Restriction Fragment Length Polymorphism Sites Research Articles

VDR Gene Polymorphisms in Kurdish Population and Its Relation to T1DM in Erbil-Iraq.

This research assessed the relationship among type 1 diabetes VDR gene polymorphisms (ApaI and TaqI) in the Kurdish population in Erbil-Iraq. Forty individuals with type 1 diabetes and thirty healthy people were recruited from the Kurdish population in Erbil, Iraq. Genomic DNA was taken from blood, being genotyped for SNP (single nucleotide polymorphisms). The distribution of VDR polymorphisms in two restriction fragment length polymorphism sites, TaqI and ApaI, was investigated in patients and controlled by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) utilizing ApaI and TaqI restriction enzymes. Using SPSS software (V15.0), the genotype dispersal and allelic incidences in patients and controls were compared. VDR polymorphism genotype dispersal and allele incidences vary dramatically among patients and controls. The results confirmed that the genotype GT in SNP ApaI was a risk factor among type 1 diabetes mellitus patients' combination that imparted the strongest susceptibility to T1DM (P=0.00023). Still, the SNP TaqI showed no relevance between cases and controls (P=0.35). Our findings indicate that VDR gene polymorphisms in the combination of genotypes are related to an increased risk of T1DM in the Kurdish community and warrant further investigation as a possible genetic risk marker for T1DM. More research is needed to corroborate this finding, particularly the VDR gene, which was studied for the first time in the Kurdish population.

Association of VDR FokI and ApaI genetic polymorphisms with parkinson's disease risk in South Western Iranian population

Background: Parkinson's disease comes second comparing to Alzheimer's disease being responsible for nerve destructing diseases; it is a complex and multifactorial disease. Gene associated studies help to identify the genetic factors that introduce the Single Nucleotide Polymorphisms in different genes as genetic risk factors for non-Mendelian Parkinson's disease in diverse populations. We intended to study the association of VDR (Vitamin D Receptor) gene polymorphisms with Parkinson's disease in south western Iranian population. Results: In the present study 150 patients with Parkinson's disease and 160 Healthy controls from an Iranian population were genotyped for two polymorphic sites. The prevalence of VDR polymorphisms in two restriction fragment length polymorphism sites including FokI and ApaI were analyzed in patients and controls. Our data demonstrated no significant association between VDR FokI polymorphism and PD, whereas the ApaI polymorphism showed a significant association with PD in Iranian patient. Also no association between the age at onset, the male-female ratio and the VDR polymorphism in the PD group was detected. Conclusions: In conclusion these results determined that VDR ApaI (TG and GG) genotype might affect development of PD in our study population. There was no association between FokI polymorphism and the risk of PD. Our results were analogous only with American/Hungarian Caucasian race.

Acute chest syndrome is associated with single nucleotide polymorphism‐defined beta globin cluster haplotype in children with sickle cell anaemia

Genetic diversity at the human β-globin locus has been implicated as a modifier of sickle cell anaemia (SCA) severity. However, haplotypes defined by restriction fragment length polymorphism sites across the β-globin locus have not been consistently associated with clinical phenotypes. To define the genetic structure at the β-globin locus more thoroughly, we performed high-density single nucleotide polymorphism (SNP) mapping in 820 children who were homozygous for the sickle cell mutation (HbSS). Genotyping results revealed very high linkage disequilibrium across a large region spanning the locus control region and the HBB (β-globin gene) cluster. We identified three predominant haplotypes accounting for 96% of the β(S) -carrying chromosomes in this population that could be distinguished using a minimal set of common SNPs. Consistent with previous studies, fetal haemoglobin level was significantly associated with β(S) -haplotypes. After controlling for covariates, an association was detected between haplotype and rate of hospitalization for acute chest syndrome (ACS) (incidence rate ratio 0·51, 95% confidence interval 0·29-0·89) but not incidence rate of vaso-occlusive pain or presence of silent cerebral infarct (SCI). Our results suggest that these SNP-defined β(S) -haplotypes may be associated with ACS, but not pain or SCI in a study population of children with SCA.

