Nine out of 19 genes encoding GABAA receptor subunits have been linked to monogenic syndromes characterized by seizures and developmental disorders. Previously, we reported the de novo variant p.(Thr300Ile) in GABRA4 in a patient with epilepsy and neurodevelopmental abnormalities. However, no new cases have been reported since then. Through an international collaboration, we collected molecular and phenotype data of individuals carrying de novo variants in GABRA4. Patients and their parents were investigated either by exome or genome sequencing, followed by targeted Sanger sequencing in some cases. All variants within the transmembrane domain, including the previously reported p.(Thr300Ile) variant, were characterized in silico and analyzed by molecular dynamics (MD) simulation studies. We identified three novel de novo missense variants in GABRA4 (NM_000809.4): c.797 C > T, p.(Pro266Leu), c.899 C > A, p.(Thr300Asn), and c.634 G > A, p.(Val212Ile). The p.(Thr300Asn) variant impacts the same codon as the previously reported variant p.(Thr300Ile) and likely arose post-zygotically as evidenced by sequencing oral mucosal cells. Overlapping phenotypes among affected individuals included developmental delay (4/4), epileptiform EEG abnormalities (3/4), attention deficits (3/4), seizures (2/4), autistic features (2/4) and structural brain abnormalities (2/4). MD simulations of the three variants within the transmembrane domain of the receptor indicate that sub-microsecond scale dynamics differ between wild-type and mutated subunits. Taken together, our findings further corroborate an association between GABRA4 and a neurological phenotype including variable neurodevelopmental, behavioral and epileptic abnormalities.

Copy number variants that duplicate distal upstream enhancer elements of the SOX9 gene cause 46,XX testicular differences of sex development (DSD) which is characterized by a 46,XX karyotype in an individual presenting with either ambiguous genitalia or genitalia with varying degrees of virilization, including those resembling typical male genitalia. Reported duplications in this region range in size from 24 to 780 kilobases (kb). Here we report a family with two affected individuals, the proband and his maternal uncle, harboring a 3.7 kb duplication of a SOX9 enhancer identified by clinical genome sequencing. Prior fluorescence in situ hybridization (FISH) for SRY and a multi-gene panel for ambiguous genitalia were non-diagnostic. The unaffected mother also carries this duplication, consistent with previously described incomplete penetrance. To our knowledge, this is the smallest duplication identified to-date, most of which resides in a 5.2 kb region that has been previously shown to possess enhancer activity that promotes the expression of SOX9. The duplication was confirmed by quantitative-PCR and shown to be in tandem by bidirectional Sanger sequencing breakpoint analysis. This finding highlights the importance of non-coding variant interrogation in suspected genetic disorders.

Purpose We evaluated clinical and genetic features enriched in patients with multiple Mendelian conditions to determine which patients are more likely to have multiple potentially relevant genetic findings (MPRF). Methods Results of the first 7698 patients who underwent exome sequencing at Ambry Genetics were reviewed. Clinical and genetic features were examined and degree of phenotypic overlap between the genetic diagnoses was evaluated. Results Among patients referred for exome sequencing, 2% had MPRF. MPRF were more common in patients from consanguineous families and patients with greater clinical complexity. The difference in average number of organ systems affected is small: 4.3 (multiple findings) vs. 3.9 (single finding) and may not be distinguished in clinic. Conclusion Patients with multiple genetic diagnoses had a slightly higher number of organ systems affected than patients with single genetic diagnoses, largely because the comorbid conditions affected overlapping organ systems. Exome testing may be beneficial for all cases with multiple organ systems affected. The identification of multiple relevant genetic findings in 2% of exome patients highlights the utility of a comprehensive molecular workup and updated interpretation of existing genomic data; a single definitive molecular diagnosis from analysis of a limited number of genes may not be the end of a diagnostic odyssey.

