Recurrent de novo mutations in PACS1 cause defective cranial-neural-crest migration and define a recognizable intellectual-disability syndrome.

We studied two unrelated boys with intellectual disability (ID) and a striking facial resemblance suggestive of a hitherto unappreciated syndrome. Exome sequencing in both families identified identical de novo mutations in PACS1, suggestive of causality. To support these genetic findings and to understand the pathomechanism of the mutation, we studied the protein in vitro and in vivo. Altered PACS1 forms cytoplasmic aggregates in vitro with concomitant increased protein stability and shows impaired binding to an isoform-specific variant of TRPV4, but not the full-length protein. Furthermore, consistent with the human pathology, expression of mutant PACS1 mRNA in zebrafish embryos induces craniofacial defects most likely in a dominant-negative fashion. This phenotype is driven by aberrant specification and migration of SOX10-positive cranial, but not enteric, neural-crest cells. Our findings suggest that PACS1 is necessary for the formation of craniofacial structures and that perturbation of its functions results in a specific syndromic ID phenotype.

Central 22q11.2 deletions.

22q11.2 deletion syndrome is one of the most common microdeletion syndromes. Most patients have a deletion resulting from a recombination of low copy repeat blocks LCR22-A and LCR22-D. Loss of the TBX1 gene is considered the most important cause of the phenotype. A limited number of patients with smaller, overlapping deletions distal to the TBX1 locus have been described in the literature. In these patients, the CRKL gene is deleted. Haploinsufficiency of this gene has also been implicated in the pathogenesis of 22q11.2 deletion syndrome. To distinguish these deletions (comprising the LCR22-B to LCR22-D region) from the more distal 22q11.2 deletions (located beyond LCR22-D), we propose the term "central 22q11.2 deletions". In the present study we report on 27 new patients with such a deletion. Together with information on previously published cases, we review the clinical findings of 52 patients. The prevalence of congenital heart anomalies and the frequency of de novo deletions in patients with a central deletion are substantially lower than in patients with a common or distal 22q11.2 deletion. Renal and urinary tract malformations, developmental delays, cognitive impairments and behavioral problems seem to be equally frequent as in patients with a common deletion. None of the patients had a cleft palate. Patients with a deletion that also encompassed the MAPK1 gene, located just distal to LCR22-D, have a different and more severe phenotype, characterized by a higher prevalence of congenital heart anomalies, growth restriction and microcephaly. Our results further elucidate genotype-phenotype correlations in 22q11.2 deletion syndrome spectrum.

Unstable transmission of a familial complex chromosome rearrangement.

Complex chromosome rearrangements (CCRs) are rare genomic structural aberrations involving three or more breakpoints on two or more chromosomes. About one-third of all CCRs are familial. Transmittance of such a CCR results either in genomic imbalance due to abnormal segregation at meiosis I or is stably passed on to the next generation. Here we present a phenotypically normal mother with a CCR involving chromosomes 1, 3, and 5 that gave birth to a phenotypically abnormal son. The boy presented with hypotonia, mild facial dysmorphisms, and severe intellectual disability. Conventional karyotyping revealed the same apparently balanced CCR as in the mother. However, by use of array-comparative genome hybridization (array-CGH) and fluorescence in situ hybridization (FISH) we discovered that one of the derivative chromosomes in the patient contained a de novo rearrangement. It appears that during transmission of the CCR, an additional de novo deletion and duplication had arisen in one of the derivative chromosomes. We speculate that this was the result of the inverted duplication with a distal deletion mechanism. We also demonstrate the importance of high-resolution breakpoint analysis in CCRs and stress that genetic counseling of a familial CCR is not straightforward. To our knowledge, this would be the first description of this mechanism operating on a structurally abnormal chromosome.

Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies.

