A rare WNT1 missense variant overrepresented in ASD leads to increased Wnt signal pathway activation.

Wnt signaling, which encompasses multiple biochemical pathways that regulate neural development downstream of extracellular Wnt glycoprotein ligands, has been suggested to contribute to major psychiatric disorders including autism spectrum disorders (ASD). We used next-generation sequencing and Sequenom genotyping technologies to resequence 10 Wnt signaling pathway genes in 198 ASD patients and 240 matched controls. Results for single-nucleotide polymorphisms (SNPs) of interest were confirmed in a second set of 91 ASD and 144 control samples. We found a significantly increased burden of extremely rare missense variants predicted to be deleterious by PolyPhen-2, distributed across seven genes in the ASD sample (3.5% in ASD vs 0.8% in controls; Fisher's exact test, odds ratio (OR)=4.37, P=0.04). We also found a missense variant in WNT1 (S88R) that was overrepresented in the ASD sample (8 A/T in 267 ASD (minor allele frequency (MAF)=1.69%) vs 1 A/T in 377 controls (MAF=0.13%), OR=13.0, Fisher's exact test, P=0.0048; OR=8.2 and P=0.053 after correction for population stratification). Functional analysis revealed that WNT1-S88R is more active than wild-type WNT1 in assays for the Wnt/ß-catenin signaling pathway. Our findings of a higher burden in ASD of rare missense variants distributed across 7 of 10 Wnt signaling pathway genes tested, and of a functional variant at the WNT1 locus associated with ASD, support that dysfunction of this pathway contributes to ASD susceptibility. Given recent findings of common molecular mechanisms in ASD, schizophrenia and affective disorders, these loci merit scrutiny in other psychiatric conditions as well.

A screening study of drug-drug interactions in cerivastatin users: an adverse effect of clopidogrel.

An analysis of a case-control study of rhabdomyolysis was conducted to screen for previously unrecognized cytochrome P450 enzyme (CYP) 2C8 inhibitors that may cause other clinically important drug-drug interactions. Medication use in cases of rhabdomyolysis using cerivastatin (n = 72) was compared with that in controls using atorvastatin (n = 287) for the period 1998-2001. The use of clopidogrel was strongly associated with rhabdomyolysis (odds ratio (OR) 29.6; 95% confidence interval (CI), 6.1-143). In a replication effort that used the US Food and Drug Administration (FDA) Adverse Event Reporting System (AERS), it was found that clopidogrel was used more commonly in patients with rhabdomyolysis receiving cerivastatin (17%) than in those receiving atorvastatin (0%, OR infinity; 95% CI = 5.2-infinity). Several medications were tested in vitro for their potential to cause drug-drug interactions. Clopidogrel, rosiglitazone, and montelukast were the most potent inhibitors of cerivastatin metabolism. Clopidogrel and its metabolites also inhibited cerivastatin metabolism in human hepatocytes. These epidemiological and in vitro findings suggest that clopidogrel may cause clinically important, dose-dependent drug-drug interactions with other medications metabolized by CYP2C8.

Functional characterization of liver enhancers that regulate drug-associated transporters.

Little is known about how genetic variations in enhancers influence drug response. In this study, we investigated whether nucleotide variations in enhancers that regulate drug transporters can alter their expression levels. Using comparative genomics and liver-specific transcription factor binding site (TFBS) analyses, we identified evolutionary conserved regions (ECRs) surrounding nine liver membrane transporters that interact with commonly used pharmaceuticals. The top 50 ECRs were screened for enhancer activity in vivo, of which five--located around ABCB11, SLC10A1, SLCO1B1, SLCO1A2, and SLC47A1--exhibited significant enhancer activity. Common variants identified in a large ethnically diverse cohort (n = 272) were assayed for differential enhancer activity, and three variants were found to have significant effects on reporter activity as compared with the reference allele. In addition, one variant was associated with reduced SLCO1A2 mRNA expression levels in human liver tissues, and another was associated with increased methotrexate (MTX) clearance in patients. This work provides a general model for the rapid characterization of liver enhancers and identifies associations between enhancer variants and drug response.

Sequencing of TNFAIP3 and association of variants with multiple autoimmune diseases.

The TNFAIP3 locus at 6q23, encoding A20, has been associated with multiple autoimmune diseases (AIDs). In this study, we sequence the coding portions of the gene to identify contributing causal polymorphisms that may explain some of the observed associations. A collection of 123 individuals from the Multiple Autoimmune Disease Genetics Consortium (MADGC) collection, each with multiple AIDs (mean=2.2 confirmed diagnoses), and 397 unrelated healthy controls were used for initial sequencing. A total of 32 polymorphisms were identified in the sequencing experiments, including 16 novel and 11 coding variants. Association testing in the entire MADGC collection (1,008 Caucasians with one or more AIDs and 770 unaffected family controls) revealed association of a novel intronic insertion-deletion polymorphism with rheumatoid arthritis (RA) (odds ratio (OR)=2.48, P=0.041). Genotyping of the most common coding polymorphism, rs2230926, in the MADGC collection and additional control individuals revealed a significant association with Sjögren's syndrome (OR=3.38, P=0.038), Crohn's disease (OR=2.25, P=0.041), psoriasis (OR=0.037, P=0.036) and RA (OR=1.9, P=0.025). Finally, haplotype and additional testing of polymorphisms revealed that cases were enriched for 5' and 3' untranslated region variants (one-sided P-value=0.04), but not specifically for common (>2% minor allele frequency), rare, exonic, intronic, non-synonymous or synonymous variants.

