Exome sequencing as a first-tier test for copy number variant detection: retrospective evaluation and prospective screening in 2418 cases.

BACKGROUND: Despite the availability of whole exome (WES) and genome sequencing (WGS), chromosomal microarray (CMA) remains the first-line diagnostic test in most rare disorders diagnostic workup, looking for copy number variations (CNVs), with a diagnostic yield of 10%-20%. The question of the equivalence of CMA and WES in CNV calling is an organisational and economic question, especially when ordering a WGS after a negative CMA and/or WES. METHODS: This study measures the equivalence between CMA and GATK4 exome sequencing depth of coverage method in detecting coding CNVs on a retrospective cohort of 615 unrelated individuals. A prospective detection of WES-CNV on a cohort of 2418 unrelated individuals, including the 615 individuals from the validation cohort, was performed. RESULTS: On the retrospective validation cohort, every CNV detectable by the method (ie, a CNV with at least one exon not in a dark zone) was accurately called (64/64 events). In the prospective cohort, 32 diagnoses were performed among the 2418 individuals with CNVs ranging from 704 bp to aneuploidy. An incidental finding was reported. The overall increase in diagnostic yield was of 1.7%, varying from 1.2% in individuals with multiple congenital anomalies to 1.9% in individuals with chronic kidney failure. CONCLUSION: Combining single-nucleotide variant (SNV) and CNV detection increases the suitability of exome sequencing as a first-tier diagnostic test for suspected rare Mendelian disorders. Before considering the prescription of a WGS after a negative WES, a careful reanalysis with updated CNV calling and SNV annotation should be considered.

First evidence of SOX2 mutations in Peters' anomaly: Lessons from molecular screening of 95 patients.

Peters' anomaly (PA) is a rare anterior segment dysgenesis characterized by central corneal opacity and irido-lenticulo-corneal adhesions. Several genes are involved in syndromic or isolated PA (B3GLCT, PAX6, PITX3, FOXE3, CYP1B1). Some copy number variations (CNVs) have also been occasionally reported. Despite this genetic heterogeneity, most of patients remain without genetic diagnosis. We retrieved a cohort of 95 individuals with PA and performed genotyping using a combination of comparative genomic hybridization, whole genome, exome and targeted sequencing of 119 genes associated with ocular development anomalies. Causative genetic defects involving 12 genes and CNVs were identified for 1/3 of patients. Unsurprisingly, B3GLCT and PAX6 were the most frequently implicated genes, respectively in syndromic and isolated PA. Unexpectedly, the third gene involved in our cohort was SOX2, the major gene of micro-anophthalmia. Four unrelated patients with PA (isolated or with microphthalmia) were carrying pathogenic variants in this gene that was never associated with PA before. Here we described the largest cohort of PA patients ever reported. The genetic bases of PA are still to be explored as genetic diagnosis was unavailable for 2/3 of patients. Nevertheless, we showed here for the first time the involvement of SOX2 in PA, offering new evidence for its role in corneal transparency and anterior segment development.

Molecular and clinical analyses with neuropsychological assessment of a case of del(10)(q26.2qter) without intellectual disability: Genomic and transcriptomic combined approach and review of the literature.

Terminal deletion of the long arm of the chromosome 10 is a rare but well known abnormality, with a large phenotypic variability. Very few data are available about subtelomeric deletion 10q26 patients without intellectual disability. Herein, we report the case of a young adult with a classical 10q26.2qter deletion. She exhibited mainly short stature at birth and in childhood/adulthood without intellectual disability or behavioral problems. After clinical and neuropsychological assessments, we performed genomic array and transcriptomic analysis and compared our results to the data available in the literature. The patient presents a 6.525 Mb heterozygous 10q26.2qter deletion, encompassed 48 genes. Among those genes, DOCK1, C10orf90, and CALY previously described as potential candidate genes for intellectual disability, were partially or completed deleted. Interestingly, they were not deregulated as demonstrated by transcriptomic analysis. This allowed us to suggest that the mechanism involved in the deletion 10qter phenotype is much more complex that only the haploinsufficiency of DOCK1 or other genes encompassed in the deletion. Genomic and transcriptomic combined approach has to be considered to understand this pathogenesis. © 2016 Wiley Periodicals, Inc.

Partial deletions of the GRN gene are a cause of frontotemporal lobar degeneration.

Mutations in the progranulin gene (GRN) are an important cause of frontotemporal lobar degeneration (FTLD). Most known GRN mutations are null mutations, such as nonsense and frameshift mutations, which create a premature stop codon resulting in loss of function of the progranulin protein. Complete or near-complete genomic GRN deletions have also been found in three families, but heterozygous partial deletions that remove only one or two exons have not been reported to date. In this study, we analysed three unrelated FTLD patients with low plasma progranulin levels but no point GRN mutations by multiplex ligation-dependent probe amplification (MLPA) and quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF). We detected two heterozygous partial GRN deletions in two patients. One deletion removed exon 1 and part of intron 1. The second deletion was complex: it removed 1,410 bp extending from the part of intron 1 to the part of exon 3, with a small 5-bp insertion at the breakpoint junction (c.-7-1121_159delinsGATCA). Our findings illustrate the usefulness of a quantitative analysis in addition to GRN gene sequencing for a comprehensive genetic diagnosis of FTLD, particularly in patients with low plasma progranulin levels.

