Allison E. Ashley-Koch

A Kinesin Heavy Chain (KIF5A) Mutation in Hereditary Spastic Paraplegia (SPG10)

We have identified a missense mutation in the motor domain of the neuronal kinesin heavy chain gene KIF5A, in a family with hereditary spastic paraplegia. The mutation occurs in the family in which the SPG10 locus was originally identified, at an invariant asparagine residue that, when mutated in orthologous kinesin heavy chain motor proteins, prevents stimulation of the motor ATPase by microtubule-binding. Mutation of kinesin orthologues in various species leads to phenotypes resembling hereditary spastic paraplegia. The conventional kinesin motor powers intracellular movement of membranous organelles and other macromolecular cargo from the neuronal cell body to the distal tip of the axon. This finding suggests that the underlying pathology of SPG10 and possibly of other forms of hereditary spastic paraplegia may involve perturbation of neuronal anterograde (or retrograde) axoplasmic flow, leading to axonal degeneration, especially in the longest axons of the central nervous system.

Identification of Significant Association and Gene-Gene Interaction of GABA Receptor Subunit Genes in Autism

Autism is a common neurodevelopmental disorder with a significant genetic component. Existing research suggests that multiple genes contribute to autism and that epigenetic effects or gene-gene interactions are likely contributors to autism risk. However, these effects have not yet been identified. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, has been implicated in autism etiology. Fourteen known autosomal GABA receptor subunit genes were studied to look for the genes associated with autism and their possible interactions. Single-nucleotide polymorphisms (SNPs) were screened in the following genes: GABRG1, GABRA2, GABRA4, and GABRB1 on chromosome 4p12; GABRB2, GABRA6, GABRA1, GABRG2, and GABRP on 5q34-q35.1; GABRR1 and GABRR2 on 6q15; and GABRA5, GABRB3, and GABRG3 on 15q12. Intronic and/or silent mutation SNPs within each gene were analyzed in 470 white families with autism. Initially, SNPs were used in a family-based study for allelic association analysis--with the pedigree disequilibrium test and the family-based association test--and for genotypic and haplotypic association analysis--with the genotype-pedigree disequilibrium test (geno-PDT), the association in the presence of linkage (APL) test, and the haplotype family-based association test. Next, with the use of five refined independent marker sets, extended multifactor-dimensionality reduction (EMDR) analysis was employed to identify the models with locus joint effects, and interaction was further verified by conditional logistic regression. Significant allelic association was found for markers RS1912960 (in GABRA4; P = .01) and HCV9866022 (in GABRR2; P = .04). The geno-PDT found significant genotypic association for HCV8262334 (in GABRA2), RS1912960 and RS2280073 (in GABRA4), and RS2617503 and RS12187676 (in GABRB2). Consistent with the allelic and genotypic association results, EMDR confirmed the main effect at RS1912960 (in GABRA4). EMDR also identified a significant two-locus gene-gene effect model involving RS1912960 in GABRA4 and RS2351299 in GABRB1. Further support for this two-locus model came from both the multilocus geno-PDT and the APL test, which indicated a common genotype and haplotype combination positively associated with disease. Finally, these results were also consistent with the results from the conditional logistic regression, which confirmed the interaction between GABRA4 and GABRB1 (odds ratio = 2.9 for interaction term; P = .002). Through the convergence of all analyses, we conclude that GABRA4 is involved in the etiology of autism and potentially increases autism risk through interaction with GABRB1. These results support the hypothesis that GABA receptor subunit genes are involved in autism, most likely via complex gene-gene interactions.

Identification of MeCP2 mutations in a series of females with autistic disorder.

