Tetranucleotide Repeat Polymorphism Research Articles

Development of a 17-Plex of Penta- and Tetra-Nucleotide Microsatellites for DNA Profiling and Paternity Testing in Horses.

Tetranucleotide and pentanucleotide short tandem repeat (hereafter termed tetraSTR and pentaSTR) polymorphisms have properties that make them desirable for DNA profiling and paternity testing. However, certain species, such as the horse, have far fewer tetraSTRs than other species and for this reason dinucleotide STRs (diSTRs) have become the standard for DNA profiling in horses, despite being less desirable for technical reasons. During our testing of a series of candidate genes as potentially underlying a heritable condition characterized by megaesophagus in the Friesian horse breed, we found that good tetraSTRs do exist in horses but, as expected, at a much lower frequency than in other species, e.g., dogs and humans. Using a series of efficient methods developed in our laboratory for the production of multiplexed tetraSTRs in other species, we identified a set of tetra- and pentaSTRs that we developed into a 17-plex panel for the horse, plus a sex-identifying marker near the amelogenin gene. These markers were tested in 128 horses representing 16 breeds as well as crossbred horses, and we found that these markers have useful genetic variability. Average observed heterozygosities (Ho) ranged from 0.53 to 0.89 for the individual markers (0.66 average Ho for all markers), and 0.62-0.82 for expected heterozygosity (He) within breeds (0.72 average He for all markers). The probability of identity (PI) within breeds for which 10 or more samples were available was at least 1.1 x 10−11, and the PI among siblings (PIsib) was 1.5 x 10−5. Stutter was ≤ 11% (average stutter for all markers combined was 6.9%) compared to the more than 30% typically seen with diSTRs. We predict that it will be possible to develop accurate allelic ladders for this multiplex panel that will make cross-laboratory comparisons easier and will also improve DNA profiling accuracy. Although we were only able to exclude candidate genes for Friesian horse megaesophagus with no unexcluded genes that are possibly causative at this point in time, the study helped us to refine the methods used to develop better tetraSTR multiplexed panels for species such as the horse that have a low frequency of tetraSTRs.

Leptin Gene Tetranucleotide Repeat Polymorphism in Obese Individuals in Egypt

Leptin is a peptide hormone secreted by the adipose tissue. Genetic mutations of the leptin gene were reported to cause severe obesity. This study was undertaken to investigate the association of the polymorphic tetranucleotide repeat locus 3' UTR of leptin gene with obesity in Egyptian cases. This study has included 120 subjects affected with obesity 57 of them were consistent with the diagnosis of metabolic syndrome (MS) while the rest (63) had simple obesity. These cases were compared to 83 normal weight healthy controls. All participants were subjected to an estimation of their body mass index (BMI), waist hip ratio (WHR), serum as well as characterization of leptin gene tetranucleotide repeat (TTTC)n polymorphism by PCR technique. Thirteen different alleles were identified in all cases of obesity versus only 5 alleles in normal controls. The most frequent allele was the 154 bp allele (57.5% in all cases of obesity vs. 92.2% in controls). Total cases with obesity showed a significantly higher carriage rate of class II alleles (I/II + II/II genotypes) compared to healthy controls (48.3% vs. 6.0%, OR=14.6, 95% CI=5.5-38.6, p=<0.0001). This was more apparent in the group with simple obesity (52.3% vs. 6.0%, OR=17.2, 95% CI=6.1-48.1, p=<0.0001) than in MS cases (43.9 % vs. 6.0 %, OR =12.19, 95% CI=4.9-30.4, p=< 0.0001). Interestingly, cases with MS did not differ from those with simple obesity regarding their class I or II allele frequencies (p> 0.05). Although serum lipids were significantly higher in obese cases compared to controls, no difference was found among obese cases with different leptin gene class genotypes (p> 0.05). Tetranucleotide repeat (TTTC)n polymorphism in the 3' UTR of the human leptin gene was associated with obesity in Egyptian obese cases showing higher class II allele carriage rate. However, the lipoprotein levels were not affected by this polymorphism.