Patients with dystonia are particularly appropriate for diagnostic exome sequencing (DES), due to the complex, diverse features and genetic heterogeneity. Personal and family history data were collected from test requisition forms and medical records from 189 patients with reported dystonia and available family members received for clinical DES. Of them, 20.2% patients had a positive genetic finding associated with dystonia. Detection rates for cases with isolated and combined dystonia were 22.4% and 25.0%, respectively. 71.4% of the cohort had co-occurring non-movement-related findings and a detection rate of 24.4%. Patients with childhood-onset dystonia trended toward higher detection rates (31.8%) compared to infancy (23.6%), adolescence (12.5%), and early-adulthood onset (16%). Uncharacterized gene findings were found in 6.7% (8/119) of cases that underwent analysis for genes without an established disease relationship. Patients with intellectual disability/developmental delay, seizures/epilepsy and/or multifocal dystonia were more likely to have positive findings (P = .0093, .0397, .0006). Four (2.1%) patients had findings in two genes, and seven (3.7%) had reclassification after the original report due to new literature, new clinical information or reanalysis request. Pediatric patients were more likely to have positive findings (P = .0180). Our observations show utility of family-based DES in patients with dystonia and illustrate the complexity of testing.

Purpose Intellectual disability (ID) and autism spectrum disorder (ASD) are genetically heterogeneous neurodevelopmental disorders. We sought to delineate the clinical, molecular, and neuroimaging spectrum of a novel neurodevelopmental disorder caused by variants in the zinc finger protein 292 gene ( ZNF292 ). Methods We ascertained a cohort of 28 families with ID due to putatively pathogenic ZNF292 variants that were identified via targeted and exome sequencing. Available data were analyzed to characterize the canonical phenotype and examine genotype–phenotype relationships. Results Probands presented with ID as well as a spectrum of neurodevelopmental features including ASD, among others. All ZNF292 variants were de novo, except in one family with dominant inheritance. ZNF292 encodes a highly conserved zinc finger protein that acts as a transcription factor and is highly expressed in the developing human brain supporting its critical role in neurodevelopment. Conclusion De novo and dominantly inherited variants in ZNF292 are associated with a range of neurodevelopmental features including ID and ASD. The clinical spectrum is broad, and most individuals present with mild to moderate ID with or without other syndromic features. Our results suggest that variants in ZNF292 are likely a recurrent cause of a neurodevelopmental disorder manifesting as ID with or without ASD.

Clinical diagnostic exome sequencing (DES) is currently infrequently used for detecting uniparental disomy (UPD). We present a patient with a dual diagnosis of GLI2 haploinsufficiency as well as UPD of chromosome 20, both identified through DES. We therefore recommend routine UPD analysis during DES to identify this genetic aberration.

Background During mouse embryonic development the protein kinase domain containing, cytoplasmic (Pkdcc) gene, also known as Vlk, is expressed in several tissues including the ventral midbrain, with particularly strong expression in branchial arches and limb buds. Homozygous Pkdcc knockout mice have dysmorphic features and shortened long bones as the most obvious morphological abnormalities. The human PKDCC gene has currently not been associated with any disorders. Objective To use clinical diagnostic exome sequencing (DES) for providing genetic diagnoses to two apparently unrelated patients with similar skeletal abnormalities comprising rhizomelic shortening of limbs and dysmorphic features. Methods Patient-parents trio DES was carried out and the identified candidate variants were confirmed by Sanger sequencing. Results Each patient had a homozygous gene disrupting variant in PKDCC considered to explain the skeletal phenotypes shared by both. The first patient was homozygous for the nonsense variant p.(Tyr217*) (NM_1 38 370 c.651C>A) expected to result in nonsense-mediated decay of the mutant transcripts, whereas the second patient was homozygous for the splice donor variant c.639+1G>T predicted to abolish the donor splice site by three in silico splice prediction algorithms. Conclusions Biallelic gene disrupting variants in PKDCC in humans, just like in mice, cause dysmorphic features and rhizomelic shortening of limbs.

In the published version of this paper, some of the columns in the last three rows of Table 3 were mistakenly transposed. The corrected table appears below. In col. 6 of the row for DNMT3A, "S3" was published in the original article. However, in the revised table for the corrigendum, it has been corrected to "S1". In col. 6 of the row for SON, "S3" was published in the original article. However, in the revised table for the corrigendum, it has been corrected to "S2".

