HindIII DNA polymorphism in the lipoprotein lipase gene and plasma lipid phenotypes and carotid artery atherosclerosis

Lipoprotein lipase (LPL) is the rate limiting enzyme in the hydrolysis of core triglyceride in chylomicron and very low density lipoprotein (VLDL) thus affecting a broad spectrum of plasma lipid levels. In this paper, we investigated the association of a HindIII polymorphism in the LPL gene with plasma lipid levels and carotid artery wall thickness measured by B-mode ultrasonography. A total of 238 Caucasian subjects were selected from the Atherosclerosis Risk In Community (ARIC) study (male = 131, female = 107) based on their fasting triglyceride and LDL-cholesterol levels: normolipidemic (n = 48), hypertriglyceridemic (n = 44), hypercholesterolemic (n = 36), and hypertriglyceridemic-hypercholesterolemic (n = 110) groups. We observed a marginally significant association between lipid phenotypes and HindIII genotypes (P = 0.04) in males, with the hypertriglyceridemic and hypercholesterolemic groups having a higher frequency (0.65) of the H+H+ genotype than the other two groups (pooled: 0.55). In males, there was also a significant association between HindIII genotypes and carotid artery wall thickness after considering the effects of age, body mass index, cigarette smoking, lipid phenotype and diabetes status (P = 0.013), with the H+H+ genotype having a higher average value of carotid artery wall thickness (0.84 ± 0.15 mm) than the other two genotype groups (0.76 ± 0.14 mm in H+H– genotype class, 0.75 ± 0.13 mm in H–H– genotype class). In females, no significant associations among LPL HindIII genotype, lipid phenotype and carotid artery wall thickness were observed. These results suggest that the LPL HindIII polymorphism influences LPL-catalyzed, triglyceride-rich lipoprotein metabolism and carotid artery atherosclerosis in a gender-specific manner. Received: 28 December 1995 / Revised: 2 May 1996     

Association of the HindIII Polymorphism of the Lipoprotein Lipase Gene with Myocardial Infarction and the Life Span in Elderly Patients with Coronary Heart Disease

Allele and genotype frequencies of the HindIII polymorphism of the lipoprotein lipase (LPL) gene were studied in patients with myocardial infarction (MI) and stable angina of effort (SAE), including long-lived people (over 90). The polymorphism proved to be associated with MI and with the life span, genotype H+/H+ being predisposing to MI and allele H– being protective. The allele and genotype frequencies of long-lived people differed significantly from the Hardy–Weinberg proportions and from those of SAE patients aged up to 90. An excess of heterozygotes in this group suggests a selective pressure which eliminates homozygotes. Possibly, heterozygotes H+/H– have an adaptive advantage, which provides for their longevity.     

Genetic variation in MDR1, LPL and eNOS genes and the response to atorvastatin treatment in ischemic stroke

Statins reduce the risk of cardiovascular events by lowering the blood cholesterol. Many genes involved in the pharmacodynamic pathway of statins have been part of pharmacogenetic research in patients with hypercholesterolemia, with an emphasis on genes involved in the cholesterol pathway. The present study was carried out with an aim to evaluate the association between the genetic variants of lipoprotein lipase gene [HindIII (+/+)/HindIII (?/?)], multiple drug resistance gene (C3435T) and endothelial nitric oxide synthase gene (4a/4b) with clinical outcome including an increased risk of recurrent stroke or death in ischemic stroke patients on atorvastatin therapy. 525 stroke patients and 500 healthy controls were involved in the study. Follow-up telephone interviews were conducted with patients post-event to determine stroke outcome. Blood samples were collected and genotypes determined by polymerase chain reaction-restriction digestion technique. A significant association of MDR1 and LPL gene variants with bad outcome in stroke patients on atorvastatin therapy was found. However, there was no significant association of 27?bp VNTR polymorphism of eNOS gene with outcome. MDR analysis was carried out to analyze gene–gene interaction involving these gene variants contributing to clinical outcome of patients on stratin therapy but no significant interaction between these variants was observed. In conclusion the individuals with HindIII (?/?) genotype of LPL and CC genotype of MDR1 gene would benefit more from atorvastatin therapy.     

Cloning of the yeast ribosomal DNA repeat unit in SstI and HindIII lambda vectors using genetic and physical size selections.