Vitamin D receptor gene polymorphisms in type 1 diabetes mellitus: a new pattern from Khorasan province, Islamic Republic of Iran

Reported associations between vitamin D receptor (VDR) polymorphism and type 1 diabetes mellitus vary across ethnic groups. We studied the association between type 1 diabetes and 4 VDR gene polymorphisms (Bb, Ff Aa and Tt) in an Iranian population. A group of 69 patients with type 1 diabetes mellitus and 45 unrelated healthy subjects were recruited. The prevalence of VDR polymorphisms in 4 restriction fragment length polymorphism sites including Bsml, Fokl, Apal and Taql were analysed in patients and controls. The frequencies of 3 genotypes (Aa, FF and Bb) were significantly higher in the patient group. The relationship between VDR gene polymorphisms and onset pattern of diabetes was not significant. There were no significant difference between the genotype frequencies and chronic complications of diabetes, but the relationship between the Ffgenotype and ketoacidosis was significant. Our results differ from previous polymorphism studies in other regions.

High-density SNP genotyping to define β-globin locus haplotypes

Five major β-globin locus haplotypes have been established in individuals with sickle cell disease (SCD) from the Benin, Bantu, Senegal, Cameroon, and Arab-Indian populations. Historically, β-haplotypes were established using restriction fragment length polymorphism (RFLP) analysis across the β-locus, which consists of five functional β-like globin genes located on chromosome 11. Previous attempts to correlate these haplotypes as robust predictors of clinical phenotypes observed in SCD have not been successful. We speculate that the coverage and distribution of the RFLP sites located proximal to or within the globin genes are not sufficiently dense to accurately reflect the complexity of this region. To test our hypothesis, we performed RFLP analysis and high-density single nucleotide polymorphism (SNP) genotyping across the β-locus using DNA samples from healthy African Americans with either normal hemoglobin A (HbAA) or individuals with homozygous SS (HbSS) disease. Using the genotyping data from 88 SNPs and Haploview analysis, we generated a greater number of haplotypes than that observed with RFLP analysis alone. Furthermore, a unique pattern of long-range linkage disequilibrium between the locus control region and the β-like globin genes was observed in the HbSS group. Interestingly, we observed multiple SNPs within the HindIII restriction site located in the Gγ-globin intervening sequence II which produced the same RFLP pattern. These findings illustrated the inability of RFLP analysis to decipher the complexity of sequence variations that impacts genomic structure in this region. Our data suggest that high-density SNP mapping may be required to accurately define β-haplotypes that correlate with the different clinical phenotypes observed in SCD.

Relationship between MT1 melatonin receptor gene polymorphism and seasonal physiological responses in Île-de-France ewes

The gene encoding the MT1 melatonin receptor in sheep has a restriction fragment length polymorphism (RFLP) site to the MnlI enzyme whose incidence is associated to the expression of seasonality in several breeds. The aim of this study was to examine the relationship between this genetic marker and the physiological effects of MT1 receptor gene polymorphism on several seasonal functions in Ile-de-France ewes. The study was performed using 12 pairs of half-sib adult Ile-de-France ewes. Within each pair, ewes were selected on the basis of their genotype at the MnlI RFLP site: group +/+ and -/- (presence and absence of MnlI restriction site, respectively). No difference in the dates of the beginning, the end or the length of the breeding season was observed between groups during the two-year study. The seasonal changes in prolactin secretion were not different between groups. Similarly, wool growth rate and primary follicle activity, measured for one year, varied with the time of the year in the same way in the two groups. Our study therefore failed to show any relationship between MT1 polymorphism and reproductive seasonality in Ile-de-France ewes. This suggests that the influence of this polymorphism on the regulation of seasonal function is dependent upon the breed and/or environmental conditions. The MT1 polymorphism can explain only a small part of the genetic variability of seasonal functions and the implication of other genes must be investigated.

