Spectrum of mutations in MMACHC, allelic expression, and evidence for genotype-phenotype correlations.

Methylmalonic aciduria and homocystinuria, cblC type, is a rare disorder of intracellular vitamin B(12) (cobalamin [Cbl]) metabolism caused by mutations in the MMACHC gene. MMACHC was sequenced from the gDNA of 118 cblC individuals. Eleven novel mutations were identified, as well as 23 mutations that were observed previously. Six sequence variants capture haplotype diversity in individuals across the MMACHC interval. Genotype-phenotype correlations of common mutations were apparent; individuals with c.394C>T tend to present with late-onset disease whereas patients with c.331C>T and c.271dupA tend to present in infancy. Other missense variants were also associated with late- or early-onset disease. Allelic expression analysis was carried out on human cblC fibroblasts compound heterozygous for different combinations of mutations including c.271dupA, c.331C>T, c.394C>T, and c.482G>A. The early-onset c.271dupA mutation was consistently underexpressed when compared to control alleles and the late-onset c.394C>T and c.482G>A mutations. The early-onset c.331C>T mutation was also underexpressed when compared to control alleles and the c.394C>T mutation. Levels of MMACHC mRNA transcript in cell lines homozygous for c.271dupA, c.331C>T, and c.394C>T were assessed using quantitative real-time RT-PCR. Cell lines homozygous for the late onset c.394C>T mutation had significantly higher levels of transcript when compared to cell lines homozygous for the early-onset mutations. Differential or preferential MMACHC transcript levels may provide a clue as to why individuals carrying c.394C>T generally present later in life.

Targeted screening of cis-regulatory variation in human haplotypes.

Regulatory cis-acting variants account for a large proportion of gene expression variability in populations. Cis-acting differences can be specifically measured by comparing relative levels of allelic transcripts within a sample. Allelic expression (AE) mapping for cis-regulatory variant discovery has been hindered by the requirements of having informative or heterozygous single nucleotide polymorphisms (SNPs) within genes in order to assign the allelic origin of each transcript. In this study we have developed an approach to systematically screen for heritable cis-variants in common human haplotypes across >1,000 genes. In order to achieve the highest level of information per haplotype studied, we carried out allelic expression measurements by using both intronic and exonic SNPs in primary transcripts. We used a novel RNA pooling strategy in immortalized lymphoblastoid cell lines (LCLs) and primary human osteoblast cell lines (HObs) to allow for high-throughput AE. Screening hits from RNA pools were further validated by performing allelic expression mapping in individual samples. Our results indicate that >10% of expressed genes in human LCLs show genotype-linked AE. In addition, we have validated cis-acting variants in over 20 genes linked with common disease susceptibility in recent genome-wide studies. More generally, our results indicate that RNA pooling coupled with AE read-out by second generation sequencing or by other methods provides a high-throughput tool for cataloging the impact of common noncoding variants in the human genome.

Differential allelic expression in the human genome: a robust approach to identify genetic and epigenetic cis-acting mechanisms regulating gene expression.

The recent development of whole genome association studies has lead to the robust identification of several loci involved in different common human diseases. Interestingly, some of the strongest signals of association observed in these studies arise from non-coding regions located in very large introns or far away from any annotated genes, raising the possibility that these regions are involved in the etiology of the disease through some unidentified regulatory mechanisms. These findings highlight the importance of better understanding the mechanisms leading to inter-individual differences in gene expression in humans. Most of the existing approaches developed to identify common regulatory polymorphisms are based on linkage/association mapping of gene expression to genotypes. However, these methods have some limitations, notably their cost and the requirement of extensive genotyping information from all the individuals studied which limits their applications to a specific cohort or tissue. Here we describe a robust and high-throughput method to directly measure differences in allelic expression for a large number of genes using the Illumina Allele-Specific Expression BeadArray platform and quantitative sequencing of RT-PCR products. We show that this approach allows reliable identification of differences in the relative expression of the two alleles larger than 1.5-fold (i.e., deviations of the allelic ratio larger than 60:40) and offers several advantages over the mapping of total gene expression, particularly for studying humans or outbred populations. Our analysis of more than 80 individuals for 2,968 SNPs located in 1,380 genes confirms that differential allelic expression is a widespread phenomenon affecting the expression of 20% of human genes and shows that our method successfully captures expression differences resulting from both genetic and epigenetic cis-acting mechanisms.

Systematic assessment of the human osteoblast transcriptome in resting and induced primary cells.