Dysregulation of FOXG1 pathway in a 14q12 microdeletion case.

"FOXG1 syndrome" includes postnatal microcephaly, severe intellectual disability with absence of language and agenesis of the corpus callosum. When the syndrome is associated with large 14q12q13 deletions, the patients present characteristic facial dysmorphism. Although all reports were based on genomic analysis, recently a FOXG1 regulatory elements deletion, associated with down regulated mRNA, suggested an implication of FOXG1 pathway. Herein, we report on a young boy with a phenotype consistent with a FOXG1 syndrome. He had a de novo translocation t(6;14)(q22.1;q12) associated with a heterozygous 14q12.2q13 deletion encompassing FOXG1. Subsequently, we investigated his transcriptomic profile on lymphoblastoïd cell lines and/or fibroblasts and showed that FOXG1 was commonly down-regulated. Moreover, several other FOXG1 pathway genes were also disturbed. Our data and review of previous reports highlight dysregulation of FOXG1 pathway as the cause of the "FOXG1 syndrome" developmental disorder.

Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS or Ondine's curse; OMIM 209880) is a life-threatening disorder involving an impaired ventilatory response to hypercarbia and hypoxemia. This core phenotype is associated with lower-penetrance anomalies of the autonomic nervous system (ANS) including Hirschsprung disease and tumors of neural-crest derivatives such as ganglioneuromas and neuroblastomas. In mice, the development of ANS reflex circuits is dependent on the paired-like homeobox gene Phox2b. Thus, we regarded its human ortholog, PHOX2B, as a candidate gene in CCHS. We found heterozygous de novo mutations in PHOX2B in 18 of 29 individuals with CCHS. Most mutations consisted of 5-9 alanine expansions within a 20-residue polyalanine tract probably resulting from non-homologous recombination. We show that PHOX2B is expressed in both the central and the peripheral ANS during human embryonic development. Our data support an essential role of PHOX2B in the normal patterning of the autonomous ventilation system and, more generally, of the ANS in humans.

Pure proximal deletion of chromosome 21 and kyphosis.

We report on two unrelated patients with a proximal deletion of the long arm of chromosome 21. The deletion encompassed 14.5Mb of DNA. Molecular studies showed that the two telomeric breakpoints were within the same DNA clone (BAC RP11-56D12). The centromeric breakpoints, however, were separated by only 250kb of DNA (BAC RP11-645E14 and RP11-324B9). The phenotype observed in the two patients was very different, as patient 2, who had the largest deletion, had severe kyphosis not observed in patient 1. Previous studies have identified a 6Mb region of chromosome 21 associated with severe kyphosis. Interestingly, this region overlaps the 250kb segment deleted in patient 2. We suggest that one gene (NT011512.4) located in this small overlapping region might be responsible for severe kyphosis.

PHOX2B gene mutation in a patient with late-onset central hypoventilation.

Congenital central hypoventilation syndrome, which is related to abnormal autonomic control of breathing and typically manifests at birth, was recently associated with PHOX2B gene mutations. In contrast, central hypoventilation with later onset constitutes a poorly defined group of unknown etiology. Here, we report on the identification of a de novo heterozygous PHOX2B mutation in a patient with central hypoventilation manifesting in childhood. This finding suggests that some of these cases may be genetically determined and allelic to congenital central hypoventilation syndrome.

Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by a BKCa channel opener molecule.

BACKGROUND: Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability and is also associated with autism spectrum disorders. Previous studies implicated BKCa channels in the neuropathogenesis of FXS, but the main question was whether pharmacological BKCa stimulation would be able to rescue FXS neurobehavioral phenotypes. METHODS AND RESULTS: We used a selective BKCa channel opener molecule (BMS-204352) to address this issue in Fmr1 KO mice, modeling the FXS pathophysiology. In vitro, acute BMS-204352 treatment (10 µM) restored the abnormal dendritic spine phenotype. In vivo, a single injection of BMS-204352 (2 mg/kg) rescued the hippocampal glutamate homeostasis and the behavioral phenotype. Indeed, disturbances in social recognition and interaction, non-social anxiety, and spatial memory were corrected by BMS-204352 in Fmr1 KO mice. CONCLUSION: These results demonstrate that the BKCa channel is a new therapeutic target for FXS. We show that BMS-204352 rescues a broad spectrum of behavioral impairments (social, emotional and cognitive) in an animal model of FXS. This pharmacological molecule might open new ways for FXS therapy.

Combined deletion of two Condensin II system genes (NCAPG2 and MCPH1) in a case of severe microcephaly and mental deficiency.