Genetic Variation and Management of Striped Bass Populations in the Coastal Rivers of South Carolina

Abstract The coastal rivers of South Carolina possess reproducing populations of striped bass Morone saxatilis. Historically, all of these populations have been augmented with hatchery stocks from the Santee–Cooper drainage. Concern has been expressed that locally adapted, reproductively isolated populations may exist in these coastal rivers. Thus, the objective of this effort was to assess the distinctiveness of reproducing striped bass populations in these coastal rivers. Tagging studies indicated that the interriver movement of striped bass was minimal. A population estimate of 406 striped bass longer than 30 cm was obtained for the Combahee River, suggesting a relatively small breeding population. Genetic assays at three nuclear restriction fragment length polymorphism sites showed highly significant allele frequency differences among seven coastal rivers, suggesting reproductive isolation. Partially or fully isolated populations can develop beneficial adaptations to local environmental conditions. Th...

Factor VIII gene polymorphisms in North Indian population: a consensus algorithm for carrier analysis of hemophilia A

Background: Hemophilia A, an X-linked recessive disorder, has the prevalence of 1 male per 7000 of the male population in the Northern states of India. The aim of the present study was to analyze the polymorphisms of the factor VIII gene in a sample population comprised of 22 families (112 persons) in order to formulate an algorithm that would be informative and accurate, yet cost-effective for carrier diagnosis. Methods: Polymerase chain reaction was used to study the polymorphisms of two intragenic restriction fragment length polymorphic sites (recognized by BclI and HindIII) and an extragenic variable number tandem repeat (VNTR) locus (St14). Results: Fifty-eight percent of the women tested were heterozygous for the BclI restriction fragment length polymorphism (RFLP) (significantly high compared to earlier reports), signifying the usefulness of this marker in carrier detection. About 64% of the families from the target population could be diagnosed using this marker alone. The other intragenic HindIII RFLP site showed a heterozygosity rate of 43% in women, and was effective in diagnosing 50% of the families studied. The population prevalence for ‘+’ alleles of BclI and HindIII were 68% and 33%, respectively. About 88% of the women tested were heterozygous for the St14 locus, and 83% of the families could be diagnosed using this marker alone. The 1390- and 1300-bp alleles were most prevalent, while novel polymorphisms of 1500 and 1345 bp were detected. Conclusions: Based on the above data, we suggest screening hemophilic families first for BclI polymorphism, followed by an analysis for HindIII polymorphism in case of homozygosity at the BclI site. When both were noninformative, analysis of St14 locus would be necessary.

Mutation analysis of the dystrophin gene in Southern French DMD or BMD families: from Southern blot to protein truncation test.

Data from 6 years of experience in molecular diagnosis of Duchenne (DMD) and Becker (BMD) muscular dystrophy in Southern France are reported. DMD and BMD patients have been extensively analyzed for deletions and for point mutations in the dystrophin gene. By scanning the whole coding sequence as reverse-transcribed from lymphocytes or muscular RNA by the protein truncation test, we have reached a minimum of an 86% detection rate for point mutations responsible for DMD; these mutations consist of nonsense, frameshifting, and splicing mutations. Four of 12 small alterations identified in our sample are novel and described in this study. We also present an improved protocol for the automated detection of fluorescently labeled duplex polymerase chain reactions of six known intragenic microsatellites (Dys II, TG 15, STRs 44, 45, 49, and 50). Accurate sizing of the alleles at each locus was performed, and we elucidated the sequence of several repeat units. Allele frequencies at each of the six microsatellite loci and at one restriction fragment length polymorphism site (intron 16/TaqI) were defined in a sample of normal, DMD, and BMD X chromosomes from Southern France. The determination of the grandparental origin of either deletions or point mutations revealed differences depending on the type of the mutation, with most of the deletions occurring in oogenesis and most of the point mutations occurring in spermatogenesis.

P-element-induced male recombination and gene conversion in Drosophila.