DOCK3 encodes the dedicator of cytokinesis 3 protein, a member of the DOCK180 family of proteins that are characterized by guanine-nucleotide exchange factor activity. DOCK3 is expressed exclusively in the central nervous system and plays an important role in axonal outgrowth and cytoskeleton reorganization. Dock3 knockout mice exhibit motor deficiencies with abnormal ataxic gait and impaired learning. We report 2 siblings with biallelic loss-of-function variants in DOCK3. Diagnostic whole-exome sequencing (WES) and chromosomal microarray were performed on a proband with severe developmental disability, hypotonia, and ataxic gait. Testing was also performed on the proband's similarly affected brother. A paternally inherited 458 kb deletion in chromosomal region 3p21.2 disrupting the DOCK3 gene was identified in both affected siblings. WES identified a nonsense variant c.382C>G (p.Gln128*) in the DOCK3 gene (NM_004947) on the maternal allele in both siblings. Common features in both affected individuals include severe developmental disability, ataxic gait, and severe hypotonia, which recapitulates the Dock3 knockout mouse phenotype. We show that complete DOCK3 deficiency in humans leads to developmental disability with significant hypotonia and gait ataxia, probably due to abnormal axonal development.

Purpose: Rett syndrome (RTT) is a neurodevelopmental disorder caused primarily by de novo mutations in MECP2 and sometimes in CDKL5 and FOXG1 . However, some RTT patients lack mutations in these genes. Methods: Twenty-two RTT patients without apparent MECP2 , CDKL5 , and FOXG1 mutations were subjected to both whole-exome sequencing and single-nucleotide polymorphism array–based copy-number variant (CNV) analyses. Results: Three patients had MECP2 mutations initially missed by clinical testing. Of the remaining 19, 17 (89.5%) had 29 other likely pathogenic intragenic mutations and/or CNVs (10 patients had 2 or more). Interestingly, 13 patients had mutations in a gene/region previously reported in other neurodevelopmental disorders (NDDs), thereby providing a potential diagnostic yield of 68.4%. These mutations were significantly enriched in chromatin regulators (corrected P = 0.0068) and moderately enriched in postsynaptic cell membrane molecules (corrected P = 0.076), implicating glutamate receptor signaling. Conclusion: The genetic etiology of RTT without MECP2 , CDKL5 , and FOXG1 mutations is heterogeneous, overlaps with other NDDs, and complicated by a high mutation burden. Dysregulation of chromatin structure and abnormal excitatory synaptic signaling may form two common pathological bases of RTT. Genet Med 19 1, 13–19.

SYNJ1 encodes a polyphosphoinositide phosphatase, synaptojanin 1, which contains two consecutive phosphatase domains and plays a prominent role in synaptic vesicle dynamics. Autosomal recessive inherited variants in SYNJ1 have previously been associated with two different neurological diseases: a recurrent homozygous missense variant (p.Arg258Gln) that abolishes Sac1 phosphatase activity was identified in three independent families with early onset parkinsonism, whereas a homozygous nonsense variant (p.Arg136*) causing a severe decrease of mRNA transcript was found in a single patient with intractable epilepsy and tau pathology. We performed whole exome or genome sequencing in three independent sib pairs with early onset refractory seizures and progressive neurological decline, and identified novel segregating recessive SYNJ1 defects. A homozygous missense variant resulting in an amino acid substitution (p.Tyr888Cys) was found to impair, but not abolish, the dual phosphatase activity of SYNJ1, whereas three premature stop variants (homozygote p.Trp843* and compound heterozygote p.Gln647Argfs*6/p.Ser1122Thrfs*3) almost completely abolished mRNA transcript production. A genetic follow-up screening in a large cohort of 543 patients with a wide phenotypical range of epilepsies and intellectual disability revealed no additional pathogenic variants, showing that SYNJ1 deficiency is rare and probably linked to a specific phenotype. While variants leading to early onset parkinsonism selectively abolish Sac1 function, our results provide evidence that a critical reduction of the dual phosphatase activity of SYNJ1 underlies a severe disorder with neonatal refractory epilepsy and a neurodegenerative disease course. These findings further expand the clinical spectrum of synaptic dysregulation in patients with severe epilepsy, and emphasize the importance of this biological pathway in seizure pathophysiology.

