Combinatorial interaction between two human serotonin transporter gene variable number tandem repeats and their regulation by CTCF

Two distinct variable number tandem repeats (VNTRs) within the human serotonin transporter gene ( SLC6A4 ) have been implicated as predisposing factors for CNS disorders. The linked polymorphic region in the 5'-promoter exists as short ( s ) and long ( l ) alleles of a 22 or 23 bp elements. The second within intron 2 (Stin2) exists as three variants containing 9, 10 or 12 copies of a 16 or 17 bp element. These VNTRs, individually or in combination, supported differential reporter gene expression in rat neonate prefrontal cortical cultures. The level of reporter gene activity from the dual VNTR constructs indicated combinatorial action between the two domains. Chromatin immunoprecipitation demonstrated that both these VNTR domains can bind the CCCTC-binding factor and this correlated with the ability of exogenously supplied CCCTC-binding factor to modulate the expression supported by these reporter gene constructs. We suggest that the potential for interaction between multiple polymorphic domains should be incorporated into genetic association studies.     

Polymerase chain reaction amplification of two polymorphic simple repeat sequences within the von Willebrand factor gene: application to family studies in von Willebrand disease

Summary We have used the polymerase chain reaction to amplify two variable number of tandem repeats (VNTRs) within a region of repetitive DNA located in intron 40 of the von Willebrand factor (vWf) gene. Heterozygosity for VNTR I was observed in 30 out of 39 normal unrelated individuals tested (77%), and for VNTR II in 29 out of 44 (66%) similar individuals. Family studies were carried out on 11 kindreds with von Willebrand disease (vWD). Ten of these families were found to be informative for one or other of the VNTRs or for a combination of data from both VNTRs. This method can be used for antenatal diagnosis and for carrier diagnosis in recessive forms of vWD. It is also useful for tracking the gene associated with vWD in type I families where there may be one or more individuals with a phenotypically uncertain diagnosis.     

IL1RN genetic variations and risk of IPF: a meta-analysis and mRNA expression study

Idiopathic pulmonary fibrosis (IPF) is a rare and devastating lung disease of unknown aetiology. Genetic variations in the IL1RN gene, encoding the interleukin-1 receptor antagonist (IL-1Ra), have been associated with IPF susceptibility. Several studies investigated the variable number tandem repeat (VNTR) or single nucleotide polymorphisms rs408392, rs419598 and rs2637988, with variable results. The aim of this study was to elucidate the influence of polymorphisms in IL1RN on IPF susceptibility and mRNA expression. We performed a meta-analysis of the five case–control studies that investigated an IL1RN polymorphism in IPF in a Caucasian population. In addition, we investigated whether IL1RN mRNA expression was influenced by IL1RN polymorphisms. The VNTR, rs408392 and rs419598 were in tight linkage disequilibrium, with D′ > 0.99. Furthermore, rs2637988 was in linkage disequilibrium with the VNTR (D′ = 0.90). A haploblock of VNTR*2 and the minor alleles of rs408392and rs419598 was constructed. Meta-analysis revealed that this VNTR*2 haploblock is associated with IPF susceptibility both with an allelic model (odds ratio = 1.42, p = 0.002) and a carriership model (odds ratio = 1.60, p = 0.002). IL1RN mRNA expression was significantly influenced by rs2637988, with lower levels found in carriers of the (minor) GG genotype (p < 0.001). From this meta-analysis, we conclude that the VNTR*2 haploblock is associated with susceptibility to IPF. In addition, polymorphisms in IL1RN influence IL-1Ra mRNA expression, suggesting that lower levels of IL-1Ra predispose to developing IPF. Together these findings demonstrate that the cytokine IL-1Ra plays a role in IPF pathogenesis.     