The size of DNA fragments complementary to ribosomal RNA was determined in SstI and HindIII restriction spectra from totally and partially cleaved yeast (Saccharomyces cerevisiae) DNA. The results indicated that the yeast ribosomal RNA gene cluster consists of 9000 base-pair long tandemly repeated units. Three different repeating units, which are overlapping with respect to their sequences, were cloned as SstI and HindIII fragments with λ vectors. The isolation of these clones was facilitated by genetic or physical preselection for those recombinant phage which contained DNA inserts in the expected size range. Both preselection methods gave about a 30-fold purification with respect to the λ-rDNA clones. A heteroduplex analysis of the clones obtained with a three-component HindIII vector showed that the center part of the λ genome carrying λ recombination and regulation genes (57 to 77% λ) can become inverted without apparent decrease of growth capacities.     

Molecular cloning and sequence of theBacillus stearothermophilus translational initiation factor IF2 gene

Summary The structural gene for theBacillus stearothermophilus initiation factor IF2 was localized to a 6 kbHindIII restriction fragment by cross-hybridization with theSstI-SmaI fragment of theEscherichia coli infB gene. This fragment corresponds to the central region of the molecule containing the GTP-binding domain which is homologous inE. coli IF2, EF-Tu, EF-G and the humanras1 oncogene protein. After cloning into pACYC177, theHindIII fragment was further analysed by restriction mapping and cross-hybridization. A smaller (2.2 kb)SphI-HindIII fragment, which showed cross-hybridization, was subcloned into M13 phage and sequenced by the dideoxy chain-terminating method. This fragment was found to contain the entire IF2 gene except for the region coding for the N-terminus. This remaining region, coding for 45 amino acids, was located by homologous hybridization on an overlappingClaI-SstI fragment which was also subcloned and sequenced. Overall, theB. stearothermophilus IF2 gene codes for a protein of 742 amino acids (M_r=82,043) whose primary sequence displays extensive homology with the C-terminal two-thirds (but little or no homology with the N-terminal one-third) of the correspondingE. coli IF2 molecule. When cloned into an expression vector under the control of the λP_L promoter, theB. stearothermophilus IF2 gene, reconstituted by ligation of the two separately cloned pieces, could be expressed at high levels inE. coli cells.     

A HindIII/BglII dystrophin gene polymorphism in the African-American population

Summary We describe a common dystrophin gene polymorphism in the black population that alters both HindIII and BglIII restriction sites.     

中国人群脂蛋白脂肪酶基因突变与高脂血症的相关性研究

为进行脂蛋白脂肪酶基因突变与中国人群高脂血症的相关性研究,采用单链构象多态性分析结合DNA序列测定的方法,对386例(其中108例高脂血症患者,278例正常对照)中国人群进行突变筛查。结果发现1个新的沉默突变L103L,1个错义突变P207L,3个剪接突变Int3/3′-ass/C(-6)→T和普遍存在的S447X多态性,其中发生在高脂血症组的P207L杂合子为亚洲首报,并对先证者的家系进行了研究,认为P207L是家族性高脂血症的病因之一,而在正常对照组中也有发现的Int3/3′-ass/C(-6)→T,对以往研究认为其是高脂血症易患因素的观点提出了相反的报告,对于普遍认为有益的多态性位点S447X,进一步研究认为其对于正常人群,特别是健康男性的保护作用更强。结论:脂蛋白脂肪酶基因变异与高脂血症的相关性十分复杂多样,大规模的人群筛查具有重要意义。     

The calcium-sensing receptor R990G polymorphism is associated with increased risk of hypertriglyceridemia in obese Chinese

Background

We have demonstrated that the calcium-sensing receptor (CaSR) is involved in lipid metabolism; however, whether CaSR polymorphisms affect lipid metabolism in obesity is still unclear. The present study aimed to determine the effects of CaSR polymorphisms on HTG risk in obese Chinese.

Methods

A total of 972 subjects with HTG and 1197 with normal triglyceride (NTG) were stratified by body mass index (BMI) into normal weight, overweight or obesity subgroups. After 12-h fasting, CaSR polymorphisms in exon 7 were determined in the blood. Serum lipids and glucose, as well as height, body weight and waist circumference were measured. The anthropometric and metabolic characteristics of the NTG subjects were re-evaluated 3 years later.