Evaluation of leptin and leptin receptor gene 3′ UTR polymorphisms in essential hypertension

Introduction: Leptin and leptin receptor gene polymorphisms have been associated with obesity; however, their association with blood pressure has not been fully elucidated. The aim of this study was to examine the effect of tetranucleotide repeat polymorphism in the 3′ flanking region of the leptin and leptin receptor gene on blood pressure in hypertensives with obesity.Methods: Two hundred and eighty hypertensives and 200 healthy controls were analyzed for a tetranucleotide repeat polymorphism of leptin and leptin receptor genes. Genotyping was done by amplifying DNA and determining the allele sizes using gel documentation system. Odds ratios were computed to predict the risk for hypertension caused by specific genotypes of leptin and leptin receptor genes and the effect of interaction between them on the development of hypertension was determined by MDR test.Results: Significant preponderance in the incidence of male sex, obese individuals and those with positive family history was observed with significant elevation in the mean levels of SBP, DBP, BMI and reduction of HDL levels in hypertensives as compared to controls. Class I/I genotypes of leptin showed significantly high risk for developing hypertension irrespective of obesity. Genotypes of leptin receptor did not confer any risk for hypertension and cohorts studied.Conclusion: Homozygotes I/I were at greater risk for developing hypertension irrespective of obesity. When leptin and leptin receptor genes were considered together, synergistic interaction was observed between the two genes leading to hypertension, while the polymorphism at leptin gene and obesity was correlated.

Association between a Tetranucleotide Repeat Polymorphism of SPAG16 Gene and Cataract in Male Children

Purpose. Studies involving genotyping of STR markers at 2q34 have repeatedly found the region to host the disease haplotype for pediatric cataract. Present study investigated the association of D2S2944 marker, in sperm associated antigen 16 (SPAG16) gene and rs2289917 polymorphism, in γ-crystallin B gene, with childhood cataract. Methods. 97 pediatric cataract cases and 110 children with no ocular defects were examined for tetranucleotide repeat marker/SNP using PCR-SSLP/RFLP techniques. Polymorphisms were assessed for association using contingency tables and linkage disequilibrium among alleles of the markers was estimated. Energy-optimization program predicted the secondary structure models of repeats of D2S2944. Results. Seven alleles of D2S2944, with 9–15 “GATA” repeats, were observed. Frequency of the longer allele of D2S2944, ≥(GATA)13 repeats, was 0.73 in cases and 0.56 in controls (P = 0.0123). Male children bearing ≥(GATA)13 repeats showed >3-fold higher risk for cataract (CI95% = 1.43–7.00, P = 0.0043, P c = 0.0086) as compared to female children (OR = 1.19, CI95% = 0.49–2.92, P = 0.70). Cases with haplotype—≥(GATA)13 of D2S2944 and “C” allele rs2289917—have a higher risk for pediatric cataract (OR = 2.952, CI95% = 1.595~5.463, P = 0.000453). >(GATA)13 repeats formed energetically more favorable stem-loop structure. Conclusion. Intragenic microsatellite repeat expansion in SPAG16 gene increases predisposition to pediatric cataract by probably interfering posttranscriptional events and affecting the expression of adjacent lens transparency gene/s in a gender bias manner.

Tetranucleotide Repeat Polymorphism in the 3' UTR of the Human Leptin Gene and Risk of Type 2 Diabetes Mellitus in a South Indian Population

Tetranucleotide Repeat Polymorphism in the 3' UTR of the Human Leptin Gene and Risk of Type 2 Diabetes Mellitus in a South Indian Population

Linkage between HPRTB STR alleles and Lesch–Nyhan syndrome inside a family: Implications in forensic casework

The use of neutral markers in the forensic field has been recommended in order to avoid ethical concerns. Therefore, genetic markers that disclose diseases or genetic risks should not be considered in forensic investigations and, for this reason, in 2005, Szibor and co-authors discarded the use of HumARA [1]. The HPRTB is a polymorphic tetranucleotide repeat located in the intron 3 of the HPRT gene, which was first reported in 1991 [2] with alleles presenting 9–17 TCTA repeat units. Together with HumARA, HPRTB was one of the first X chromosome specific STRs used in forensic genetics and it is still widely used because of its inclusion in a commercial kit Investigator Argus X-12 Kit (Qiagen) (e.g.