Osteoblasts are key players in bone remodeling. The accessibility of human primary osteoblast-like cells (HObs) from bone explants makes them a lucrative model for studying molecular physiology of bone turnover, for discovering novel anabolic therapeutics, and for mesenchymal cell biology in general. Relatively little is known about resting and dynamic expression profiles of HObs, and to date no studies have been conducted to systematically assess the osteoblast transcriptome. The aim of this study was to characterize HObs and investigate signaling cascades and gene networks with genomewide expression profiling in resting and bone morphogenic protein (BMP)-2- and dexamethasone-induced cells. In addition, we compared HOb gene expression with publicly available samples from the Gene Expression Omnibus. Our data show a vast number of genes and networks expressed predominantly in HObs compared with closely related cells such as fibroblasts or chondrocytes. For instance, genes in the insulin-like growth factor (IGF) signaling pathway were enriched in HObs (P = 0.003) and included the binding proteins (IGFBP-1, -2, -5) and IGF-II and its receptor. Another HOb-specific expression pattern included leptin and its receptor (P < 10(-8)). Furthermore, after stimulation of HObs with BMP-2 or dexamethasone, the expression of several interesting genes and pathways was observed. For instance, our data support the role of peripheral leptin signaling in bone cell function. In conclusion, we provide the landscape of tissue-specific and dynamic gene expression in HObs. This resource will allow utilization of osteoblasts as a model to study specific gene networks and gene families related to human bone physiology and diseases.

Genome-wide analysis of transcript isoform variation in humans.

We have performed a genome-wide analysis of common genetic variation controlling differential expression of transcript isoforms in the CEU HapMap population using a comprehensive exon tiling microarray covering 17,897 genes. We detected 324 genes with significant associations between flanking SNPs and transcript levels. Of these, 39% reflected changes in whole gene expression and 55% reflected transcript isoform changes such as splicing variants (exon skipping, alternative splice site use, intron retention), differential 5' UTR (initiation of transcription) use, and differential 3' UTR (alternative polyadenylation) use. These results demonstrate that the regulatory effects of genetic variation in a normal human population are far more complex than previously observed. This extra layer of molecular diversity may account for natural phenotypic variation and disease susceptibility.

Heritability of alternative splicing in the human genome.

Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genome-wide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblastoid cell lines (LCLs) derived from the CEPH HapMap population. We show the identification of transcripts containing sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. A number of novel alternative splicing events with no previous annotations in either the RefSeq and EST databases were identified, indicating that we are able to discover de novo splicing events. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes: OAS1, CAST, and CRTAP. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. In one candidate, we identified a regulatory polymorphism that disrupts a 5' splice site of an exon in the CAST gene, resulting in its exclusion in the mutant allele. This report illustrates that our approach can detect both annotated and novel alternatively spliced variants, and that such variation among individuals is heritable and genetically controlled.

Vitamin D receptor 3' haplotypes are unequally expressed in primary human bone cells and associated with increased fracture risk: the MrOS Study in Sweden and Hong Kong.

UNLABELLED: The VDR is a prime candidate gene for osteoporosis. Here, we studied three common VDR haplotypes in relation to bone phenotypes in 5014 participants of the global MrOS Study. We also studied the relative expression of the haplotypes in human bone cells. One haplotype was associated with increased fracture risk and differently expressed in primary human bone cells. INTRODUCTION: Vitamin D plays an essential role in skeletal metabolism by binding to its nuclear steroid receptor, the vitamin D receptor (VDR). The heritability of BMD is well established, and the VDR gene is considered a prime candidate suggested to partially account for genetically controlled BMD variance in the population. MATERIALS AND METHODS: Here, we reconstructed common haplotypes in the VDR 3' untranslated region (UTR) and studied the association to BMD and risk of vertebral fractures in elderly men from Sweden (n = 3014) and Hong Kong (n = 2000), all participants of the global MrOS Study. To assess any functional implications of the VDR polymorphisms, we studied allele-specific expressions of the different VDR 3' UTR haplotypes in the normal chromosomal context of 70 unrelated human trabecular bone samples. This was performed by quantitative genotyping of coding polymorphisms in RNA samples and in corresponding DNA samples isolated from the bone samples. RESULTS: Three major haplotypes were reconstructed and in agreement with the previously well-defined baT, BAt, and bAT haplotypes, herein denoted Hap1, Hap2, and Hap3. The Hap1 haplotype was independently associated with increased risk of vertebral fractures in Swedish men (OR, 1.655; 95% CI, 1.146-2.391; p < 0.01) and with lower lumbar spine BMD in elderly men from Sweden (p < 0.01) and Hong Kong (p < 0.05). The VDR gene was also shown to exhibit a 3' UTR haplotype dependent allelic imbalance, indicating that the VDR Hap1 allele was overexpressed in human trabecular bone samples. CONCLUSIONS: The results indicate that the relatively overexpressed VDR Hap1 haplotype could be considered a risk allele for osteoporosis regardless of ethnicity.

