Strong bias in the location of functional promoter polymorphisms.

A considerable proportion of heritable human phenotypic variation is thought to result from altered gene expression. Unfortunately, it is currently impossible to use bioinformatic analysis to discriminate between DNA sequence variants that are likely to influence gene expression and those that are not. In an attempt to define some of the characteristics of promoter polymorphisms with functional effects on gene expression, we examined 674 haplotypes representing 247 unique gene promoters using a standardized reporter gene assay system. Sequence variants that altered gene expression by 1.5-fold or more were strongly biased toward a location in the core and proximal promoter regions, 50% being within the first 100 bases 5' to the transcription start site. No bias was seen in the allele frequencies of functional and nonfunctional sequence variants. Only 33% of the functional variants were found in known consensus transcription factor binding sequences or motifs, which suggests that either there are many unknown transcription factor binding motifs or other, unknown mechanisms are involved. The genes with functional polymorphisms that are reported here for the first time include AGTRL2, CAT, CHRNA5, CTSG, CYP2D6, DLD, ERCC1, GABRA1, GABRP, HNRPH3, HIP1, IGKV1-9, KCNJ15, KCNK6, KLK1, MSMB, MYOC, NPY2R, NOTCH4, ORM2, PEDF, PTPRCAP, ST16 (IL24), SULT1A1, and TSHR.

A high proportion of polymorphisms in the promoters of brain expressed genes influences transcriptional activity.

There is increasing interest in the possibility that polymorphisms affecting gene expression are responsible for a significant proportion of heritable human phenotypic variation, including human disease. We have sought to determine if polymorphisms in the promoters of brain expressed genes are commonly functional. We screened for polymorphism 56 genes previously reported to be differentially expressed in the brains of schizophrenics [Y. Hakak, J.R. Walker, C. Li, W.H. Wong, K.L. Davis, J.D. Buxbaum, V. Haroutunian, A.A. Fienberg, Genome-wide expression analysis reveals dysregulation of myelination-related genes in chronic schizophrenia. Proc. Natl. Acad. Sci. 98 (2001) 4746-4751.]. We found 60 variants distributed across 31 of the genes. A total of 77 haplotypes representing 28 different putative promoters were analyzed in a reporter gene assay in two cell lines. Of a total of 54 sequence variants represented in the haplotypes, 12 (or around 22%) were functional according to a highly conservative definition. These were found in the promoters of eight genes: NPY, PCSK1, NEFL, KIAA0513, LMO4, HSPA1B, TF and MDH1. We therefore estimate that around 20-25% of promoter polymorphisms in brain expressed genes are functional, and this is likely to be an underestimate. Our data therefore provide for the first time empirical evidence that promoter element polymorphisms, at least in brain expressed genes, should be afforded a high priority for molecular genetic studies.

Low gene expression conferred by association of an allele of the 5-HT2C receptor gene with antipsychotic-induced weight gain.

OBJECTIVE: Association has been reported between the C allele of a -759C/T polymorphism in the promoter of the 5-HT2C receptor gene (HTR2C) and antipsychotic-induced weight gain, suggesting that polymorphic HTR2C expression influences this phenotype. The authors tested this polymorphism, and other promoter variants, for effects on HTR2C transcription. METHOD: Six HTR2C promoter haplotypes constructed from four polymorphisms were cloned into a luciferase reporter gene plasmid. Their transcriptional activities were then compared in two human cell lines. RESULTS: All haplotypes containing the -759C allele showed less transcriptional activity than haplotypes containing the -759T allele. The A allele of a -997G/A polymorphism was also associated with reduced expression. CONCLUSIONS: These findings suggest that the -759C allele is functional and results in relative underexpression of HTR2C. Reduced expression of HTR2C mRNA may underlie vulnerability to weight gain following antipsychotic treatment.

Functional analysis of polymorphisms in the promoter regions of genes on 22q11.