Evolution of a VNTR located within the promoter region of the thiopurine methyltransferase gene: inferences from population and sequence data

The promoter region of the human thiopurine methyltransferase (TPMT) gene contains a variable number of tandem repeats (VNTR) with three kind of motifs (A, B, and C) differing by the length of the unit core and nucleotide sequence. We have studied the structural variation within the VNTR alleles in two human populations and in samples from gorillas and chimpanzees. In humans, no intermingling of motifs was detected within the VNTR, and the sequences of the three core motifs remained remarkably unchanged, differences between alleles corresponding essentially to variations in the number of A and B repeats. The variation pattern in humans is consistent with an interpretation in which two contiguous genetic units (repeats A and B) behave evolutionarily according to the stepwise mutation model, as inferred from the population distribution profiles and from the molecular phylogenetic relationships among the VNTR alleles. However, the observation of a strong negative correlation between the numbers of A and B repeats also suggests that the regularity and/or independence of the mutational process has been disrupted to some extent by interactions between the A and B stretches. Selective pressure (the VNTR plays some role, although minor, in the TPMT function) or biased mutation are possible explanations. In gorillas and chimpanzees, several A-, B-, or C-like motifs were detected, but their arrangement within the VNTR alleles did not followed the regular pattern registered in humans and, particularly for the B-like motifs, a considerable sequence hypervariability was registered. Furthermore, the structural differences among non-human alleles were sufficiently numerous to render more plausible the assumption of the infinite allele model.     

Molecular diversity in Bacillus anthracis

Molecular typing of Bacillus anthracis has been extremely difficult due to the lack of polymorphic DNA markers. We have identified nine novel variable number tandemly repeated loci from previously known amplified fragment length polymorphism markers or from the DNA sequence. In combination with the previously known vrrA locus, these markers provide discrimination power to genetically characterize B. anthracis isolates. The variable number tandem repeat (VNTR) loci are found in both gene coding (genic) and non-coding (non-genic) regions. The genic differences are 'in frame' and result in additions or deletion of amino acids to the predicted proteins. Due the rarity of molecular differences, the VNTR changes represent a significant portion of the genetic variation found within B. anthracis. This variation could represent an important adaptive mechanism. Marker similarity and differences among diverse isolates have identified seven major diversity groups that may represent the only world-wide B. anthracis clones. The lineages reconstructed using these data may reflect the dispersal and evolution of this pathogen.     

The association between Interleukin (IL)-4 gene intron 3 VNTR polymorphism and alopecia areata (AA) in Turkish population

Objective

Alopecia areata (AA) is hypothesized to be an organ-specific autoimmune disease of hair follicles mediated by T cells. As immunological and genetic factors have been implicated in the pathogenesis of AA, the purpose of the present study was to investigate possible associations between the functional Interleukin (IL)-4 gene intron 3 VNTR polymorphism and AA susceptibility and disease progression in Turkish population.

Methods

The study group consisted of 116 unrelated patients with AA and 125 unrelated healthy controls. Genomic DNA was isolated and IL-4 gene 70 bp VNTR polymorphism determined by using polymerase chain reaction (PCR) with specific primers.

Results

No association was observed between AA patients and controls according to genotype distribution (p = 0.051). The allele distribution of IL-4 gene intron 3 VNTR polymorphism was statistically different between AA patients and control group (p = 0.026). The frequency of P1 allele in patients was significantly higher than that in the control group. When the P2P2 genotype was compared with P1P2 + P1P1 genotypes, a statistically significant difference was observed between patients and controls (p = 0.036). Intron 3 VNTR polymorphism in the IL-4 gene was found to be associated with AA susceptibility in Turkish population.

Conclusion

The results suggest that IL-4 VNTR polymorphism in the intron 3 region may be a risk factor for the development of AA among Turkish population. This is the first to report that intron 3 VNTR polymorphism in the IL-4 gene is associated with AA susceptibility.     