1201 Whole Exon Deletion in the Claudin 16 Gene, a Novel Mutation in Familial Hypomagnesemia/Hypercalciuria/Nephrocalcinosis (FHHNC) and Sensorineural Deafness (SND)

BackgroundFHHNC is a rare autosomal recessive tubulopathy of the thick ascending limb due to inactivating mutations in the Claudin-16 and Claudin-19 genes which are responsible for the paracellular reabsorption of...

Identification of a highly polymorphic tetranucleotide repeat locus (DXpS) at Xp and development of a DXpS/HUMARA biplex methylation-based PCR assay that enhances detection of X-chromosome inactivation

Identification of a highly polymorphic tetranucleotide repeat locus (DXpS) at Xp and development of a DXpS/HUMARA biplex methylation-based PCR assay that enhances detection of X-chromosome inactivation

Identification of a highly polymorphic tetranucleotide repeat locus (DXpS) at Xp and development of a DXpS/HUMARA biplex methylation-based PCR assay that enhances detection of X-chromosome inactivation

AbstractThe methylation-based PCR assay at the polymorphic (CAG)n repeat in exon 1 of the human androgen receptor _AR_ gene (HUMARA) is a standard method for determination of the methylation state of alleles in active X (Xa) and inactive X (Xi) chromosomes. HUMARA assay is endowed with heterozygosity rates ~85% worldwide. This means that in a proportion of females it is uninformative. The HUMARA genotype is not neutral, being linked to Kennedy´s disease. Moreover, allele designation and quantification from trinucleotide repeats demand normalizing for minor (stutter) products differing from the original template by multiples of the repeat unit. Here, we report on the identification of a highly polymorphic tetranucleotide repeat (named DXpS) mapping to within a CpG island on Xp. This island is 191 bp downstream from the start of the repeat element, and contains sites for the HhaI, HpaII and BstUI methyl-sensitive restriction enzymes. We developed the DXpS and the HUMARA markers into a biplex methylation-based quantitative fluorescent PCR assay. For DXpS we observed twelve alleles with negligible stuttering. DXpS exhibited a heterozygosity rate of 87% (n = 60), matching that of HUMARA. The combined informativeness of the biplex assay was 98%. Random and nonrandom X-inactivation patterns inferred with DXpS in phenotypically normal females and haemophiliac females carrying a defective F8 gene were highly concordant (r^2^ = 0.96) with HUMARA patterns. DXpS represents a notable advancement in detecting X-chromosome inactivation due to the observed high rate of heterozygosity, negligible stuttering, concordance with HUMARA, and the apparent neutrality of allelic variants. Financial support: FAPESP (09/10615-7), FAPERJ, CNPq.

Age-related macular degeneration with discordant late stage phenotypes in monozygotic twins

Purpose: Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors. Monozygotic twins with late stage AMD provide an opportunity to evaluate the role of environmental factors on a genetically uniform background.Methods: Monozygosity was determined in four twin pairs by genotyping 15 highly polymorphic tetranucleotide repeat markers. Clinically, the monozygotic twin pairs were evaluated by ophthalmologic examination, fundus autofluorescence (FAF) imaging, and, upon suspicion of choroidal neovascular membranes, by fluorescein angiography. Twin pairs were followed over a period of 4–8 years.Results: Genetically confirmed monozygotic twins with early stages of AMD reveal striking symmetry of early macular pathology. Upon progression to late stage AMD each twin pair developed a discordant phenotype with respect to disease onset and specific phenotype as choroidal neovascularization (CNV) and geographic atrophy (GA).Conclusions: Genetic risk variants are known to predispose to the development of AMD. Course and visual outcome of the disease, however, appear to be influenced by environmental factors rather than genetic determinants.

A long AAAG repeat allele in the 5′ UTR of the ERR-γ gene is correlated with breast cancer predisposition and drives promoter activity in MCF-7 breast cancer cells

We sequenced the 5' UTR of the estrogen-related receptor gamma gene (ERR-γ) in ~500 patient and volunteer samples and found that longer alleles of the (AAAG)(n) microsatellite were statistically and significantly more likely to exist in the germlines of breast cancer patients when compared to healthy volunteers. This microsatellite region contains multiple binding sites for a number of transcription factors, and we hypothesized that the polymorphic AAAG-containing sequence in the 5' UTR region of ERR-γ might modulate expression of ERR-γ. We found that the 369 bp PCR product containing the AAAG repeat drove expression of a reporter gene in estrogen receptor positive breast cancer cells. Our results support a role for the 5' UTR region in ERR-γ expression, which is potentially mediated via binding to the variable tandem AAAG repeat, the length of which correlates with breast cancer pre-disposition. Our study indicates that the AAAG tetranucleotide repeat polymorphism in ERR-γ gene 5' UTR region may be a new biomarker for genetic susceptibility to breast cancer.