Rett disorder and autistic disorder are both pervasive developmental disorders. Recent studies indicate that at least 80% of Rett Disorder cases are caused by mutations in the methyl-CpG-binding protein 2 (MeCP2) gene. Since there is some phenotypic overlap between autistic disorder and Rett disorder, we analyzed 69 females clinically diagnosed with autistic disorder for the presence of mutations in the MeCP2 gene. Two autistic disorder females were found to have de novo mutations in the MeCP2 gene. These data provide additional evidence of variable expression in the Rett disorder phenotype and suggest MeCP2 testing may be warranted for females presenting with autistic disorder.

Epidemiologic and genetic aspects of spina bifida and other neural tube defects.

The worldwide incidence of neural tube defects (NTDs) ranges from 1.0 to 10.0 per 1,000 births with almost equal frequencies between two major categories: anencephaly and spina bifida (SB). Epidemiological studies have provided valuable insight for (a) researchers to identify nongenetic and genetic factors contributing to etiology, (b) public health officials to design and implement policies to prevent NTD pregnancies, and (c) individuals to take precautions to reduce the chance of having an NTD-affected pregnancy. Despite extensive research, our knowledge of the genetic etiology of human NTDs is limited. Although more than 200 small animal models with NTDs exist, most of these models do not replicate the human disease phenotype. Over a hundred candidate genes have been examined for risk association to human SB. The candidate genes studied include those important in folic acid metabolism, glucose metabolism, retinoid metabolism, and apoptosis. Many genes that regulate transcription in early embryogenesis and maintain planar cell polarity have also been tested as candidates. Additionally, genes identified through mouse models of NTDs have been explored as candidates. We do not know how many genes in the human genome may confer risk for NTDs in human. Less than 20% of the studied candidate genes have been determined to confer even a minor effect on risk association. Many studies have provided conflicting conclusions due to limitations in study design that potentially affect the power of statistical analysis. Future directions such as genomewide association studies (GWAS) and whole exome or even whole genome sequencing are discussed as possible avenues to identify genes that affect risk for human NTDs.

Mutations in the novel mitochondrial protein REEP1 cause hereditary spastic paraplegia type 31

Hereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous diseases that affect the upper motor neurons and their axonal projections. For the novel SPG31 locus on chromosome 2p12, we identified six different mutations in the receptor expression-enhancing protein 1 gene (REEP1). REEP1 mutations occurred in 6.5% of the patients with HSP in our sample, making it the third-most common HSP gene. We show that REEP1 is widely expressed and localizes to mitochondria, which underlines the importance of mitochondrial function in neurodegenerative disease.

Analysis of the RELN gene as a genetic risk factor for autism

Several genome-wide screens have indicated the presence of an autism susceptibility locus within the distal long arm of chromosome 7 (7q). Mapping at 7q22 within this region is the candidate gene reelin (RELN). RELN encodes a signaling protein that plays a pivotal role in the migration of several neuronal cell types and in the development of neural connections. Given these neurodevelopmental functions, recent reports that RELN influences genetic risk for autism are of significant interest. The total data set consists of 218 Caucasian families collected by our group, 85 Caucasian families collected by AGRE, and 68 Caucasian families collected at Tufts University were tested for genetic association of RELN variants to autism. Markers included five single-nucleotide polymorphisms (SNPs) and a repeat in the 5′-untranslated region (5′-UTR). Tests for association in Duke and AGRE families were also performed on four additional SNPs in the genes PSMC2 and ORC5L, which flank RELN. Family-based association analyses (PDT, Geno-PDT, and FBAT) were used to test for association of single-locus markers and multilocus haplotypes with autism. The most significant association identified from this combined data set was for the 5′-UTR repeat (PDT P-value=0.002). These analyses show the potential of RELN as an important contributor to genetic risk in autism.

Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus

Central corneal thickness (CCT) is associated with eye conditions including keratoconus and glaucoma. We performed a meta-analysis on >20,000 individuals in European and Asian populations that identified 16 new loci associated with CCT at genome-wide significance (P < 5 × 10−8). We further showed that 2 CCT-associated loci, FOXO1 and FNDC3B, conferred relatively large risks for keratoconus in 2 cohorts with 874 cases and 6,085 controls (rs2721051 near FOXO1 had odds ratio (OR) = 1.62, 95% confidence interval (CI) = 1.4–1.88, P = 2.7 × 10−10, and rs4894535 in FNDC3B had OR = 1.47, 95% CI = 1.29–1.68, P = 4.9 × 10−9). FNDC3B was also associated with primary open-angle glaucoma (P = 5.6 × 10−4; tested in 3 cohorts with 2,979 cases and 7,399 controls). Further analyses implicate the collagen and extracellular matrix pathways in the regulation of CCT.

Self-Regulation of Emotion, Functional Impairment, and Comorbidity Among Children With AD/HD

Objective: This study investigated the role of self-regulation of emotion in relation to functional impairment and comorbidity among children with and without AD/HD. Method: A total of 358 probands and their siblings participated in the study, with 74% of the sample participants affected by AD/HD. Parent-rated levels of emotional lability served as a marker for self-regulation of emotion. Results: Nearly half of the children affected by AD/HD displayed significantly elevated levels of emotional lability versus 15% of those without this disorder. Children with AD/HD also displayed significantly higher rates of functional impairment, comorbidity, and treatment service utilization. Emotional lability partially mediated the association between AD/HD status and these outcomes. Conclusion: Findings lent support to the notion that deficits in the self-regulation of emotion are evident in a substantial number of children with AD/HD and that these deficits play an important role in determining functional impairment and comorbidity outcomes.

Fetal hemoglobin in sickle cell anemia: genome-wide association studies suggest a regulatory region in the 5′ olfactory receptor gene cluster

In a genome-wide association study of 848 blacks with sickle cell anemia, we identified single nucleotide polymorphisms (SNPs) associated with fetal hemoglobin concentration. The most significant SNPs in a discovery sample were tested in a replication set of 305 blacks with sickle cell anemia and in subjects with hemoglobin E or beta thalassemia trait from Thailand and Hong Kong. A novel region on chromosome 11 containing olfactory receptor genes OR51B5 and OR51B6 was identified by 6 SNPs (lowest P = 4.7E-08) and validated in the replication set. An additional olfactory receptor gene, OR51B2, was identified by a novel SNP set enrichment analysis. Genome-wide association studies also validated a previously identified SNP (rs766432) in BCL11A, a gene known to affect fetal hemoglobin levels (P = 2.6E-21) and in Thailand and Hong Kong subjects. Elements within the olfactory receptor gene cluster might play a regulatory role in gamma-globin gene expression.