Multiple DNA Variant Association Analysis: Application to the Insulin Gene Region in Type 1 Diabetes

Association and linkage studies have shown that at least one of the genetic factors involved in susceptibility to insulin-dependent diabetes mellitus (IDDM) is contained within a 4.1-kb region of the insulin gene. Sequence analysis has led to the identification of 10 DNA variants in this region that are associated with increased risk for IDDM. These variants are in strong linkage disequilibrium with each other, and previous studies have failed to distinguish between the variant(s) that cause increased susceptibility to IDDM and others that are associated with the disease because of linkage disequilibrium. To address this problem, we have undertaken a large population study of French diabetics and controls and have analyzed genotype patterns for several of the variant sites simultaneously. This has led to the identification of a subset consisting of four variants (−2733AC, −23HphI, −365VNTR, and +1140AC), at least one of which appears to be directly implicated in disease susceptibility. The multiple-DNA-variant association-analysis approach that is applied here to the problem of identifying potential susceptibility variants in IDDM is likely to be important in studies of many other multifactorial diseases.     

Evolutionary History of the PER3 Variable Number of Tandem Repeats (VNTR): Idiosyncratic Aspect of Primate Molecular Circadian Clock

The PER3 gene is one of the clock genes, which function in the core mammalian molecular circadian system. A variable number of tandem repeats (VNTR) locus in the 18th exon of this gene has been strongly associated to circadian rhythm phenotypes and sleep organization in humans, but it has not been identified in other mammals except primates. To better understand the evolution and the placement of the PER3 VNTR in a phylogenetical context, the present study enlarges the investigation about the presence and the structure of this variable region in a large sample of primate species and other mammals. The analysis of the results has revealed that the PER3 VNTR occurs exclusively in simiiforme primates and that the number of copies of the primitive unit ranges from 2 to 11 across different primate species. Two transposable elements surrounding the 18th exon of PER3 were found in primates with published genome sequences, including the tarsiiforme Tarsius syrichta, which lacks the VNTR. These results suggest that this VNTR may have evolved in a common ancestor of the simiiforme branch and that the evolutionary copy number differentiation of this VNTR may be associated with primate simiiformes sleep and circadian phenotype patterns.     

Association of variable number of tandem repeats polymorphism in the IL-4 gene with end-stage renal disease in Malaysian patients

Variable number of tandem repeats (VNTR) polymorphism in the interleukin 4 (IL-4) gene has been associated with end-stage renal disease (ESRD) subjects in many different populations, although with conflicting results. We determined the 70 bp of VNTR polymorphism at intron 3 of the IL-4 gene in Malaysian ESRD subjects. Buccal cells were collected from 160 case and 160 control subjects; genomic DNA was amplified using PCR, followed by agarose gel electrophoresis. There were significant differences in genotypes and alleles of the IL-4 gene. We conclude that VNTR polymorphism of the IL-4 gene is a risk factor for the development of ESRD among Malaysians.     

Mucin variable number tandem repeat polymorphisms and severity of cystic fibrosis lung disease: significant association with MUC5AC

Variability in cystic fibrosis (CF) lung disease is partially due to non-CFTR genetic modifiers. Mucin genes are very polymorphic, and mucins play a key role in the pathogenesis of CF lung disease; therefore, mucin genes are strong candidates as genetic modifiers. DNA from CF patients recruited for extremes of lung phenotype was analyzed by Southern blot or PCR to define variable number tandem repeat (VNTR) length polymorphisms for MUC1, MUC2, MUC5AC, and MUC7. VNTR length polymorphisms were tested for association with lung disease severity and for linkage disequilibrium (LD) with flanking single nucleotide polymorphisms (SNPs). No strong associations were found for MUC1, MUC2, or MUC7. A significant association was found between the overall distribution of MUC5AC VNTR length and CF lung disease severity (p = 0.025; n = 468 patients); plus, there was robust association of the specific 6.4 kb HinfI VNTR fragment with severity of lung disease (p = 6.2×10(-4) after Bonferroni correction). There was strong LD between MUC5AC VNTR length modes and flanking SNPs. The severity-associated 6.4 kb VNTR allele of MUC5AC was confirmed to be genetically distinct from the 6.3 kb allele, as it showed significantly stronger association with nearby SNPs. These data provide detailed respiratory mucin gene VNTR allele distributions in CF patients. Our data also show a novel link between the MUC5AC 6.4 kb VNTR allele and severity of CF lung disease. The LD pattern with surrounding SNPs suggests that the 6.4 kb allele contains, or is linked to, important functional genetic variation.     