Mapping common regulatory variants to human haplotypes.

Inter-individual variation in gene expression has proven to be in part governed by genetic determinants, which may be trans- or cis-acting. The underlying cause of cis-acting regulatory variation has been identified in only a handful of the hundreds of genes shown to display differential allelic expression. In this report, we describe a systematic effort to map common cis-acting variants in 64 genes, using association methods in HapMap samples. We identified 16 loci (25%), each of which harbors common haplotypes that affect total expression of a gene, and a further 17 loci (27%) with evidence of haplotypes affecting relative allelic expression in heterozygote samples. Our survey suggests that detailed mapping of allele-specific in vivo expression will provide a rich source of regulatory SNPs or haplotypes that should be given high priority in association studies of human phenotypes.

Survey of allelic expression using EST mining.

Cis-acting allelic variation in gene regulation is a source of phenotypic variation. Consequently, recent studies have experimentally screened human genes in an attempt to initiate a catalog of genes possessing cis-acting variants. In this study, we use human EST data in dbEST as the source of allelic expression data, and the HapMap database to provide expected allele frequencies in human populations. We demonstrate a greater concordance of allele frequencies estimated from human ESTs in dbEST with those derived from the CEPH HapMap sample representing Caucasians from northern and western Europe, than population samples obtained in Asia and Africa. Deviations between allele frequencies observed in EST databases and the ones obtained from the CEPH HapMap samples may result from common heritable cis-acting variants altering the relative allele distribution in RNA. We provide in silico as well as experimental evidence that this strategy does allow significant enrichment of genes harboring common heritable cis-acting polymorphisms in linkage disequilibrium with expressed alleles.

A survey of genes differentially expressed in subcutaneous and visceral adipose tissue in men.

Adipose tissue located within the abdominal cavity has been suggested to be functionally and metabolically distinct from that of the subcutaneous compartment. These differences could play a role in obesity-related complications. The aim of this study was to compare gene expression profiles of subcutaneous and visceral adipose tissues of 10 nondiabetic, normolipidemic obese men. Affymetrix human U133A arrays (10 arrays for subcutaneous fat samples and 10 arrays for visceral fat samples) were used. Differential gene expression was confirmed by real-time polymerase chain reaction in a subset of genes. A total of 5894 transcripts were detected in both depots in all 10 subjects, and 409 transcripts representing 347 encoded genes were differentially expressed. Of these, 131 genes were expressed at higher levels in subcutaneous adipose tissue, and 216 were expressed more abundantly in visceral fat. Differentially expressed profiles included genes of the Wnt signaling pathway, as well as CEPBA and HOX genes. In addition, genes involved in lipolytic stimuli and cytokine secretion were differentially expressed. The identification of a consistent and rather uniform pattern of differentially expressed genes between the two fat depots using multiple array replicates (10 arrays per fat compartment) generated new perspectives for future research on regional differences in adipose tissue biology.

A survey of genetic and epigenetic variation affecting human gene expression.

The identification of human sequence polymorphisms that regulate gene expression is key to understanding human genetic diseases. We report a survey of human genes that demonstrate allelic differences in gene expression, reflecting the presence of putative allele-specific cis-acting factors of either genetic or epigenetic nature. The expression of allelic transcripts in heterozygous samples is assessed directly by relative quantitation of intragenic marker alleles in messenger or heteronuclear RNA derived from cells or tissues. This survey used 193 single-nucleotide polymorphisms (SNPs) from 129 genes expressed in lymphoblastoid cell lines, to identify 23 genes (18%) with common allele-specific transcripts whose expression deviated from the expected equimolar ratio. A subset of these deviations, or "allelic imbalances," can be observed in multiple samples derived from reference CEPH ("Centre d'Etude du Polymorphisme Humain") pedigrees and demonstrate a spectrum of patterns of transmission, including cosegregation of allelic skewing across generations compatible with Mendelian inheritance as well as random monoallelic expression for three genes (IL1A, HTR2A, and FGB). Additional studies for BTN3A2 provide evidence of SNPs and haplotypes in complete linkage disequilibrium with high- and low-expressing transcripts. The pipeline described herein offers tools for efficient identification and characterization of allelic expression allowing identification of regulatory sequence variants as well as epigenetic variation affecting human gene expression.