Multiple rare SAPAP3 missense variants in trichotillomania and OCD

Obsessive–compulsive disorder (OCD) and the spectrum of associated conditions, such as trichotillomania (TTM), Tourette syndrome and body dysmorphic disorder, affect about 2–4% of the world population. 1,2 Clinically OCD spectrum disorders are characterized by persistent intrusive thoughts (obsessions), repetitive actions (compulsions) and excessive anxiety. Although heritability studies in OCD have shown a 3–12 times increased risk for first-degree relatives and twin studies revealed higher concordance amongst monozygotic twins (80–90%) compared to dizygotic twins (47–50%), the identification of the underlying risk-conferring genetic variation by means of classic genetic association studies has proven to be difficult.3 Recently, it has been shown that mice deficient of the postsynaptic synapse-associated protein 90 (SAP90)/postsynaptic density-95 (PSD95)-associated protein 3 (SAPAP3, also known as Dlgap3) develop an OCD-like phenotype, which includes compulsive grooming and increased anxiety. Interestingly, the phenotype of Sapap3 knock-out mice can be rescued by administering selective serotonin reuptake inhibitors.4 We hypothesized that rare variants in the human orthologue SAPAP3 could contribute to disorders in the OCD spectrum. To test this, we resequenced SAPAP3 in three case populations, including 77 unrelated TTM probands collected at Duke University, 44 OCD with TTM probands from National Institute of Mental Health (NIMH), and 44 OCD cases without TTM from NIMH.5,6 Controls were 48 OCD spectrum-negative subjects from NIMH6 and a psychiatric comparison sample of 138 subjects screened for depression but not specifically for OCD from Duke University.7 A board-certified psychiatrist saw all patients and controls and diagnoses met Diagnostic and Statistical Manual of Mental Disorders, 4th edn. criteria. Samples were collected under approved institutional review board protocols. In 165 cases and 178 controls the complete coding region and flanking intronic sequence of SAPAP3 was resequenced using standard capillary sequencing methods (Applied Biosystems, Foster City, CA, USA). We detected seven novel nonsynonymous heterozygous variants, with all but A189V occurring only once (Table 1; Figure 1). Thus, in total, heterozygous SAPAP3 variants were present in 4.2% of diagnosed TTM/OCD patients, but only in 1.1% of controls (two changes in Duke control samples, with one developing depression subsequent to entry into the study). The majority of changes presented missense mutations; one variant was an in-frame insertion of five amino acids, A148insGPAGA. In silico analysis of the missense variants applying PMut and PolyPhen predicted two, or three, respectively, variants as of functional relevance (Table 1). The remaining polymorphisms were considered benign, including the two changes detected in controls. Further, we genotyped 6 of the identified variants in the TTM/OCD subjects in an additional sample of 281 OCD cases and in 751 general population controls.6 R13C and P606T were found in one control each, whereas A189V was present in three controls. This suggests that these specific variants are not by themselves disease-causing abnormalities, but still leaves open the possibility that an aggregate of susceptibility variants may prove contributory to disease, as suggested for some other disorders including autism as well as OCD. The combined analyses of 2766 alleles showed that all changes are very rare, with minor allele frequencies between 0.00036 (T523K, K910R) and 0.002 (A189V). Figure 1 Identified rare nonsynonymous polymorphisms in synapse-associated protein 90/postsynaptic density-95-associated protein 3 (SAPAP3). (a) Schematic of SAPAP3, which consists of 10 coding exons (blue boxes). Seven rare changes were identified in trichotillomania ... Table 1 Identified rare variants in SAPAP3 and predicted functional relevance Available pedigrees from TTM/OCD mutation carriers were enriched for a diverse set of psychiatric conditions, including panic disorder, attention deficit hyperactivity disorder (ADHD), depression, bipolar disorder and substance abuse as well as OCD spectrum disorders (details are given in Supplementary Figure 1 and Supplementary Table 1). This situation is quite typical for psychiatric genetic studies and complicates allele segregation studies. Cosegregation of genotype and phenotype is also confounded by phenotypic penetrance rates, limited psychometric instruments and assortative mating. Thus, we consider it more significant to study the combined mutation load of SAPAP3 comparing cases to controls. Similar approaches were recently adopted by other studies.8 We observed a significant case–control association in our moderately sized sample (Fisher’s one-sided exact test P = 0.045). With generally still limited abilities to determine functional consequences of genetic variants, we speculate that the predicted moderate functional consequences (Table 1) are not detrimental for protein function but rather increase susceptibility for OCD spectrum behavior, possibly through permissive or epistatic interactions with additional genetic and environmental factors. A recent study estimated that up to 70% of low-frequency missense alleles in humans have mildly deleterious effects.9 The excess of rare mildly deleterious variants in any OCD risk gene could be promoted by an inefficient evolutionary selection against OCD risk alleles, which is supported by the high OCD spectrum frequency of 2–4% in the population, early disease onset and normal reproductive fitness. In summary, on the background of an intriguing Sapap3-OCD mouse model we suggest that the present data support a role for SAPAP3 in TTM and OCD. Expansion of our approach and modeling of rare genetic variants in SAPAP3 will be essential to further test this hypothesis.