Functional promoter polymorphisms of macrophage migration inhibitory factor in peptic ulcer diseases

Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator, which plays a pivotal role in inflammatory and immune diseases. We attempted to clarify the association of functional polymorphisms of MIF gene promoter with the development of gastro-duodenal ulcer. The study was performed in 471 stocked DNAs obtained from the subjects, including 93 healthy volunteers, with no evidence of gastric malignancy. We employed the PCR-SSCP method to detect gene polymorphisms. In all 471 DNAs, 92 and 43 were obtained from gastric and duodenal ulcer patients, respectively. By an unadjusted analysis, infection with Helicobacter pylori (H. pylori), male gender and non-steroidal anti-inflammatory drug (NSAID/aspirin) use were significantly associated with a risk for developing a gastric ulcer, whereas MIF promoter polymorphisms were not. On the other hand, infection with H. pylori, male gender and 7-CATT repeat at position -794 were significantly associated with the development of a duodenal ulcer, whereas NSAID/ aspirin use was not. By the analysis after adjustment for age, gender, NSAID/aspirin use and H. pylori infection status, 7/7-CATT homozygote had a significantly increased risk for the development of duodenal ulcers (OR, 6.31; 95% CI, 1.50-26.6; p=0.012). No factors were significantly associated with the development of peptic ulcers in NSAID/aspirin users. Our results suggested that tetranucleotide repeat polymorphism of MIF gene promoter might be associated with the development of duodenal ulcers.

Polymorphisms of Renin-Angiotensin System and Natriuretic Peptide Receptor A Genes in Patients of Greek Origin with a History of Myocardial Infarction

We assessed the association between (CA)n repeat polymorphism of angiotensinogen (AGT), 250 base pair (bp) insertion/deletion (I/D) of angiotensin-converting enzyme (ACE), tetranucleotide repeat polymorphism (TCTG)n of renin (REN), (CT)n repeat polymorphism of the natriuretic peptide receptor A (NPRA) genes, and the presence and extent of coronary artery disease (CAD) in Greek patients with a history of myocardial infarction (MI). A total of 158 post-MI patients referred for coronary angiography were compared with 144 controls. The SS genotype of the AGT gene was related with an increased risk for 3-vessel CAD (odds ratio [OR], 1.94; 95% confidence interval [CI], 1.05-3.61; P = .041), whereas the SL genotype was related with a decreased risk (OR, 0.44; 95% CI, 0.22-0.87; P = .019). Moreover, there was a trend for the SL genotype of the REN gene toward increased risk for CAD. There was a significant association between (CA)n polymorphism of the AGT gene and the extent of CAD in Greek patients with a history of MI.

Association of renin-angiotensin system and natriuretic peptide receptor A gene polymorphisms with hypertension in a Hellenic population

Hypertension results from the interaction of genetic and environmental factors. Since the renin-angiotensin and the natriuretic peptide systems contribute to blood pressure regulation, variations in the relative genes are candidates for the development of hypertension. In 194 hypertensives and 304 controls of Hellenic origin, the possible association between the (CA)n repeat polymorphism of angiotensinogen (AGT), the 250 bp insertion/deletion (I/D) of angiotensin-converting enzyme (ACE), the tetranucleotide repeat polymorphism (TCTG)n of renin, and the (CT)n repeat polymorphism of the natriuretic peptide receptor A (NPRA) and hypertension was assessed. No association between AGT and NPRA polymorphisms and hypertension was observed. The presence of ID or DD genotype of ACE was associated with an increased risk for hypertension compared with the II genotype (OR: 1.782 [95% CI: 1.032-3.077]), whereas the LL genotype of the renin gene was associated with a decreased risk compared with the SS genotype (OR: 0.174 [95% CI: 0.044-0.689]). However, after adjustment for confounding factors only the latter association remained. In the present study conducted in a homogeneous Hellenic population, no associations between AGT,ACE, and NPRA gene polymorphisms and hypertension were found. The presence of a significant negative association between the LL polymorphism of the renin gene and hypertension requires further confirmation.