Idiopathic generalized epilepsies account for 30% of all epilepsies. Despite a predominant genetic aetiology, the genetic factors predisposing to idiopathic generalized epilepsies remain elusive. Studies of structural genomic variations have revealed a significant excess of recurrent microdeletions at 1q21.1, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 in various neuropsychiatric disorders including autism, intellectual disability and schizophrenia. Microdeletions at 15q13.3 have recently been shown to constitute a strong genetic risk factor for common idiopathic generalized epilepsy syndromes, implicating that other recurrent microdeletions may also be involved in epileptogenesis. This study aimed to investigate the impact of five microdeletions at the genomic hotspot regions 1q21.1, 15q11.2, 16p11.2, 16p13.11 and 22q11.2 on the genetic risk to common idiopathic generalized epilepsy syndromes. The candidate microdeletions were assessed by high-density single nucleotide polymorphism arrays in 1234 patients with idiopathic generalized epilepsy from North-western Europe and 3022 controls from the German population. Microdeletions were validated by quantitative polymerase chain reaction and their breakpoints refined by array comparative genomic hybridization. In total, 22 patients with idiopathic generalized epilepsy (1.8%) carried one of the five novel microdeletions compared with nine controls (0.3%) (odds ratio = 6.1; 95% confidence interval 2.8-13.2; chi(2) = 26.7; 1 degree of freedom; P = 2.4 x 10(-7)). Microdeletions were observed at 1q21.1 [Idiopathic generalized epilepsy (IGE)/control: 1/1], 15q11.2 (IGE/control: 12/6), 16p11.2 IGE/control: 1/0, 16p13.11 (IGE/control: 6/2) and 22q11.2 (IGE/control: 2/0). Significant associations with IGEs were found for the microdeletions at 15q11.2 (odds ratio = 4.9; 95% confidence interval 1.8-13.2; P = 4.2 x 10(-4)) and 16p13.11 (odds ratio = 7.4; 95% confidence interval 1.3-74.7; P = 0.009). Including nine patients with idiopathic generalized epilepsy in this cohort with known 15q13.3 microdeletions (IGE/control: 9/0), parental transmission could be examined in 14 families. While 10 microdeletions were inherited (seven maternal and three paternal transmissions), four microdeletions occurred de novo at 15q13.3 (n = 1), 16p13.11 (n = 2) and 22q11.2 (n = 1). Eight of the transmitting parents were clinically unaffected, suggesting that the microdeletion itself is not sufficient to cause the epilepsy phenotype. Although the microdeletions investigated are individually rare (<1%) in patients with idiopathic generalized epilepsy, they collectively seem to account for a significant fraction of the genetic variance in common idiopathic generalized epilepsy syndromes. The present results indicate an involvement of microdeletions at 15q11.2 and 16p13.11 in epileptogenesis and strengthen the evidence that recurrent microdeletions at 15q11.2, 15q13.3 and 16p13.11 confer a pleiotropic susceptibility effect to a broad range of neuropsychiatric disorders.

Genes for hereditary sensory and autonomic neuropathies: a genotype-phenotype correlation.

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders characterized by axonal atrophy and degeneration, exclusively or predominantly affecting the sensory and autonomic neurons. So far, disease-associated mutations have been identified in seven genes: two genes for autosomal dominant (SPTLC1 and RAB7) and five genes for autosomal recessive forms of HSAN (WNK1/HSN2, NTRK1, NGFB, CCT5 and IKBKAP). We performed a systematic mutation screening of the coding sequences of six of these genes on a cohort of 100 familial and isolated patients diagnosed with HSAN. In addition, we screened the functional candidate gene NGFR (p75/NTR) encoding the nerve growth factor receptor. We identified disease-causing mutations in SPTLC1, RAB7, WNK1/HSN2 and NTRK1 in 19 patients, of which three mutations have not previously been reported. The phenotypes associated with mutations in NTRK1 and WNK1/HSN2 typically consisted of congenital insensitivity to pain and anhidrosis, and early-onset ulcero-mutilating sensory neuropathy, respectively. RAB7 mutations were only found in patients with a Charcot-Marie-Tooth type 2B (CMT2B) phenotype, an axonal sensory-motor neuropathy with pronounced ulcero-mutilations. In SPTLC1, we detected a novel mutation (S331F) corresponding to a previously unknown severe and early-onset HSAN phenotype. No mutations were found in NGFB, CCT5 and NGFR. Overall disease-associated mutations were found in 19% of the studied patient group, suggesting that additional genes are associated with HSAN. Our genotype-phenotype correlation study broadens the spectrum of HSAN and provides additional insights for molecular and clinical diagnosis.