Construction and phenotypic analysis of mice carrying a duplication of the major histocompatibility class I (MHC-I) locus

Copy number variation (CNV) has been associated increasingly with altered susceptibility to human disease. Large CNVs are likely to incur disease risk or resilience via predictable changes in gene dosage that are relatively straightforward to model using chromosomal engineering in mice. The classical class I major histocompatibility locus (MHC-I) contains a dense set of genes essential for innate immune system function in vertebrates. MHC-I genes are highly polymorphic and genetic variation in the region is associated with altered susceptibility to a wide variety of common diseases. Here we investigated the role of gene dosage within MHC-I on susceptibility to disease by engineering a mouse line carrying a 1.9-Mb duplication of this region [called Dp(MHC-I)]. Extensive phenotypic analysis of heterozygous (3N) Dp(MHC-I) animals did not reveal altered blood and stem cell parameters, susceptibility to high-fat diet, death by cancer, or contact dermatitis. However, several measures of disease severity in a model of atherosclerosis were improved, suggesting dosage-sensitive modulators of cardiovascular disease. Homozygous Dp(MHC-I)/Dp(MHC-I) mice demonstrated embryonic lethality. These mice serve as a model for studying the consequences of targeted gene dosage alteration in MHC-I with functional and evolutionary implications.     

Interaction between insulin (VNTR) and hepatic lipase (LIPC-514C>T) variants on the response to an oral glucose tolerance test in the EARSII group of young healthy men

Insulin resistance is polygenic in origin, and can be observed at an early age. We have shown that variations in APOC3-482T>C and hepatic lipase (LIPC)-514C>T), individually (APOC3 alone) and interactively, modulate insulin and glucose levels after an OGTT in young healthy men participating in the European Atherosclerosis Research Study II (EARSII). Variation in the insulin gene (INS) variable number tandem repeat (VNTR) has been found to predispose to type 1 and type 2 diabetes. We have evaluated the HphI site 23 bp upstream of the INS gene (a surrogate marker for the VNTR) in EARSII (n=822), to determine if variation in INS contributes to insulin resistance. Carriers of the INS VNTR class III (HphI-) allele (frequency=0.29 (95%CI 0.27, 0.31)) had significantly higher 60-min insulin concentrations after the OGTT (P=0.014) and a marginally higher AUC insulin (P=0.07), compared to class I (HphI+) homozygotes. However, this effect on AUC insulin was modified by the level of physical activity, displaying significant gene:environment interaction (P=0.03). We tested for gene:gene interaction between the INS VNTR and both the LIPC-514C>T and APOC3-482T>C. While there was a significant interaction between INS VNTR and LIPC-514C>T on AUC glucose (P=0.013) and on AUC insulin (P=0.015), there was no interaction with APOC3-482T>C. Thus, despite a modest effect of the INS VNTR alone, the influence of this variant on insulin sensitivity was modified by gene:environment and gene:gene interactions, illustrating the biological complexity of insulin resistance.     

Significant association of interleukin-4 gene intron 3 VNTR polymorphism with susceptibility to knee osteoarthritis

Objective

Interleukin-4 (IL-4) is a strong chondroprotective cytokine and polymorphisms within this gene may be a risk factor for osteoarthritis (OA). We aimed to investigate genotype and allele frequencies of IL-4 gene intron 3 variable number of tandem repeats (VNTR) polymorphism in patients with knee OA in a Turkish population.

Methods

The study included 202 patients with knee OA and 180 healthy controls. Genomic DNA was isolated and IL-4 gene 70 bp VNTR polymorphism determined by using polymerase chain reaction (PCR) with specific primers followed by restriction fragment length polymorphism (RFLP) analysis.