Leptin and Leptin Receptor Gene Polymorphisms in Obstructive Sleep Apnea Syndrome

Obstructive sleep apnea is common in obese people. Leptin is an adipocyte-derived signaling factor that has an important role in metabolic control. There is growing evidence that leptin regulation is altered in obstructive sleep apnea syndrome (OSAS). The aim of this study was to investigate the relation between polymorphisms of the leptin and leptin receptor (LEPR) genes and OSAS. The study population consisted of 130 patients with OSAS and 50 healthy control subjects. All the subjects were Japanese. Diagnostic polysomnography was performed in all patients and control subjects. A highly polymorphic tetranucleotide repeat polymorphism in the 3'-flanking region of the leptin gene and three single nucleotide polymorphisms (SNPs) [Lys109Arg (A/G) in exon 4, Gln223Arg (A/G) in exon 6, and Lys656Asn (G/C) in exon 14] in the LEPR gene were examined. There were no significant differences in allelic frequencies and genotype distributions of the examined polymorphisms of the leptin and LEPR genes between OSAS patients and control subjects. For the LEPR gene, the wild-type alleles of the Gln223Arg and Lys656Asn SNPs had a marginally significant effect on mild OSAS, which was defined as an apnea-hypopnea index from 10 and 20 events/h in the dominant model. The tetranucleotide repeat polymorphism of the leptin gene and the Lys109Arg, Gln223Arg, and Lys656Asn SNPs in the LEPR gene were not associated with OSAS in the Japanese population. Further studies are required to confirm the association of the wild types of Gln223Arg and Lys656Asn SNPs with the severity of OSAS.

Polymorphisms and haplotypes of the gene encoding the estrogen-metabolizing CYP19 gene in Korean women: no association with advanced-stage endometriosis

A variety of factors affect the development of endometriosis, including hormonal status and genetic factors. The growth of endometriosis is stimulated by local estrogen production in conjunction with circulating estrogen. The CYP19 gene encodes a steroid aromatase that catalyses the conversion of C-19 androgens to estrogens. This study investigated whether polymorphisms of the CYP19 gene are associated with the risk of advanced endometriosis in Korean women. Blood samples were collected from 224 female patients with endometriosis of stages III and IV, as diagnosed by both pathologic and laparoscopic findings, and from a control group comprising of 188 women undergoing laparoscopic surgery or laparotomy for nonmalignant lesions. Single-nucleotide polymorphisms, restriction fragment length polymorphisms, and tetranucleotide tandem repeat polymorphisms were discriminated by the polymerase chain reaction (PCR). Haplotype analysis was also performed. CYP19 115T>C, 240G>A, and 1531C>T polymorphisms and [TTTA]n tetranucleotide repeat polymorphisms in the CYP19 gene and their haplotypes were not significantly associated with the risk of endometriosis. The risk of endometriosis also did not increase significantly with the number of higher risk alleles of the CYP19 gene. In conclusion, our findings suggest that CYP19 genetic polymorphisms are not associated with advanced-stage endometriosis in Korean women.

Replication study for the association of TCF7L2 with susceptibility to type 2 diabetes in a Japanese population

The transcription factor 7-like 2 gene (TCF7L2) has been shown to be strongly associated with an increased risk of type 2 diabetes in white populations. To further investigate the involvement of TCF7L2 in conferring susceptibility to type 2 diabetes, we examined the association of TCF7L2 polymorphisms with type 2 diabetes in a Japanese population. We analysed four SNPs (rs12255372, rs7903146, rs7901695 and rs11196205) and one tetranucleotide repeat polymorphism (DG10S478) in 1,630 Japanese subjects with type 2 diabetes and 1,064 control subjects. All investigated polymorphisms were significantly associated with type 2 diabetes, and rs12255372 showed the strongest association (T vs G, chi2 = 9.20, p = 0.0024, odds ratio = 1.70, 95% CI = 1.20-2.41), although the frequency of the risk allele in our population was much lower than that in white populations. The microsatellite polymorphism showed an almost complete linkage disequilibrium to rs1255372 when the alleles with longer repeats (+8, +12) were considered as minor alleles and showed an association with type 2 diabetes (chi2 = 5.34, p = 0.021, odds ratio = 1.50, 95% CI = 1.06-2.12). These results indicate that TCF7L2 might be a strong candidate for conferring susceptibility to type 2 diabetes across different ethnicities.