Comprehensive clinical and molecular assessment of 32 probands with congenital contractural arachnodactyly: report of 14 novel mutations and review of the literature.

Beals-Hecht syndrome or congenital contractural arachnodactyly (CCA) is a rare, autosomal dominant connective tissue disorder characterized by crumpled ears, arachnodactyly, contractures, and scoliosis. Recent reports also mention aortic root dilatation, a finding previously thought to differentiate the condition from Marfan syndrome (MFS). In many cases, the condition is caused by mutations in the fibrillin 2 gene (FBN2) with 26 mutations reported so far, all located in the middle region of the gene (exons 23-34). We directly sequenced the entire FBN2 gene in 32 probands clinically diagnosed with CCA. In 14 probands, we found 13 new and one previously described FBN2 mutation including a mutation in exon 17, expanding the region in which FBN2 mutations occur in CCA. Review of the literature showed that the phenotype of the FBN2 positive patients was comparable to all previously published FBN2-positive patients. In our FBN2-positive patients, cardiovascular involvement included mitral valve prolapse in two adult patients and aortic root enlargement in three patients. Whereas the dilatation regressed in one proband, it remained marked in a child proband (z-score: 4.09) and his father (z-score: 2.94), warranting echocardiographic follow-up. We confirm paradoxical patellar laxity and report keratoconus, shoulder muscle hypoplasia, and pyeloureteral junction stenosis as new features. In addition, we illustrate large intrafamilial variability. Finally, the FBN2-negative patients in this cohort were clinically indistinguishable from all published FBN2-positive patients harboring a FBN2 mutation, suggesting locus heterogeneity.

Clinical and cytogenetic characterization of 13 Dutch patients with deletion 9p syndrome: Delineation of the critical region for a consensus phenotype.

The deletion 9p syndrome is caused by a constitutional monosomy of part of the short arm of chromosome 9. It is clinically characterized by dysmorphic facial features (trigonocephaly, midface hypoplasia, and long philtrum), hypotonia and mental retardation. Deletion 9p is known to be heterogeneous and exhibits variable deletion sizes. The critical region for a consensus phenotype has been reported to be located within a approximately 4-6 Mb interval on 9p22. In the present study, deletion breakpoints were determined in 13 Dutch patients by applying fluorescence in situ hybridization (FISH) and in some specific cases by array-based comparative genomic hybridization (array CGH). No clear genotype-phenotype correlation could be established for various developmental features. However, we were able to narrow down the critical region for deletion 9p syndrome to approximately 300 kb. A functional candidate gene for trigonocephaly, the CER1 gene, appeared to be located just outside this region. Sequence analysis of this gene in nine additional patients with isolated trigonocephaly did not reveal any pathogenic mutations.

Loss-of-function variants of SCN8A in intellectual disability without seizures.

OBJECTIVE: To determine the functional effect of SCN8A missense mutations in 2 children with intellectual disability and developmental delay but no seizures. METHODS: Genomic DNA was analyzed by next-generation sequencing. SCN8A variants were introduced into the Nav1.6 complementary DNA by site-directed mutagenesis. Channel activity was measured electrophysiologically in transfected ND7/23 cells. The stability of the mutant channels was assessed by Western blot. RESULTS: Both children were heterozygous for novel missense variants that altered conserved residues in transmembrane segments of Nav1.6, p.Gly964Arg in D2S6 and p.Glu1218Lys in D3S1. Both altered amino acids are evolutionarily conserved in vertebrate and invertebrate channels and are predicted to be deleterious. Neither was observed in the general population. Both variants completely prevented the generation of sodium currents in transfected cells. The abundance of Nav1.6 protein was reduced by the Glu1218Lys substitution. CONCLUSIONS: Haploinsufficiency of SCN8A is associated with cognitive impairment. These observations extend the phenotypic spectrum of SCN8A mutations beyond their established role in epileptic encephalopathy (OMIM#614558) and other seizure disorders. SCN8A should be considered as a candidate gene for intellectual disability, regardless of seizure status.

