PCR-based Restriction Fragment Length Research Articles

Association of Genetic Polymorphisms of TERT with Telomere Length in Coke Oven Emissions-Exposed Workers

ABSTRACT We explored the association between variations in the telomere maintenance genes and change in telomere length (TL) in workers. The TL of peripheral blood leukocytes from 544 coke oven workers and 238 controls were detected using the Real-time PCR method. Variations in four genes were then detected using the PCR based restriction fragment length polymorphism. The effects of environmental and genetic factors on TL were subsequently analyzed through covariance analysis and a generalized linear model .The TL of subjects with GG genotypes were longer than those with AG genotype in the TERT rs2736098 locus amongst the controls (P = .032). The combined effect of COEs exposure and AG+AA genotypes had a significant effect on TL (P < .001). The interaction between the COEs exposure factor and the rs2736098AG+AA genotypes had a significant effect on the TL (P < .05). The TL in coke oven workers is associated with the interactions between TERT rs2736098 AG+AA and COEs exposure.

Interaction of Interleukin 7 Receptor (IL7R) and IL6 Gene Polymorphisms with Smoking Associated with Susceptibility to Asthma in Chinese Han Adults

ABSTRACT Background the aim of this study was to investigate the relationship between the risk of asthma and multiple single nucleotide polymorphisms (SNPs) in interleukin 7 receptor (IL7R) and IL6 genes, as well as the gene- environment interactions. Methods This is a hospital- based case- control study. A total of 430 patients with asthma were continuously recruited. Four SNPs within IL7R and IL6 gene were genotyped by PCR based restriction fragment length polymorphism. The Hardy- Weinberg balance of all participants was tested by SNPstats. The best interaction combination of four SNPs in IL7R and IL6 genes and smoking was screened by generalized multifactor dimensionality reduction (GMDR). Logistic regression was used to test the association between four SNPs and asthma, and stratified analysis for rs1800795 gene-smoking interaction, synergy index (SI) was calculated. Results The rs1494558-G and rs1800795-C were associated with an increased risk of asthma, adjusted ORs (95% CI) was 1.81 (1.29–2.42) and 1.75 (1.20–2.28), respectively. GMDR indicated that the test accuracy for two-locus model involving rs1800795 and smoking was 0.5721, and the p = .011, the results providing evidence for rs1800795 gene-smoking interaction. The asthma risk was higher in smokers with GC or CC genotype than the sum of risks in subjects with smoking or GC or CC genotype alone, compared to the never smokers with GG genotype, the OR (95%CI) was 4.97 (3.01–7.24), and the synergy index (SI) was 1.68 (1.08–2.60). Conclusions The rs1494558-G and rs1800795-C alleles, gene- environment interaction between rs1800795 and smoking were all associated with increased asthma risk.

Impact of ATM and SLC22A1 Polymorphisms on Therapeutic Response to Metformin in Iranian Diabetic Patients.

Metabolic syndrome and its pathological sequel, type 2 diabetes are considered as important global health problems. Metformin is the most common drug prescribed for patients with this disorder. Consequently, understanding the genetic pathways involved in pharmacokinetics and pharmacodynamics of this drug can have a considerable effect on the personalized treatment of type 2 diabetes. In this study, we evaluated the association between rs11212617 polymorphism of ATM gene and rs628031 of SLC22A1 gene with response to treatment in newly diagnosed type 2 diabetes patients. We genotyped rs11212617 and rs628031 polymorphism by PCR based restriction fragment length polymorphism (RFLP) and assessed the role of this polymorphisms on response to treatment in 140 patients who have been recently diagnosed with type 2 diabetes and were under monotherapy with metformin for 6 months. Response to metformin was defined by HbA1c and fasting blood sugar (FBS) values. Based on such evaluations, patients were divided into two groups: responders (n= 63) and non-responders (n= 77). No significant association was found between these polymorphisms and response to treatment (OR= 0.86, [95% CI 0.52-1.41], P= 0.32) for rs11212617 and (OR= 0.45, [95% CI 0.64-1.76], P= 0.45) for rs 628031. The reported gene variants in ATM and SLC22A1 are not significantly associated with metformin treatment response in type 2 diabetic patients in an Iranian population.

Genetic variation in vascular endothelial growth factor gene and its association with recurrent spontaneous abortion.

Vascular endothelial growth factor (VEGF) plays a main role in fetal and placental angiogenesis and is secreted by different cells of endometrium and placenta. In the present study we investigated the association of VEGF gene polymorphisms with recurrent spontaneous abortion (RSA). A case-control study of 100 women with at least two consecutive pregnancy losses before 20 weeks of gestational age and 100 fertile controls was performed to evaluate four VEGF gene polymorphisms including +936C/T (rs3025039), -154G>A (rs1570360), rs3025010 and +5092A/C (rs2146323). Genotyping was performed by PCR based restriction fragment length polymorphism (PCR-RFLP) analysis. Haplotype frequency was estimated for three SNPs' genotypes. Analysis of genetic STRUCTURE and K means clustering were performed to estimate genetic variation. We found an association between -154G/A heterozygous genotype (GA) and RSA. The VEGF single nucleotide polymorphism (SNP) in intron region (rs3025010) in different inheritance models was also associated with RSA. Linkage disequilibrium analysis revealed that VEGF SNP in intron 5 (rs3025010) was linked to promoter region SNP (rs1570360). Cluster analysis including Neighbor Joining and K-means clustering supported genetic differentiation of women with RSA and controls. Allelic polymorphisms in common VEGF SNPs was associated with RSA samples and haplotypes with at least one minor allele showing an association with RSA pathogenesis (Tab. 8, Fig. 2, Ref. 35).