A P-element insertion flanked by 13 restriction fragment length polymorphism (RFLP) marker sites was used to examine male recombination and gene conversion at an autosomal site. The great majority of crossovers on chromosome arm 2R occurred within the 4-kb region containing the P element and RFLP sites. Of the 128 recombinants analyzed, approximately two-thirds carried duplications or deletions flanking the P element. These rearrangements are described in more detail in the accompanying report. In a parallel experiment, we examined 91 gene conversion tracts resulting from excision of the same autosomal P element. We found the average tract length was 1463 bp, which is essentially the same as found previously at the white locus. The distribution of conversion tract endpoints was indistinguishable from the distribution of crossover points among the nonrearranged male recombinants. Most recombination events can be explained by the "hybrid element insertion" model, but, for those lacking a duplication or deletion, a second step involving double-strand gap repair must be postulated to explain the distribution of crossover points.

Analysis of the BglI restriction fragment length polymorphism in the human factor VIII gene using "virtual PCR"--a novel approach employing the polymerase chain reaction in the absence of sequence information for the locus.

The BglI restriction fragment length polymorphism (RFLP) of the human factor VIII (FVIII) gene is potentially useful in linkage studies in haemophilia A. The sequence at the RFLP locus is not known, therefore it is not amenable to analysis by the polymerase chain reaction (PCR) and Southern blotting is required. We present a novel approach for analysis of the BglI RFLP using the PCR targeted to known sequence downstream in exon 26 of the FVIII gene. Briefly, the size of the genomic restriction fragment carrying the PCR target depends upon whether the RFLP site is present or absent. If fragments of the required size are isolated from a genomic digest and used as substrates in the exon 26 PCR, the generation of a product in one or other fraction indicates the upstream RFLP status. We have called this approach "virtual PCR", since PCR is used to obtain information about the RFLP without amplifying the locus itself.

The Atrial Natriuretic Peptide Gene in Patients with Familial Primary Open-Angle Glaucoma

Family history is a major risk factor in the development of primary open-angle glaucoma. The atrial natriuretic peptide system has been implicated in the underlying pathophysiology of the disease. This study looked for any alterations in the ANP gene and 5′ proximal promoter regions of the ANP gene, in 53 patients from familial primary open-angle glaucoma families. The ANP gene was amplified by a long-PCR technique from peripheral blood DNA. Gross insertions or deletions in the gene were sought and allelic frequencies at two restriction fragment length polymorphism (RFLP) sites within the gene (ScaI,HpaII) were compared with allelic frequencies obtained from 60 normal controls with no known family history of glaucoma or ocular hypertension. PCR-based single strand conformation polymorphism analysis was then used to search for possible point mutations in the 5′ proximal promoter region of the ANP gene, which is known to contain regulatory elements which modify gene transcription. No gross alterations in the ANP gene or differences in allelic frequencies at the RFLP sites within the gene were observed. PCR-SSCP analysis of the 5′ proximal promoter region of the gene revealed mutations in 10 patients in the −595 to −384bp region (19% of patients). Mutations in the 5′ proximal promoter region of the ANP gene may contribute to altered ANP transcription in at least a proportion of patients with familial glaucoma.

Population haplotype analysis and evolutionary relations of the COL2A1 gene.

We have determined the allele frequencies and pairwise linkage disequilibria of restriction fragment length polymorphisms (RFLPs) distributed over the entire COL2A1 gene (spanning 23.6 kb) in a population of unrelated Dutch Caucasians. Pairwise linkage disequilibrium analysis of RFLP sites between exon 5B and 51 indicated a high degree of partly positive (the rare alleles of both loci are associated) and partly negative (the rare allele is associated with the common allele) linkage disequilibrium. The high degree of linkage disequilibrium enabled the assignment of 13 out of 128 possible haplotypes with 7 RFLPs. An evolutionary tree of these haplotypes was derived using a minimum spanning tree approach, indicating at least two ancestral haplotypes. Our data indicate that disease related population studies involving the COL2A1 gene should include a minimum of 4 RFLPs (D9, A9, H33, P51) to obtain 98% of possible haplotypes occurring.

Frequent loss of heterozygosity at the deleted in colorectal carcinoma gene locus and its association with histologic phenotypes in breast carcinoma.