Mutations in GABRB3: From febrile seizures to epileptic encephalopathies.

OBJECTIVE: To examine the role of mutations in GABRB3 encoding the ß3 subunit of the GABAA receptor in individual patients with epilepsy with regard to causality, the spectrum of genetic variants, their pathophysiology, and associated phenotypes. METHODS: We performed massive parallel sequencing of GABRB3 in 416 patients with a range of epileptic encephalopathies and childhood-onset epilepsies and recruited additional patients with epilepsy with GABRB3 mutations from other research and diagnostic programs. RESULTS: We identified 22 patients with heterozygous mutations in GABRB3, including 3 probands from multiplex families. The phenotypic spectrum of the mutation carriers ranged from simple febrile seizures, genetic epilepsies with febrile seizures plus, and epilepsy with myoclonic-atonic seizures to West syndrome and other types of severe, early-onset epileptic encephalopathies. Electrophysiologic analysis of 7 mutations in Xenopus laevis oocytes, using coexpression of wild-type or mutant ß3, together with α5 and γ2s subunits and an automated 2-microelectrode voltage-clamp system, revealed reduced GABA-induced current amplitudes or GABA sensitivity for 5 of 7 mutations. CONCLUSIONS: Our results indicate that GABRB3 mutations are associated with a broad phenotypic spectrum of epilepsies and that reduced receptor function causing GABAergic disinhibition represents the relevant disease mechanism.

Generalized Epilepsy and Myoclonic Seizures in 22q11.2 Deletion Syndrome.

Prompted by the observations of juvenile myoclonic epilepsy (JME) in 22q11.2 deletion syndrome (22q11DS) and recurrent copy number variants in genetic generalized epilepsy (GGE), we searched for further evidence supporting a possible correlation of 22q11DS with GGE and with myoclonic seizures. Through routine diagnostics, we identified 3 novel individuals with the seemingly uncommon combination of 22q11DS and JME. We subsequently screened the literature for reports focussing on the epilepsy phenotype in 22q11DS. We additionally screened a database of 173 22q11DS patients and identified a fourth individual with JME as well as 2 additional cases with GGE. We describe 6 novel and 22 published cases with co-occurrence of 22q11DS and GGE. In many patients, GGE was associated with myoclonic seizures allowing for a diagnosis of JME in at least 6 individuals. Seventeen of the 173 22q11DS cases (10%) had a diagnosis of either focal or generalized epilepsy. In these cases, focal epilepsy could often be attributed to syndrome-associated hypocalcaemia, cerebral bleeds, or structural brain anomalies. However, the cause of GGE remained unclear. In this study, we describe and review 28 individuals with 22q11DS and GGE (especially JME), showing that both disorders frequently co-occur. Compared to the reported prevalence of 15-21%, in our case series only 10% of 22q11DS individuals were found to have epilepsy, often GGE. Since 22q11.2 does not contain convincing GGE candidate genes, we discuss the possibility of an aetiological correlation through a possibly disturbed interaction with the GABAB receptor.

Remarkable Phenytoin Sensitivity in 4 Children with SCN8A-related Epilepsy: A Molecular Neuropharmacological Approach.

Mutations in SCN8A are associated with epilepsy and intellectual disability. SCN8A encodes for sodium channel Nav1.6, which is located in the brain. Gain-of-function missense mutations in SCN8A are thought to lead to increased firing of excitatory neurons containing Nav1.6, and therefore to lead to increased seizure susceptibility. We hypothesized that sodium channel blockers could have a beneficial effect in patients with SCN8A-related epilepsy by blocking the overactive Nav1.6 and thereby counteracting the effect of the mutation. Herein, we describe 4 patients with a missense SCN8A mutation and epilepsy who all show a remarkably good response on high doses of phenytoin and loss of seizure control when phenytoin medication was reduced, while side effects were relatively mild. In 2 patients, repeated withdrawal of phenytoin led to the reoccurrence of seizures. Based on the findings in these patients and the underlying molecular mechanism we consider treatment with (high-dose) phenytoin as a possible treatment option in patients with difficult-to-control seizures due to an SCN8A mutation.