Segmental aneusomy, which includes chromosome 22 deletion syndrome (del(22)(q11.2q11.2)), has been associated with DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), conotruncal anomaly face (CAF) syndrome, cat-eye syndrome (CES), der(22) syndrome, and duplication of the del(22)(q11.2q11.2) syndrome's typically deleted region. Adults with del(22)(q11.2q11.2) may develop psychiatric illnesses, including schizophrenia, schizoaffective disorder, and bipolar disorder, suggesting that lower gene dosage leads to a predisposition to these illnesses. In a bid to identify important regulatory polymorphisms (SNPs) that may emulate changes in gene dosage of the genes within the common deletion, we have analyzed the promoter region of 47 genes (44 of which encode a protein with known function) encoding proteins in and around 22q11 for sequence variants. A total of 33 of the promoters contained polymorphisms. Of those, 25 were cloned into a reporter gene vector, pGL3. The relative ability of each promoter haplotype to promote transcription of the luciferase gene was tested in each of two human cell lines (HEK293t and TE671), using a cotransfected CMV-SPAP plasmid as an internal control. Five genes (PRODH, DGCR14, GSTT2, SERPIND1, and a gene tentatively called DKFZP434P211) showed activity differences between haplotypes of greater than 1.5-fold. Of those, PRODH, which encodes proline dehydrogenase, has previously been highlighted in relation to schizophrenia, and the functional promoter polymorphism reported here may be involved in pathogenic mechanisms.

The human hyaluronan synthase genes: genomic structures, proximal promoters and polymorphic microsatellite markers.

The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5' cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3' end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5'-untranslated region (5'-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.

Promoter polymorphisms in glutathione-S-transferase genes affect transcription.

The glutathione-S-transferases are a group of enzymes that play a major role in detoxification and defence against toxic, carcinogenic and other compounds. We analysed the proximal promoters of 14 genes encoding glutathione-S-transferase for polymorphism. Ten of the promoters contained sequence variants, nine of which we were able to clone into a reporter gene vector, pGL3. The relative ability of each haplotype to promote transcription of the luciferase gene was tested in each of two human cell lines (HEK293t and TE671) using a cotransfected CMV-SEAP plasmid as a control. Four genes (GSTA1, GSTA2, GSTM4 and GSTT2) showed activity differences greater than 1.5-fold between haplotypes, and a fifth gene (MGST1) showed a 1.4-fold difference. The promoter sequence variants in these genes may therefore play a role in human variation, susceptibility to diseases and the effects of drugs.

Functional analysis of human promoter polymorphisms.

The potential importance of gene regulation in disease susceptibility and other inherited phenotypes has been underlined by the observation that the human genome contains fewer protein coding genes than expected. Promoter sequences are potential sources of polymorphism affecting gene expression, although to date there are no large-scale systematic studies that have determined how frequently such variants occur. We have used denaturing high performance liquid chromatography to screen the first 500 bp of the 5' flanking region of 170 opportunistically selected genes identified from the Eukaryotic Promoter Database (EPD) for common polymorphisms. Using a screening set of 16 chromosomes, single-nucleotide polymorphisms were found in approximately 35% of genes. It was attempted to clone each of these promoters into a T-vector constructed from the reporter gene vector pGL3. The relative ability of each promoter haplotype to promote transcription of the luciferase gene was tested in each of three human cell lines (HEK293, JEG and TE671) using a co-transfected SEAP-CMV plasmid as a control. The findings suggest that around a third of promoter variants may alter gene expression to a functionally relevant extent.

Identification and analysis of the promoter region of the human hyaluronan synthase 2 gene.

Hyaluronan (HA) is a linear glycosaminoglycan of the vertebrate extracellular matrix that is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2 and -3. The regulation of HA synthesis has been implicated in a variety of extracellular matrix-mediated and pathological processes, including renal fibrosis. We have recently described the genomic structures of each of the human HAS genes. In the present study, we analyzed the HAS2 promoter region. In 5'-rapid amplification of cDNA ends analysis of purified mRNA from human renal epithelial proximal tubular cells, we detected an extended sequence for HAS2 exon 1, relocating the transcription initiation site 130 nucleotides upstream of the reference HAS2 mRNA sequence, GenBank accession number NM_005328. A luciferase reporter gene assay of nested fragments spanning the 5' terminus of NM_005328 demonstrated the constitutive promoter activity of sequences directly upstream of the repositioned transcription initiation site but not of the newly designated exonic nucleotides. Using reverse transcription-PCR, expression of this extended HAS2 mRNA was demonstrated in a variety of human cell types, and orthologous sequences were detected in mouse and rat kidney. Alignment of human, murine, and equine genomic DNA sequences upstream of the repositioned HAS2 exon 1 provided evidence for the evolutionary conservation of specific transcription factor binding sites. The location of the HAS2 promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts as well as in developmental models in which HAS2 null animals have an embryonic lethal phenotype.