Deletions of 16p13.11 have been associated with a variety of phenotypes, and have also been found in normal individuals. We report on two unrelated patients with severe microcephaly, agenesis of the corpus callosum, scalp rugae, and a fetal brain disruption (FBD)-like phenotype with inherited deletions of 16p13.11. The first patient was subsequently found on whole exome sequencing to have a nonsense mutation (p.R44X) in NDE1 on the non-deleted chromosome 16 homolog. We then undertook copy number studies of 16p13.11 and sequencing of NDE1 in nine additional patients with a similar severe microcephaly, agenesis of the corpus callosum, and FBD-like phenotype. The second patient was found to have an inherited deletion of the entire NDE1 gene combined with a frameshift mutation (c.1020-1021het_delGA) in the non-deleted NDE1. These observations broaden the phenotype seen in NDE1-related microcephaly to include FBD. These data also represent the second described syndrome, after Bernard-Soulier syndrome, where an autosomal recessive condition combines an inherited segmental duplication mediated deletion with a mutation in a gene within the non-deleted homolog. Finally, we performed informatics analysis of the 16p13.11 gene content, and found that there are many genes within the region with evidence for role(s) in brain development. Sequencing of other candidate genes in this region in patients with deletion 16p13.11 and more severe neurophenotypes may be warranted.

Agenesis of the corpus callosum (ACC), cerebellar hypoplasia (CBLH), and polymicrogyria (PMG) are severe congenital brain malformations with largely undiscovered causes. We conducted a large-scale chromosomal copy number variation (CNV) discovery effort in 255 ACC, 220 CBLH, and 147 PMG patients, and 2,349 controls. Compared to controls, significantly more ACC, but unexpectedly not CBLH or PMG patients, had rare genic CNVs over one megabase (p = 1.48×10⁻³; odds ratio [OR] = 3.19; 95% confidence interval [CI] = 1.89-5.39). Rare genic CNVs were those that impacted at least one gene in less than 1% of the combined population of patients and controls. Compared to controls, significantly more ACC but not CBLH or PMG patients had rare CNVs impacting over 20 genes (p = 0.01; OR = 2.95; 95% CI = 1.69-5.18). Independent qPCR confirmation showed that 9.4% of ACC patients had de novo CNVs. These, in comparison to inherited CNVs, preferentially overlapped de novo CNVs previously observed in patients with autism spectrum disorders (p = 3.06×10⁻⁴; OR = 7.55; 95% CI = 2.40-23.72). Interestingly, numerous reports have shown a reduced corpus callosum area in autistic patients, and diminished social and executive function in many ACC patients. We also confirmed and refined previously known CNVs, including significantly narrowing the 8p23.1-p11.1 duplication present in 2% of our current ACC cohort. We found six novel CNVs, each in a single patient, that are likely deleterious: deletions of 1p31.3-p31.1, 1q31.2-q31.3, 5q23.1, and 15q11.2-q13.1; and duplications of 2q11.2-q13 and 11p14.3-p14.2. One ACC patient with microcephaly had a paternally inherited deletion of 16p13.11 that included NDE1. Exome sequencing identified a recessive maternally inherited nonsense mutation in the non-deleted allele of NDE1, revealing the complexity of ACC genetics. This is the first systematic study of CNVs in congenital brain malformations, and shows a much higher prevalence of large gene-rich CNVs in ACC than in CBLH and PMG.

Introduction The mature cerebellum coordinates movement and has additional essential roles in other central nervous system functions including learning, attention, and memory formation (Schmahmann 2004). Since normal cerebellar development is required to achieve normal function, an appreciation of cerebellar development is essential to understand dysfunction. Several features of the cerebellum make it particularly amenable to experimental manipulation in model organisms and these have led to our current understanding of cerebellar development. Importantly, loss of cerebellar function signifi cantly impacts on motor coordination, resulting in abnormal posture (ataxia), but does not cause a complete paralysis. Mice with cerebellar dysfunction are therefore viable and mutants are often easily identifi able based on their motor phenotypes. As a result, there are over 100 ataxic spontaneous mutant strains, and more than 350 transgenic or engineered ataxic mutants (www.informatics.jax.org). Additionally, the cerebellum exhibits a fairly simple architecture in contrast to the cerebral cortex. The cerebellum is comprised of seven to nine primary neuronal subtypes each displaying a unique morphology and arranged in stereotypical laminae and circuits. This architecture facilitates analysis of cerebellar mutants since developmental disruptions of lamination and neuronal morphology are relatively easy to identify. In this chapter, we will discuss four major, overlapping stages of cerebellar development. We will fi rst describe how the cerebellar territory of the neural tube is defi ned and patterned by the expression of a series of transcription factors and signaling molecules. Then we will defi ne the three germinal zones which arise within this territory and undergo extensive proliferation to give rise to all the neurons of the adult cerebellum. Extensive migration follows as the developing cerebellar neurons acquire their fi nal positions within the cerebellum. Finally, we will review several developmental aspects of neuronal connectivity as the differentiating cerebellar neurons form synaptic contacts to produce the mature circuitry of the cerebellum. Defects in one or more of these developmental processes can result in signifi cant neurologic dysfunction and congenital human cerebellar malformations. For example, Dandy-Walker malformation (DWM), the most common congenital malformation of the cerebellum, is caused by a disconnection in signaling between the developing cerebellum and adjacent posterior fossa mesenchyme (Parisi and Dobyns 2003; Aldinger et al. 2009). Disruption of neuronal cilia causes the granule cell progenitor proliferation defects of Joubert syndrome and related disorders (JSRD). Additionally, disruption of GABAergic neuron production culminates in cerebellar agenesis (Sellick et al. 2004; Hoshino et al. 2005). Defects in neuronal migration can cause cerebellar hypoplasia accompanied by lissencephaly (Hong et al. 2000). Degenerative processes during development can cause pontocerebellar hypoplasia (Edvardson et al. 2007; Budde et al. 2008). Even spinocerebellar ataxias (SCAs), usually considered to be degenerative disorders of the mature cerebellum, appear to be developmentally based (Serra et al. 2006). Other neurodevelopmental disorders such as autism are increasingly linked with subtle cerebellar developmental abnormalities (Courchesne et al. 1994; Belmonte et al. 2004). Thus, not only are the mechanisms driving cerebellar development relevant to basic science, they are also of signifi cant clinical interest.