Mate pair sequencing for the detection of chromosomal aberrations in patients with intellectual disability and congenital malformations.

Recently, microarrays have replaced karyotyping as a first tier test in patients with idiopathic intellectual disability and/or multiple congenital abnormalities (ID/MCA) in many laboratories. Although in about 14-18% of such patients, DNA copy-number variants (CNVs) with clinical significance can be detected, microarrays have the disadvantage of missing balanced rearrangements, as well as providing no information about the genomic architecture of structural variants (SVs) like duplications and complex rearrangements. Such information could possibly lead to a better interpretation of the clinical significance of the SV. In this study, the clinical use of mate pair next-generation sequencing was evaluated for the detection and further characterization of structural variants within the genomes of 50 ID/MCA patients. Thirty of these patients carried a chromosomal aberration that was previously detected by array CGH or karyotyping and suspected to be pathogenic. In the remaining 20 patients no causal SVs were found and only benign aberrations were detected by conventional techniques. Combined cluster and coverage analysis of the mate pair data allowed precise breakpoint detection and further refinement of previously identified balanced and (complex) unbalanced aberrations, pinpointing the causal gene for some patients. We conclude that mate pair sequencing is a powerful technology that can provide rapid and unequivocal characterization of unbalanced and balanced SVs in patient genomes and can be essential for the clinical interpretation of some SVs.

Structural genomic variation in childhood epilepsies with complex phenotypes.

A genetic contribution to a broad range of epilepsies has been postulated, and particularly copy number variations (CNVs) have emerged as significant genetic risk factors. However, the role of CNVs in patients with epilepsies with complex phenotypes is not known. Therefore, we investigated the role of CNVs in patients with unclassified epilepsies and complex phenotypes. A total of 222 patients from three European countries, including patients with structural lesions on magnetic resonance imaging (MRI), dysmorphic features, and multiple congenital anomalies, were clinically evaluated and screened for CNVs. MRI findings including acquired or developmental lesions and patient characteristics were subdivided and analyzed in subgroups. MRI data were available for 88.3% of patients, of whom 41.6% had abnormal MRI findings. Eighty-eight rare CNVs were discovered in 71 out of 222 patients (31.9%). Segregation of all identified variants could be assessed in 42 patients, 11 of which were de novo. The frequency of all structural variants and de novo variants was not statistically different between patients with or without MRI abnormalities or MRI subcategories. Patients with dysmorphic features were more likely to carry a rare CNV. Genome-wide screening methods for rare CNVs may provide clues for the genetic etiology in patients with a broader range of epilepsies than previously anticipated, including in patients with various brain anomalies detectable by MRI. Performing genome-wide screens for rare CNVs can be a valuable contribution to the routine diagnostic workup in patients with a broad range of childhood epilepsies.

Van Maldergem syndrome: further characterisation and evidence for neuronal migration abnormalities and autosomal recessive inheritance.

We present six patients from five unrelated families with a condition originally described by Van Maldergem et al and provide follow-up studies of the original patient. The phenotype comprises a distinctive facial appearance that includes blepharophimosis, maxillary hypoplasia, telecanthus, microtia and atresia of the external auditory meatus, intellectual disability, digital contractures and skeletal anomalies together with subependymal and subcortical neuronal heterotopia. Affected patients typically have neonatal hypotonia, chronic feeding difficulties and respiratory problems. In our cohort, we have observed one instance of sibling recurrence and parental consanguinity in three of the families, indicating that autosomal recessive inheritance is likely.

Dominant missense mutations in ABCC9 cause Cantú syndrome.

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (K(ATP)) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the K(ATP) channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.