Results

There were no genotypic or allelic distribution differences for the A986S or Q1011E polymorphisms between the NTG and HTG groups. However, the G/G genotypic and G allelic distributions of the CaSR R990G polymorphism in the HTG group were higher than the NTG group (p < 0.001). After stratification, in obese subjects, the homozygous (G/G) distribution of the CaSR R990G polymorphism in the HTG group was significantly higher than in the NTG group (p = 0.001), and showed an increased risk of HTG at baseline [OR = 2.55, 95% CI = 1.65–3.92, p < 0.006]. Interaction of the CaSR R990G polymorphism with BMI was associated with increased risk of HTG (β = 0.927, p < 0.001). Re-evaluation of the NTG subjects revealed significantly increased serum triglyceride levels in obese homozygous versus wildtype carriers (p < 0.05).

Conclusions

These results suggest that the CaSR R990G polymorphism is associated with increased risk of HTG, especially in obese Chinese, and may be a potential genetic predictor of diseases related to HTG.     

MspI andHindIII restriction fragment length polymorphisms at the human Na,K-ATPase β-subunit (ATP1B) gene locus

The authors report on four restriction fragment length polymorphisms detected at the ATP1B gene locus with the restriction endonucleasesHindIII andMspI.     

An apolipoprotein CIII marker associated with hypertriglyceridemia in Caucasians also confers increased risk in a west Japanese population

Polymorphisms and haplotypes at the adjacent apolipoprotein (apo) AI and CIII gene loci were investigated in 61 Japanese patients with triglycerides greater than 350 mg/dl and in 66 unrelated normolipidemic subjects. The polymorphic sites were the SstI site in the apoCIII 3 untranslated region, whose presence has previously been shown to be associated with hypertriglyceridemia (HTG) in Caucasians, and the MspI site in the third intron of the apoAI gene. The frequencies of the SstI minor allele (S2) were 0.48 in HTG patients and 0.25 in normolipidemic subjects (P < 0.00015). The frequencies of the MspI minor allele (M2) were 0.61 in HTG patients and 0.33 in normolipidemic subjects (P < 0.00001). The two polymorphic sites were in strong linkage disequilibrium, and maximum likelihood analysis supported the existence of three of the four possible haplotypes: S1-M1, S1-M2, and S2-M2. Since all S2 alleles were estimated to be present on M2-bearing chromosomes, the HTG-associated S2-M2 haplotype conferred the same approximate relative risk as the S2 allele alone when compared with the other two haplotypes (odds ratio 2.8). This study demonstrates that the S2 allele is a marker for HTG among west Japanese subjects as well as among Caucasians. The results suggest that S2-M2 chromosomes carry HTG susceptibility sequences that predate the separation of the Asian and Caucasian races.     

Genetic linkage between Huntington disease and the D4S10 locus in South African families: further evidence against non-allelic heterogeneity

Summary A study of genetic linkage between Huntington disease (HD) and the D4S10 locus (G8) has been undertaken in 10 South African (SA) families originating from the black, white and mixed acestry population groups. Allele frequencies at the D4S10 locus have been established in the non-Caucasoid population groups. There are significant differences in the allele frequencies at the D4S10 locus between the various SA populations. Clearly, information about population-specific frequencies for all polymorphisms is essential prior to the implementation of predictive testing in different population groups. Linkage has been demonstrated within this mixed group of HD families in SA using the HindIII, EcoRI and MspI polymorphisms, detected by G8. A maximum lod score of 8.14 at a recombination fraction of 0.00 (confidence limit 0–0.058) has been calculated using a combined haplotype of the HindIII and MspI polymorphisms. Taking into account the diverse ethnic backgrounds of the different SA population groups in this investigation, the data obtained from the study provide further evidence that there is probably only a single HD locus.     