Eukaryotic genomic DNA is combined with histones, nonhistone proteins, and RNA to form chromatin, which is extensively packaged hierarchically to fit inside a cell's nucleus. The nucleosome—comprising a histone octamer with 147 base pairs of DNA wrapped around it—is the initial level and the repeating unit of chromatin packaging, which electron microscopy first made visible to the human eye as “beads on a string” nearly four decades ago. The mechanism and nature of chromatin packaging are still under intense research. Recently, classic methods like chromatin immunoprecipitation and digestion with deoxyribonuclease and micrococcal nuclease have been combined with high-throughput sequencing to provide detailed nucleosome occupancy maps, and chromosome conformation capture and its variants have revealed that higher-order chromatin structure involves long-range loop formation between distant genomic elements. This review discusses the methods for identifying higher-order chromatin structure and the information they have provided on this important topic.

Dlx5, a homeobox transcription factor, plays a key role in the development of many organ systems. It is a candidate gene for human split-hand/split-foot type 1 malformation associated with sensorineural hearing loss. A deletion of one of its enhancers has been implicated in human craniofacial defects/hearing loss and it has also been associated with autism. However, little is known of how Dlx5 exerts its regulatory effects. We identified direct targets of Dlx5 in the mouse inner ear by gene expression profiling wild-type and Dlx5 null otic vesicles from embryonic stages E10 and E10.5. Four hundred genes were differentially expressed. We examined the genomic DNA sequences in the promoter regions of these genes for (i) previously described Dlx5 binding sites, (ii) novel 12 bp long motifs with a canonical homeodomain element shared by two or more genes and (iii) 100% conservation of these motifs in promoters of human orthologs. Forty genes passed these filters, 12 of which are expressed in the otic vesicle in domains that overlap with Dlx5. Chromatin immunoprecipitation using a Dlx5 antibody confirmed direct binding of Dlx5 to promoters of seven of these (Atbf1, Bmper, Large, Lrrtm1, Msx1, Ebf1 and Lhx1) in a cell line over-expressing Dlx5. Bmper and Lrrtm1 were up-regulated in this cell line, further supporting their identification as targets of Dlx5 in the inner ear and potentially in other organs. These direct targets support a model in which Bmp signaling is downstream of Dlx5 in the early inner ear and provide new insights into how the Dlx5 regulatory cascade is initiated.

The list of genes that when mutated cause disruptions in cerebellar development is rapidly increasing. The study of both spontaneous and engineered mouse mutants has been essential to this progress, as it has revealed much of our current understanding of the developmental processes required to construct the mature cerebellum. Improvements in brain imaging, such as magnetic resonance imaging (MRI) and the emergence of better classification schemes for human cerebellar malformations, have recently led to the identification of a number of genes which cause human cerebellar disorders. In this review we argue that synergistic approaches combining classical molecular techniques, genomics, and mouse models of human malformations will be essential to fuel additional discoveries of cerebellar developmental genes and mechanisms.