Von Willebrand Factor Short Sequence Repeat Locus 2 (Intron 40) Consists of Three Polymorphic Subloci

Currently, identification of alleles within the short sequence repeat locus (SSR locus) of intron 40 of the von Willebrand factor gene ( previously known as VNTR II) is based on the size of PCR products that only predicts a certain number of repeats. Through cloning and sequencing, we demonstrated the complexity of nucleotide sequence structure of this region by describing three polymorphic tetranucleotide repeat subloci SSR ‘a’, SSR ‘b’ and SSR ‘c’ within the same originally described locus: the original TCTA (9–14 repeats), a small TCTA repeat locus (2 or 3 repeats) located at the 5′ end and 30 nucleotides upstream from the original locus and a TGTA repeat locus (5 or 6 repeats), adjacent to the 2/3 repeat locus. Sequencing of 54 VWF alleles has revealed 14 different sequence combinations in this region while the size variability in the region only amounts to a 7-allele system. While the determination of VNTR alleles by PCR remains a practical methodology in linkage analysis, this may not be applicable in forensic medicine since not all alleles of identical length based on PCR are the same. Our study reveals the important implication of the identification of SSR 2 subloci and concludes that at least in some situations, there may be a necessity to take into consideration these polymorphic subloci.

Lack of association between the tetranucleotide repeat polymorphism in the 3′-flanking region of the leptin gene and hypertension in severly obese patients

Conflicting data suggest an association between leptin gene polymorphisms and essential hypertension independently of obesity. The aim of this study was to evaluate, in severely obese subjects, the role of one of these polymorphic markers in relation to the development of hypertension. The study included 325 obese patients with mean body mass index (BMI) of 46+/-6.94 kg/m2. One hundred sixty-six were hypertensive and 159 normotensive. In both groups, the presence of a tetranucleotide repeat in the 3' flanking region of the Ob gene was investigated using polymerase chain reaction (PCR). Due to the genetic variant, in the region studied it is possible to distinguish two alleles with different size distribution: Class I (shorter one) and Class II (longer one). Class I and Class II allele frequencies were not significantly different in obese patients when analyzed according to the presence or absence of hypertension. The results presented herein do not support a significant association of this Ob gene polymorphism with hypertension. These findings are in contrast with that reported in other populations. However, we cannot rule out that different ethnicity and/or phenotypic variability might mask small effects.

Positive association of schizophrenia to JARID2 gene

Dysbindin (DTNBP1) is a positional candidate gene for 6p22.3-linked schizophrenia (SZ). However, so far, no disease-causing alleles have been identified. DTNBP1 is immediately adjacent to JARID2, a member of the ARID (AT-rich interaction domain) family of transcription modulators. We have previously suggested that proteins which bind to AT-rich domains could play a role in SZ pathogenesis. Consequently, we explored the possibility that JARID2 itself could be a candidate gene for 6p22.3-linked SZ. We used a case control design to analyze single nucleotide polymorphisms (SNPs) and insertion/deletion variants affecting AT-rich domains in both the DTNBP1 and JARID2 genes. Three of the DTNBP1 SNPs analyzed had previously been shown to be associated with SZ. We did not detect any significant difference in allele, genotype or haplotype distribution for any of these DTNBP1 markers. However, we did detect a significant difference in allele distribution for a tetranucleotide repeat polymorphism in the JARID2 gene that affects an AT-rich domain. A significant increase in short alleles (less than 11 repeats) was found in patients with SZ (chi(2) = 7.02; P = 0.008). No other JARID2 marker displayed statistically significant allele and genotype distributions. Our findings suggest that JARID2 should be viewed as a candidate gene for 6p22.3-linked SZ.