Loss of heterozygosity (LOH) at the deleted in colorectal carcinoma gene (DCC), a tumour suppressor gene that encodes a protein with high homology to the neural cell adhesion molecule, was investigated in 42 surgical specimens of primary breast carcinoma. LOH was analysed in breast carcinoma by amplifying the DNA, spanning a variable number of tandem repeats site and a restriction fragment length polymorphism site within DCC, using the polymerase chain reaction (PCR). Cell sorting was used to enrich carcinoma cells. The expression of the DCC gene was also investigated using a reverse transcription-PCR method followed by Southern blot hybridization. LOH at the DCC locus was detected in 15 (51.7%) of 29 informative cases and 10 of 13 cases having DCC-LOH showed distinct reduction or loss of DCC expression. The DCC-LOH was closely associated with certain histological phenotypes: DCC-LOH was more frequent in scirrhous carcinomas than in solid-tubular ones (P < 0.05), and was also more frequent in carcinomas with infiltration into fat tissue over the mammary gland than in those without infiltration (P < 0.05). DCC-LOH was detected in invasive lobular carcinomas (2/2), but in none of the noninvasive ductal carcinomas (0/2). These observations suggest that malignant histological phenotypes are associated with DCC-LOH.

High lysozyme activity in a Norwegian bovine family co-segregates with a restriction fragment length polymorphism.

Analysis of restriction fragment length polymorphism (RFLP) of the lysozyme gene cluster was performed in a Norwegian bovine family segregating a single dominant Mendelian factor for high lysozyme activity in serum. An RFLP site with allelic bands of 16kb and 5.9 kb turned out to be linked to the locus for the high lysozyme activity factor with a lod score of 6.8 at a recombination fraction of 3.4%. This implies that we have revealed a genetic marker for the high lysozyme activity trait.

A single polymorphic STR system in the human phenylalanine hydroxylase gene permits rapid prenatal diagnosis and carrier screening for phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive genetic disorder caused by phenylalanine hydroxylase (PAH) deficiency. Individuals afflicted with PKU develop irreversible mental retardation that can be largely prevented by the administration of a low-phenylalanine diet. A number of restriction fragment-length polymorphisms (RFLPs) have been identified in the PAH gene. Combinations of RFLPs constitute unique haplotypes that can be used to identify mutant PAH chromosomes for prenatal diagnostic purpose in PKU families. Unfortunately, the utility of haplotype analysis is limited in populations with a single predominant haplotype. We have identified a novel short tandem repeat (STR) within the PAH gene that has an average level of heterozygosity of about 75% in Orientals and about 80% in European Caucasian populations. This single marker is as informative as haplotype analysis in Europeans and nearly twice as informative as haplotype analysis in Orientals. Although there is statistically significant disequilibrium between STR alleles and RFLP-based haplotypes, there is a relatively low degree of disequilibrium between STR alleles and certain RFLP sites. Nevertheless, the combined use of the STR and RFLP haplotype systems increases the informativity of linkage-based tests for prenatal diagnosis and carrier screening in PKU families.

Haplotype analysis at the low density lipoprotein receptor locus: application to the study of familial hypercholesterolemia in Israel.

Familial hypercholesterolemia (FH) results from mutations in the low density lipoprotein (LDL) receptor gene. It has been shown that restriction fragment length polymorphisms (RFLPs) associated with this gene may be used for family and population studies. The present investigation is a population-based study of 19 Jewish families with hypercholesterolemia representing 9 different countries of origin. Ten RFLP sites were used to construct 24 different haplotypes from 112 chromosomes. These haplotypes vary in frequency from 0.9% to 28.6%. Five previously undescribed haplotypes, which comprise 8.1% of the sample, are reported here. The six most common haplotypes account for 70% of the sample. Segregation analysis reveals that, in Israel, distinct LDL receptor haplotypes are associated with hypercholesterolemia in 12 (63%) out of the 19 Jewish families. Five LDL receptor haplotypes co-segregate with hypercholesterolemia. Two of these haplotypes seem to be unique to specific population groups in Israel and may therefore represent founder mutations.