Heat-inducible translationally controlled tumor protein of Trichinella pseudospiralis: cloning and regulation of gene expression.

To elucidate the mechanism of inducing translationally controlled tumor protein (TCTP) in stress adaptation of adenophorean nematodes, the complete coding sequence of TCTP of the infective-stage larvae of Trichinella pseudospiralis was characterized. Two cDNA clones with different 3' untranslated region were identified. Tp-TCTP contained an open reading frame of 534 bp encoding 177 residues. The gene with five introns was expressed as histidine-tagged fusion protein having a molecular mass of 17.5 kDa. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that TCTP RNA was not accumulated when the infective-stage larvae were heat-shocked for 1 h at 45 or 60 degrees C. Using enzyme-linked immunosorbent assay and antiserum against the fusion protein, the expression of TCTP was found to be up-regulated at the translational level. The data suggest that translational regulation of TCTP may play an important role in the early heat-stress adaptation of the trichinellid. Cluster analysis demonstrated that the TCTP sequence of T. pseudospiralis is closely related to that of T. spiralis, but is diverged from the secernentean species.

ATP2A2 mutations in Darier's disease and their relationship to neuropsychiatric phenotypes.

Darier's disease (DD) is a rare, dominantly inherited disorder that affects the skin producing a variety of types of lesion. Close examination of lesional DD skin shows the presence of abnormal keratinization (epidermal differentiation) and acantholysis (loss of cohesion) of keratinocytes. A number of clinical studies have described the co-occurrence of various neurological and psychiatric symptoms with DD, including mood disorders, epilepsy, mental retardation and a slowly progressive encephalopathy. A single locus for DD has been mapped to chromosome 12q23-q24.1, and a variety of missense, nonsense, frameshift and splicing mutations in the ATP2A2 gene have been described recently in families with DD. This gene encodes the sarcoplasmic/endoplasmic reticulum calcium-pumping ATPase SERCA2, which has a central role in intra-cellular calcium signalling. In this study, we performed mutation analysis on ATP2A2 in 19 unrelated DD patients, of whom 10 had neuropsychiatric phenotypes. We identified and verified 17 novel mutations predicting conservative and non-conservative amino acid changes, potential premature translation terminations and potential altered splicing. Our findings confirm that mutations in ATP2A2 are associated with DD. In neuropsychiatric cases, there was a non-random clustering of mutations in the 3' end of the gene ( P = 0.01), and a predominance of the missense type (70% versus 38% in DD patients). This supports the hypothesis that the DD gene has pleiotropic effects in brain and that mutations in SERCA2 are implicated in the pathogenesis of neuropsychiatric disorders.

Efficient approach to unique single-nucleotide polymorphism discovery.

Single-nucleotide polymorphisms (SNPs) are the most frequently found DNA sequence variations in the human genome. It has been argued that a dense set of SNP markers can be used to identify genetic factors associated with complex disease traits. Because all high-throughput genotyping methods require precise sequence knowledge of the SNPs, any SNP discovery approach must involve both the determination of DNA sequence and allele frequencies. Furthermore, high-throughput genotyping also requires a genomic DNA amplification step, making it necessary to develop sequence-tagged sites (STSs) that amplify only the DNA fragment containing the SNP and nothing else from the rest of the genome. In this report, we demonstrate the utility of a SNP-screening approach that yields the DNA sequence and allele frequency information while screening out duplications with minimal cost and effort. Our approach is based on the use of a homozygous complete hydatidiform mole (CHM) as the reference. With this homozygous reference, one can identify and estimate the allele frequencies of common SNPs with a pooled DNA-sequencing approach (rather than having to sequence numerous individuals as is commonly done). More importantly, the CHM reference is preferable to a single individual reference because it reveals readily any duplicated regions of the genome amplified by the PCR assay before the duplicated sequences are found in GenBank. This approach reduces the cost of SNP discovery by 60% and eliminates the costly development of SNP markers that cannot be amplified uniquely from the genome.

Fluorescence energy transfer detection as a homogeneous DNA diagnostic method.

A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.

The homozygous complete hydatidiform mole: a unique resource for genome studies.

The most frequent type of complete hydatidiform mole is a 46, XX homozygote formed by the fertilization of an empty ovum by a single haploid sperm that later duplicates its chromosomes to give a diploid tumor. The homozygous nature of these complete hydatidiform moles makes them unique resources for human genome studies. They can serve as homozygous controls in the development of single nucleotide polymorphism (SNP) markers and provide a way to obtain long-range haplotypes that are useful in population studies. The use of a homozygous control makes it possible to estimate the allele frequencies of the SNP markers in any population by sequencing pooled DNA samples. In this report, we present evidence of homozygosity of a complete hydatidiform mole using 20 diallelic markers distributed across the genome. Furthermore, its usefulness as a homozygous control in SNP development and as a resource for long-range haplotype determination is demonstrated using 11 newly discovered loci in the BRCA2 region on chromosome 13q12-q13.