Neurogenetics

Exome sequencing is an effective way to identify genetic causes of etiologically heterogeneous conditions such as developmental delay and intellectual disabilities. Using exome sequencing, we have identified four patients with similar phenotypes of developmental delay, intellectual disability, failure to thrive, hypotonia, ataxia, and tooth enamel defects who all have the same de novo R331W missense variant in C-terminal binding protein 1 (CTBP1). CTBP1 is a transcriptional regulator critical for development by coordinating different regulatory pathways. The R331W variant found in these patients is within the C-terminal portion of the PLDLS (Pro-Leu-Asp-Leu-Ser) binding cleft, which is the domain through which CTBP1, interacts with chromatin-modifying enzymes and mediates chromatin-dependent gene repression pathways. This is the first report of mutations within CTBP1 in association with any human disease.

Spinocerebellar ataxia type 10 (SCA10), an autosomal dominant neurodegenerative disorder, is the result of a non-coding, pentanucleotide repeat expansion within intron 9 of the Ataxin 10 gene. SCA10 patients present with pure cerebellar ataxia; yet, some families also have a high incidence of epilepsy. SCA10 expansions containing penta- and heptanucleotide interruption motifs, termed “ATCCT interruptions,” experience large contractions during germline transmission, particularly in paternal lineages. At the same time, these alleles confer an earlier age at onset which contradicts traditional rules of genetic anticipation in repeat expansions. Previously, ATCCT interruptions have been associated with a higher prevalence of epileptic seizures in one Mexican-American SCA10 family. In a large cohort of SCA10 families, we analyzed whether ATCCT interruptions confer a greater risk for developing seizures in these families. Notably, we find that the presence of repeat interruptions within the SCA10 expansion confers a 6.3-fold increase in the risk of an SCA10 patient developing epilepsy (6.2-fold when considering patients of Mexican ancestry only) and a 13.7-fold increase in having a positive family history of epilepsy (10.5-fold when considering patients of Mexican ancestry only). We conclude that the presence of repeat interruptions in SCA10 repeat expansion indicates a significant risk for the epilepsy phenotype and should be considered during genetic counseling.

Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutations of survival motor neuron 1 (SMN1) but retention of one or more copies of the paralog SMN2. Within the SMA population, there is substantial variation in SMN2 copy number (CN); in general, those individuals with SMA who have a high SMN2 CN have a milder disease. Because SMN2 functions as a disease modifier, its accurate CN determination may have clinical relevance. In this study, we describe the development of array digital PCR (dPCR) to quantify SMN1 and SMN2 CNs in DNA samples using probes that can distinguish the single nucleotide difference between SMN1 and SMN2 in exon 8. This set of dPCR assays can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient–derived cell lines, the assay confirmed a strong inverse correlation between SMN2 CN and disease severity. We can detect SMN1–SMN2 gene conversion events in DNA samples by comparing CNs at exon 7 and exon 8. Partial deletions of SMN1 can also be detected with dPCR by comparing CNs at exon 7 or exon 8 with those at intron 1. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 CNs from SMA samples as well as identify gene conversion events and partial deletions of SMN1.

Alterations of the gamma-aminobutyric acid (GABA) signaling system has been strongly linked to the pathophysiology of autism spectrum disorder (ASD). Genetic associations of common variants in GABA receptor subunits, in particular GABRA4 on chromosome 4p12, with ASD have been replicated by several studies. Moreover, molecular investigations have identified altered transcriptional and translational levels of this gene and protein in brains of ASD individuals. Since the genotyped common variants are likely not the functional variants contributing to the molecular consequences or underlying ASD phenotype, this study aims to examine rare sequence variants in GABRA4, including those outside the protein coding regions of the gene. We comprehensively re-sequenced the entire protein coding and noncoding portions of the gene and putative regulatory sequences in 82 ASD individuals and 55 developmentally typical pediatric controls, all homozygous for the most significant previously associated ASD risk allele (G/G at rs1912960). We identified only a single common, coding variant, and no association of any single marker or set of variants with ASD. Functional annotation of noncoding variants identified several rare variants in putative regulatory sites. Finally, a rare variant unique to ASD cases, in an evolutionary conserved site of the 3′UTR, shows a trend toward decreasing gene expression. Hence, GABRA4 rare variants in noncoding DNA may be variants of modest physiological effects in ASD etiology.

As monogenic forms of episodic nervous system disorders are often caused by ion channel mutations, we looked for features of human central nervous system (CNS) expressed ion channels that further our understanding of those phenotypes. To this end, we compared human ion channels with other CNS-expressed genes, which we categorized according to the existence of transmembrane domains. When looking at the phylogenetic distribution of these genes, we observed an increased percentage of ion channels that exist in vertebrate genomes while missing in invertebrate genomes. Because we hypothesized that this pattern may relate to a more specific expression, we searched for characteristics of ion channels that indicate a tighter expression regulation. We found that ion channels have longer intron and protein sequences, features typical of genes with more specific expression. In addition, ion channels have increased human–rodent conservation around their transcription start site, as indicated by a higher fraction of conserved noncoding regions. This points to a high relevance of mutations that regulate ion channel expression. When we finally asked whether vertebrate-specific diversification is also displayed by non-ion channel genes with important roles in the CNS, we found a similar phylogenetic distribution. This concordant phylogenetic pattern suggests that vertebrate-specific adaptations may account for a large part of the shared genetic basis of episodic CNS disorders, including monogenic and genetically complex disease manifestations. Consequently, this phylogenetic pattern may contribute to the prioritization of candidate genes in human genetic studies of episodic CNS disorders.