Linkage disequilibrium relationships among four polymorphisms within the human fibrinogen gene cluster

The extent of linkage equilibrium was estimated among four recently characterized human fibrinogen restriction fragment length polymorphisms (RFLPs) using a randomly selected group of 110 individuals from California. Two coding region RFLPs, RsaI and MnlI (FGA codon 312 and FGB codon 448, respectively), and two RFLPs located in the 5 flanking region of the FGB gene, AluI (HindIII) and HaeIII, were analyzed. Maximum likelihood estimates based on genotypic data indicated that the RsaI polymorphism in the FGA gene was at apparent linkage equilibrium with the MnlI, AluI, and HaeIII sites in the FGB gene, but strong linkage disequilibrium was noted for the MnlI-AluI, MnlI-HaeIII, and AluI-HaeIII RFLP pairs within the latter gene. The discrepancy in disequilibrium relationships among these closely linked RFLPs may indicate a region of increased recombination between the FGA and FGB RFLP loci. The FGA RsaI polymorphism, when used in conjunction with any of the FGB sites examined, will provide more detailed linkage or association data than analyses that would utilize only FGB sites. Effective use of polymorphisms within the fibrinogen locus will aid analysis of the relationships between fibrinogen genotype, plasma fibrinogen levels, and risk of cardiovascular disease.     

Identification of three RFLPs at the HCK locus on chromosome 20

New HindIII, RsaI and TaqI restriction fragment length polymorphisms (RFLPs) within the haemopoietic cell kinase gene in chromosome band 20q11.2 are described. These RFLPs provide a useful marker for linkage analysis in proximal 20q.     

Use of RFLPs larger than 100 kbp to map the position and internal organization of the nucleolus organizer region on chromosome 2 in Arabidopsis thaliana

In Arabidopsis thaliana ribosomal RNA genes (rRNA genes or rDNA) are grouped in two nucleolus organizer regions (NORs) that together comprise approximately 6% of the genome. The map positions of the NORs relative to other genetic markers are unknown. It was found that the restriction endonuclease HindIII cuts once in some but not all rRNA genes to yield strain-specific RFLPs of 100–700 kb that could be visualized by pulsed-field gel electrophoresis and Southern blotting. The HindIII RFLPs of the A. thaliana strains Columbia and Landsberg segregated among recombinant inbred lines derived from a cross between these two strains. Linkage analysis placed the NOR bearing the polymorphic HindIII sites to the top of the upper arm of chromosome 2. The name NOR2 is suggested for this locus. HindIII-bearing rRNA genes are interspersed with clusters of HindIII-less genes throughout NOR2. The observed clustering is most consistent with unequal crossing-over, or nearest-neighbor gene conversion, as the mechanism(s) that spread rRNA gene variants throughout an NOR. No meiotic cross-over events yielding a ‘hybrid’ NOR with multiple RFLPs from both parents were observed among the 47 recombinant inbred lines examined. However, the appearance of novel HindIII profiles in approximately 40% of the recombinant inbred lines demonstrates that fluctuations in the distribution of rRNA gene variants occur frequently and can be readily detected on pulsed-field gels.     

Polymorphisms in the lipoprotein lipase and hepatic lipase genes and plasma lipid values in the Czech population

We have determined the genotypes of two common polymorphisms in the lipoprotein lipase (S447X) and hepatic lipase (-480C/T) genes in a cohort of 285 representative selected Czech probands (131 male and 154 female), examined in 1988 and reinvestigated in 1996. The genotype distributions of both polymorphisms were in Hardy-Weinberg equilibrium and did not differ between male and female subjects. The rare allele frequency of the lipoprotein lipase polymorphism did not differ significantly from the other European populations. Compared to the German populations, the frequency of the hepatic lipase -480T allele was significantly higher in the Czech group (20% vs. 36%, p<0.0001). There were no significant associations between the lipoprotein lipase gene variants and lipid parameters measured either in 1988, or in 1996 or with changes of lipid parameters over the 8-year period. The carriers of the T-480 allele of the hepatic lipase polymorphism were found to have higher HDL cholesterol levels (p=0.02). However, this difference was confined to female subjects only. The male carriers of the -480T allele had higher concentrations of total cholesterol (p=0.03) as compared to CC-480 subjects. Both associations were observed in 1996 only. In the Slavic Czech population, a common polymorphism in the hepatic lipase gene (-480C/T), but not in the lipoprotein lipase gene (S447X), is a significant determinant of plasma HDL cholesterol in females and plasma total cholesterol in males and indicates the importance of gender-associated effects in the genetic determinations of plasma lipids.     