Results

Our result show that there was statistically significant difference between knee OA patients and control group with respect to IL-4 genotype distribution and allele frequencies (p = 0.000, OR: 0.20, 95% CI: 0.10–0.41, OR: 0.22, 95% CI: 0.12–0.42, respectively).

Conclusions

Our findings suggest that there is an association of IL-4 gene intron 3 VNTR polymorphism with susceptibility of a person for development of knee OA. As a result, IL-4 gene intron 3 VNTR polymorphism could be a genetic marker in OA in a Turkish study population. This is the first association study that evaluates the associations between IL-4 gene VNTR polymorphism and knee OA.     

The VNTR polymorphism in the human dopamine transporter gene: improved detection and absence of association of VNTR alleles with attention-deficit hyperactivity disorder

The human dopamine transporter (DAT1) gene contains a variable number tandem repeat (VNTR) in its 3'-untranslated region because of repetition of a 40-bp core sequence. Methods available for the diagnosis of this polymorphism are limited in number. We have developed a new polymerase chain reaction (PCR) test, which is similar to that described originally by Vandenbergh's group, but provides a better detection of the VNTR alleles in the human DAT1 gene. Using two independent PCR methods, we have determined the distribution of VNTR alleles in 110 healthy Omani subjects, and in 92 children with attention-deficit hyperactivity disorder (ADHD). The frequency of the risk allele (DAT1*10) was similar in the healthy subjects and ADHD cases, indicating absence of association of this allele with ADHD in Oman.     

Pnlip encoding pancreatic lipase is possible candidate for obesity QTL in the OLETF rat

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat exhibits polygenic obesity, and one of quantitative trait loci (QTLs) responsible for a susceptibility to obesity in the OLETF, Nidd6/of, has been mapped to the approximately 10-cM genomic region between D1Rat166 and D1Rat90 on chromosome 1 in (OLETF x normal) F2 intercross. In this study, we have attempted to identify the causal gene for the Nidd6/of QTL. A Nidd6/of congenic strain, constructed by introgressing the OLETF allele on the mapped Nidd6/of region in the normal F344 rat strain, confirmed the existence of the Nidd6/of as obesity QTL. The Nidd6/of region was refined to a approximately 2.3-cM genomic region between D1Rat225 and D1Rat90, using informative recombinants selected from (Nidd6/of congenic x F344) F1 x Nidd6/of congenic backcross progenies. Among 46 genes located within the approximately 2.3-cM region, pancreatic lipase gene, Pnlip, was regarded as the most prominent and physiologically relevant positional candidate for the Nidd6/of QTL. We found that Pnlip possesses an OLETF allele-specific increase of mRNA levels in the pancreas, and that the OLETF allele is longer in variable number of tandem repeat (VNTR) within the 5'-flanking region than normal alleles. We further showed that the Nidd6/of QTL completely cosegregates with Pnlip VNTR in the informative recombinants from (Nidd6/of congenic x F344) F1 x Nidd6/of congenic backcross progenies. These results suggest that Pnlip is possible candidate for the Nidd6/of QTL.     

Novel interleukin-1 receptor antagonist exon polymorphisms and their use in allele-specific mRNA assessment

A variable number of tandem repeats (VNTR) polymorphism has been described in intron 2 of the interleukin-1 receptor antagonist gene. Allele 2 of this polymorphism is associated with many chronic inflammatory diseases. Using direct sequencing of polymerase chain reaction products from individuals of known genotype for the VNTR, we have identified four single base change polymorphisms in exons 1_ic and 2 and one upstream of exon 1_ic, all of which are probably in linkage disequilibrium with the intron 2 VNTR. The exonic polymorphisms do not alter the encoded amino acid sequence. Using the exon 2 polymorphism as a marker for the intron 2 disease-associated allele, we have been able to analyse allele-specific mRNA in heterozygotic keratinocyte cell lines. The disease-associated allele shows no difference from other alleles in this cell type with respect to mRNA accumulation. Received: 17 July 1995 / Revised: 4 December 1995