hearing-impaired patients selected on the basis of the presence of biallelic mutations in GJB2 or the association of 1 GJB2 mutation with the GJB6 deletion (GJB6-D13S1830)del. MAIN OUTCOME MEASURES: The prevalence of GJB2 mutations and the GJB6 deletion and audiometric phenotypes related to the most frequent genotypes. RESULTS: Twenty-nine different GJB2 mutations were identified. Allelic frequency of 35delG was 69%, and the other common mutations, 313del14, E47X, Q57X, and L90P, accounted for 2.6% to 2.9% of the variants. Concerning GJB6, (GJB6-D13S1830)del accounted for 5% of all mutated alleles and was observed in 25 of 93 compound heterozygous patients. Three novel GJB2 mutations, 355del9, V95M, and 573delCA, were identified. Hearing impairment was frequently less severe in compound heterozygotes 35delG/L90P and 35delG/N206S than in 35delG homozygotes. Moderate or mild hearing impairment was more frequent in patients with 1 or 2 noninactivating mutations than in patients with 2 inactivating mutations. Of 93 patients, hearing loss was stable in 73, progressive in 21, and fluctuant in 2. Progressive hearing loss was more frequent in patients with 1 or 2 noninactivating mutations than in those with 2 inactivating mutations. In 49 families, hearing loss was compared between siblings with similar genotypes, and variability in terms of severity was found in 18 families (37%). CONCLUSION: Genotype may affect deafness severity, but environmental and other genetic factors may also modulate the severity and evolution of GJB2-GJB6 deafness.

Large deletions encompassing the TCOF1 and CAMK2A genes are responsible for Treacher Collins syndrome with intellectual disability.

Mandibulofacial dysostosis is part of a clinically and genetically heterogeneous group of disorders of craniofacial development, which lead to malar and mandibular hypoplasia. Treacher Collins syndrome is the major cause of mandibulofacial dysostosis and is due to mutations in the TCOF1 gene. Usually patients with Treacher Collins syndrome do not present with intellectual disability. Recently, the EFTUD2 gene was identified in patients with mandibulofacial dysostosis associated with microcephaly, intellectual disability and esophageal atresia. We report on two patients presenting with mandibulofacial dysostosis characteristic of Treacher Collins syndrome, but associated with unexpected intellectual disability, due to a large deletion encompassing several genes including the TCOF1 gene. We discuss the involvement of the other deleted genes such as CAMK2A or SLC6A7 in the cognitive development delay of the patients reported, and we propose the systematic investigation for 5q32 deletion when intellectual disability is associated with Treacher Collins syndrome.

A novel TFAP2A mutation in familial Branchio-Oculo-Facial Syndrome with predominant ocular phenotype.

INTRODUCTION: Branchio-Oculo-Facial Syndrome (BOFS) is a rare autosomal dominant congenital disorder defined by branchial defects, ocular anomalies and craniofacial malformations, including variable degrees of cleft lip with or without cleft palate. In addition, temporal bone anomalies, renal and ectodermal manifestations can be present. Mutations in the TFAP2A gene have been reported in patients with BOFS, prompting phenotype-genotype studies because of the variable clinical spectrum. MATERIALS AND METHODS: We report on a family (a mother, her daughter and son) with BOFS and significant variability in clinical expression. The daughter presents predominantly with an ocular phenotype of unilateral microphthalmia and bilateral chorioretinal colobomas, whereas her brother is more severely affected contrasting with the paucisymptomatic mother. TFAP2A molecular analysis revealed a novel frameshift mutation. DISCUSSION: We confirm the wide clinical spectrum of BOFS. The importance of upper lip examination in mild and paucisymptomatic cases is underlined. TFAP2A mutation spectrum is discussed and broadened by the report of the second frameshift mutation in this gene. CONCLUSION: Patients with BOFS and predominant ocular phenotypes can be underdiagnosed. In such cases, upper lip examination can be of important diagnostic value. TFAP2A analysis provides diagnostic confirmation and improves genetic counselling.

Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis.

BACKGROUND: Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool. METHODS: We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3). RESULTS: Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel. CONCLUSIONS: Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.

Notable contribution of large CFTR gene rearrangements to the diagnosis of cystic fibrosis in fetuses with bowel anomalies.

Grade III fetal bowel hyperechogenicity and/or loop dilatation observed at the second trimester of pregnancy can be due to several disease conditions, including cystic fibrosis (CF). Screening for frequent CF mutations is performed as a first step and, in certain situations, such as when a frequent CF mutation is found in the fetus, the increased risk of CF justifies an in-depth study of the second allele. To determine the contribution of large CFTR gene rearrangements in such cases, detected using a semiquantitative fluorescent multiplex PCR (QFM-PCR) assay, we collated data on 669 referrals related to suspicion of CF in fetuses from 1998 to 2009. Deletions were found in 5/70 cases in which QFM-PCR was applied, dele19, dele22_23, dele2_6b, dele14b_15 and dele6a_6b, of which the last three remain undescribed. In 3/5 cases, hyperechogenicity was associated with dilatation and/or gallbladder anomalies. Of the total cases of CF recognized in the subgroup of first-hand referrals, deletions represent 16.7% of CF alleles. Our study thus strengthens the need to consider large CFTR gene rearrangements in the diagnosis strategy of fetal bowel anomalies, in particular in the presence of multiple anomalies.

Molecular screening of the TGIF gene in holoprosencephaly: identification of two novel mutations.

Holoprosencephaly (HPE) is the most common severe brain anomaly in humans, which results from incomplete cleavage of the forebrain during early embryogenesis. The aetiology of HPE is very heterogeneous. Among the genetic factors, TGIF ( TG-interacting factor), which codes for a transcription factor modulating the signalling pathway of TGF-beta, was previously implicated. We investigated 127 HPE probands by sequencing their TGIF gene and identified the first nonsense mutation reported so far and also a novel missense mutation, in two families that presented a large range of disease severity. The low number of mutations in TGIF suggests that this gene has no major contribution to the aetiology of HPE and our study confirms the wide clinical heterogeneity of the disease.

Large deletion of the GJB6 gene in deaf patients heterozygous for the GJB2 gene mutation: genotypic and phenotypic analysis.

Recent investigations identified a large deletion of the GJB6 gene in trans to a mutation of GJB2 in deaf patients. We looked for GJB2 mutations and GJB6 deletions in 255 French patients presenting with a phenotype compatible with DFNB1. 32% of the patients had biallelic GJB2 mutations and 6% were a heterozygous for a GJB2 mutation and a GJB6 deletion. Biallelic GJB2 mutations and combined GJB2/GJB6 anomalies were more frequent in profoundly deaf children. Based on these results, we are now assessing GJB6 deletion status in cases of prelingual hearing loss.