Molecular defects in haemophilia B: detection by direct restriction enzyme analysis

The common restriction fragment length polymorphisms (RFLPs) associated with the FIX gene: 5' BamH I, Dde I, BamH I (2), Taq I and 3' Hha I were absent or of low incidence in Southern Chinese and are therefore not useful for linkage analysis. No deletion was detected amongst seven consecutive unrelated haemophilia B patients, but one had an insertion of a 15 kb Pvu II fragment containing exon d. Using an alternate strategy of polymerase chain reaction (PCR) amplification and direct sequencing, the molecular defect in the other six patients was defined. The four novel mutations characterized were: nucleotide (nt) 6410 G----C (Gly12----Ala); nt 31261 delta T (stop codon 31 bp downstream); nt 31260 C----G (Thr380----Ser) and nt 31122 C----A (Ala34----Asp). Two patients had the same mutation at nt 6365, G----A (Arg-4----Gln), identical to one previously described in other ethnic groups, suggesting that this is a hotspot for mutation. Each of the mutations was found to affect an enzyme recognition site and could thus be identified by direct visualization of abnormal restriction fragments in amplified genomic DNA. This allows rapid and accurate DNA diagnosis of haemophilia B in an ethnic group which otherwise shows little or no polymorphism for the common RFLP sites.

DNA haplotype analysis suggests linkage disequilibrium in the human insulin receptor gene

Haplotypes of the insulin receptor gene were resolved in parents from Scandinavian nuclear families by studying the segregation of seven restriction fragment length polymorphisms (RFLPs). Of 97 unrelated parents, 41 had non-insulin-dependent diabetes mellitus (NIDDM). Considerable linkage disequilibrium in the region of the insulin receptor gene was found. Pairwise non-random associations were found between proximate RFLP sites, indicating the absence of recombinational hot spots between these sites. Thus, association studies between DNA polymorphisms at this locus and disease susceptibility genes could well be feasible in this population. Differences in the distribution of insulin receptor haplotypes were examined between NIDDM patients and healthy subjects. However, the differences observed were not statistically significant.

Frequency of the F508 deletion in cystic fibrosis patients from the European part of the USSR

The frequency of the F508 deletion (delta F508) has been analyzed in 189 cystic fibrosis (CF) patients from the European part of the USSR, viz. 127 northern Slavonians (Leningrad region), 30 southern Slavonians (the Ukraine), 10 central Slavonians (Moscow region), 14 Moldavians (Kishenev region) and 8 Lithuanians (Vilnius region). The distribution of CF+ chromosomes with and without delta F508 varied significantly in the different ethnic groups studied and correlated with the clinical manifestation of CF. The overall frequency of delta F508 in Slavonian patients is equal to 62.5%, approximately 90% of them being heterozygous or homozygous for this mutation. The frequency of the deletion among 99 Slavonian patients with severe disease manifestation (pancreatic insufficiency, PI) is equal to 67.5%, only 12 patients having pancreatic sufficiency (PS, 17.5%). The highest value of delta F508 (77.4%) is registered in PI/CF patients of the southern Slavonian group; it is much less frequent (about 57%) in relevant groups of Slavonians from the northern and central parts of the country. Unusually low frequencies (24% and 26%) of delta F508 are detected in a few samples of Lithuanian and Moldavian CF patients, respectively. All delta F508+CF-chromosomes of Slavonian origin are associated with haplotypes 2.2.2. defined by the restriction fragment length polymorphism sites KM19/PstI, CS.7/Hin6I and MP6d-9/MspI, although a high proportion (about 25%) of unknown mutations is associated with the same haplotype. Haplotype B (allele 1XV2c/TaqI; allele 2 KM19/PstI) accounts for 91% of delta F508+CF chromosomes. Our data are consistent with the hypothesis of a single origin and subsequent diffusion of this major CF mutation; however, its interpopulational dissemination in Eastern Europe does not follow the suggested south-east to north-west gradient in Western Europe. The significance of these data for prenatal diagnosis and carrier screening of CF mutations is briefly discussed.