7qter deletion syndrome includes prenatal and/or postnatal growth retardation, microcephaly, psychomotor delay or mental retardation and a characteristic dysmorphism. If clinical features are well described, the molecular mechanisms underlying the 7qter deletion syndrome remain unknown. Those deletions usually arise de novo. Here, we describe a young boy with an abnormal phenotype consistent with a 7qter deletion syndrome. High resolution genomic analysis (Affymetrix Human Genome Wide SNP 6.0) revealed a 7q36.3 deletion encompassing NCAPG2, ESYT2, WDR60 and VIPR2, inherited from his asymptomatic father and paternal grandfather. In addition, the patient also harbored a MCPH1 deletion inherited from his healthy mother. Combined NCAPG2 and MCPH1 deletions were correlated with low mRNA levels and protein expression in the patient. MCPH1 and NCAPG2 proteins interaction is known to control chromosome structure and we thus propose that double heterozygosity for null mutations of those two genes of the Condensin II system contribute to mental deficiency with severe microcephaly phenotype.

PHOX2B genotype allows for prediction of tumor risk in congenital central hypoventilation syndrome.

The Phox2b gene is necessary for autonomic nervous-system development. Phox2b-/- mice die in utero with absent autonomic nervous system circuits, since autonomic nervous system neurons either fail to form or degenerate. We first identified the Phox2b human ortholog, PHOX2B, as the gene underlying congenital central hypoventilation syndrome (CCHS, or Ondine curse), with an autosomal dominant mode of inheritance and de novo mutation at the first generation. We have subsequently shown that heterozygous mutations of PHOX2B may account for several combined or isolated disorders of autonomic nervous-system development--namely, tumors of the sympathetic nervous system (TSNS), such as neuroblastoma and late-onset central hypoventilation syndrome. Here, we report the clinical and molecular assessments of a cohort of 188 probands with CCHS, either isolated or associated with Hirschsprung disease and/or TSNS. The mutation-detection rate was 92.6% (174/188) in our series, and the most prevalent mutation was an in-frame duplication leading to an expansion of +5 to +13 alanines in the 20-alanine stretch at the carboxy terminal of the protein. Such findings suggest PHOX2B mutation screening as a simple and reliable tool for the diagnosis of CCHS, independent of the clinically variable phenotype. In addition, somatic mosaicism was detected in 4.5% of parents. Most interestingly, analysis of genotype-phenotype interactions strongly supports the contention that patients with CCHS who develop malignant TSNS will harbor either a missense or a frameshift heterozygous mutation of the PHOX2B gene. These data further highlight the link between congenital malformations and tumor predisposition when a master gene in development is mutated.

X-linked mental retardation and autism are associated with a mutation in the NLGN4 gene, a member of the neuroligin family.

A large French family including members affected by nonspecific X-linked mental retardation, with or without autism or pervasive developmental disorder in affected male patients, has been found to have a 2-base-pair deletion in the Neuroligin 4 gene (NLGN4) located at Xp22.33. This mutation leads to a premature stop codon in the middle of the sequence of the normal protein and is thought to suppress the transmembrane domain and sequences important for the dimerization of neuroligins that are required for proper cell-cell interaction through binding to beta-neurexins. As the neuroligins are mostly enriched at excitatory synapses, these results suggest that a defect in synaptogenesis may lead to deficits in cognitive development and communication processes. The fact that the deletion was present in both autistic and nonautistic mentally retarded males suggests that the NLGN4 gene is not only involved in autism, as previously described, but also in mental retardation, indicating that some types of autistic disorder and mental retardation may have common genetic origins.

Noradrenergic neuronal development is impaired by mutation of the proneural HASH-1 gene in congenital central hypoventilation syndrome (Ondine's curse).

Congenital central hypoventilation syndrome (CCHS, Ondine's curse) is a rare disorder of the chemical control of breathing. It is frequently associated with a broad spectrum of dysautonomic symptoms, suggesting the involvement of genes widely expressed in the autonomic nervous system. In particular, the HASH-1-PHOX2A-PHOX2B developmental cascade was proposed as a candidate pathway because it controls the development of neurons with a definitive or transient noradrenergic phenotype, upstream from the RET receptor tyrosine kinase and tyrosine hydroxylase. We recently showed that PHOX2B is the major CCHS locus, whose mutation accounts for 60% of cases. We also studied the proneural HASH-1 gene and identified a heterozygous nucleotide substitution in three CCHS patients. To analyze the functional consequences of HASH-1 mutations, we developed an in vitro model of noradrenergic differentiation in neuronal progenitors derived from the mouse vagal neural crest, reproducing in vitro the HASH-PHOX-RET pathway. All HASH-1 mutant alleles impaired noradrenergic neuronal development, when overexpressed from adenoviral constructs. Thus, HASH-1 mutations may contribute to the CCHS phenotype in rare cases, consistent with the view that the abnormal chemical control of breathing observed in CCHS patients is due to the impairment of noradrenergic neurons during early steps of brainstem development.