Loss ofHindIII cleavage sites in thed-amino acid oxidase gene in some inbred strains of mice

Summary d-Amino acid oxidase cDNA was amplified by a polymerase chain reaction using RNA extracted from the mouse kidney. When digested withHindIII, the cDNAs of the BALB/c and ddY/DAO^– mice were cleaved into two fragments whereas the cDNA of the ddY/DAO⁺ mice was not. Sequencing revealed that nucleotide-471 of the cDNAs was G in the BALB/c and ddY/DAO^– mice whereas it was substituted for C in the ddY/DAO⁺ mice. This base substitution was the cause of the failure of the cleavage of the cDNA of the ddY/DAO⁺ mice.Examination of other strains of inbred mice showed thatd-amino-acid oxidase cDNAs of A/J, AKR, C57BL/6, CD-1, CF#1, ICR, DBA/2, NZB and NZW mice were cleaved withHindIII into two fragments whereas those of C3H/He, CBA/J and NC mice were not. Genomic DNAs extracted from the mice of these 15 strains were digested withHindIII and hybridized withd-amino-acid oxidase cDNA. A 18.2-kb fragment hybridized with the probe in the C3H/He, CBA/J, ddY/DAO⁺ and NC mice whereas two fragments of 12 kb and 6.2 kb hybridized in the other mice. These results are consistent with those of the cDNAs, confirming the loss of theHindIII cleavage site in the C3H/He, CBA/J, ddY/DAO⁺ and NC mice. The Southern hybridization revealed a loss of a differentHindIII cleavage site in the A/J, AKR, C57BL/6, DBA/2, ICR and NZB mice.The substitution at nucleotide-471 should cause a substitution of an amino acid residue. However, this substitution did not affect catalytic activity ofd-amino acid oxidase.     

SstII polymorphic sites in the promoter region of the human cystatin C gene

Summary By direct sequencing of polymerase chain reaction (PCR) amplified DNA from different individuals, three point mutations have been found in a 220-bp fragment from the promoter region of the human cystatin C gene. The three mutations are all localized within a short segment of 85 bp on the same allele. One of the base substitutions results in the generation of a novel SstII restriction site and another in the loss of the commonly occurring SstII restriction site. A PCR-based assay for analysis of the two SstII sites was designed and used to demonstrate Mendelian inheritance of the polymorphism. This SstII restriction fragment lenght polymorphism offers a new probe-independent marker for chromosome 20 linkage studies.     

Characterization of ard B and ard C actin gene loci of Physarum polycephalum

Summary Physarum polycephalum (strain M₃CVIII) contains four unlinked actin gene loci, each with two alleles (ard A1, ard A2, ard B1, ard B2, ard C1, ard C2, ard D1 and ard D2). The 4.8 kbp HindIII component of the ard C2 locus was isolated as a recombinant phage-, after HindIII fragments of Physarum DNA ranging from 4.3 kbp to 5.5 kbp were cloned into phage- NM1149. The fraction of Physarum DNA cloned contained the ard C locus, and no other actin locus. Small inserts were favoured to reduce the probability of cloning a complete repetitive element, because such elements have been found to adversely affect the stability of recombinants.The coding sequences of the actin gene (approximately 1.1 kbp) spanned more than 3 kbp indicating the presence of introns. A 1.6 kbp HindIII/EcoRI fragment of the ard C locus, which contained some coding sequences, hybridized extensively with HindIII fragments of genomic DNA indicating the presence of repetitive sequences. A 2.3 kbp HindIII/EcoRI fragment containing most of the coding sequences of the C2 allele of the ard C locus hybridized with the C1, allele and both alleles of the ard B locus, but not with the ard A locus or ard D locus. This distinction was used to establish for the ard B and ard C loci the relationship between the EcoRI and HindIII fragments that define an ard locus. The ability to distinguish between ard loci may facilitate studies of the expression of particular actin loci.     

Heterozygosity and localisation of normal allelic fragments for an alpha1-antitrypsin homologous sequence

Summary Approximately 10 kb downstream of the alpha₁-antitrypsin (AAT) gene is a homologous sequence. Two polymorphisms detected with the restriction enzymes Bg1II and EcoRI have been reported previously. We describe two additional polymorphisms with the restriction endonucleases TaqI and HindIII and, for all four restriction enzymes, we have mapped the fragments corresponding to the normal alleles in a cosmid clone.