Association of kinase insert domain-containing receptor (KDR) gene polymorphism/ haplotypes with recurrent spontaneous abortion and genetic structure

Recurrent spontaneous abortion is one of the diseases that can lead to physical, psychological, and, economical problems for both individuals and society. Recently a few numbers of genetic polymorphisms in kinase insert domain-containing receptor (KDR) gene are examined that can endanger the life of the fetus in pregnant women. The risk of KDR gene polymorphisms was investigated in Iranian women with idiopathic recurrent spontaneous abortion (RSA). A case controlled study was performed. One hundred idiopathic recurrent spontaneous abortion patients with at least two consecutive pregnancy losses before 20 weeks of gestational age with normal karyotypes were included in the study. Also, 100 healthy women with at least one natural pregnancy were studied as control group. Two functional SNPs located in KDR gene; rs1870377 (Q472H), and rs2305948 (V297I) as well as one tag SNP in the intron region (rs6838752) were genotyped by using PCR based restriction fragment length polymorphism (PCR-RFLP) technique. Haplotype frequency was determined for these three SNPs' genotypes. Analysis of genetic STRUCTURE and K means clustering were performed to study genetic variation. Functional SNP (rs1870377) was highly linked to tag SNP (rs6838752) (D´ value=0. 214; χ(2) = 16.44, p<0. 001). K means clustering showed that k = 8 as the best fit for the optimal number of genetic subgroups in our studied materials. This result was in agreement with Neighbor Joining cluster analysis. In our study, the allele and genotype frequencies were not associated with RSA between patient and control individuals. Inconsistent results in different populations with different allele frequencies among RSA patients and controls may be due to ethnic variation and used sample size.

COMT Val158Met and 5-HT1A-R -1019 C/G polymorphisms: effects on the negative symptom response to clozapine.

Clozapine is still considered the gold standard for treatment-resistant schizophrenia patients; however, up to 40% of patients do not respond adequately. Identifying potential predictors of clinical response to this last-line antipsychotic could represent an important goal for treatment. Among these, functional polymorphisms involved in dopamine system modulation, known to be disrupted in schizophrenia, may play a role. We examined the COMT Val158Met polymorphism, which plays a key role in dopamine regulation at the prefrontal level, and the 5-HT1A-R -1019 C/G polymorphism, a target of clozapine activity involved in the interaction between the serotonin and dopamine systems. 107 neuroleptic-refractory, biologically unrelated Italian patients (70 males and 37 females) with a DSM-IV diagnosis of schizophrenia who were being treated with clozapine were recruited. Psychopathology was assessed by the Positive and Negative Symptoms Scale (PANSS) at the beginning of treatment, and at weeks 8 and 12. Genomic DNA was extracted from venous blood samples. COMT rs4680 (Val158Met) and 5-HT1A-R rs6295 (-1019 C/G) polymorphisms were analyzed by PCR-based restriction fragment length and direct sequencing, respectively. We found a significant effect of COMT and 5-HT1A-R on the PANSS Negative Subscale variation, with greater improvement among COMT Val/Val and 5-HT1A-R G/G subjects. The findings support the hypothesis that COMT rs4680 and 5-HT1A-R rs6295 polymorphisms could influence the negative symptom response to clozapine, probably through modulation of the dopaminergic system.

CYP1A1 Genetic Polymorphisms and Risk for Esophageal Cancer: a Case-control Study in Central China

The purpose of this study was to evaluate the associations of CYP1A1 genetic polymorphisms with the risk of developing esophageal cancer (EC). A case-control study was carried out in a Chinese population in which 157 hospital based EC cases and 157 population based healthy controls with 1:1 match by age and sex were included. PCR based restriction fragment length polymorphisms (PCR-RFLP) were used to detect genotypes in case and control groups. For the CYP1A1 Ile/Val polymorphism, comparing with wild genotype Ile/Ile, both the heterozygote genotype Ile/Val and the combined variant genotype Ile/Val+Val/Val increased the risk of esophageal cancer (OR: 2.05, 95%CI: 1.19-3.54, OR: 1.86, 95%CI: 1.11-3.12). No significant association was found between the CYP1A1 MspI polymorphism and EC. According to analysis of combined genotypes, the TC/AG combined genotype which contained both variant alleles of these two polymorphisms increased the risk of developing EC (OR: 2.12, 95%CI: 1.16-3.85). Our results suggested that genetic polymorphisms of CYP1A1 may increase the susceptibility to EC.

The role of IL6 and ESR1 gene polymorphisms as immunological factors of pregnancy maintenance

Aim. The study is aimed at the evaluation of the association of IL6 gene -174G/C polymorphism and ESR1 gene -397C/T polymorphism with recurrent pregnancy loss (RPL) pathogenesis and at the investigation of the ESR1 gene -397C/T variant regulatory significance for the IL6 gene function. Methods. A case group of 75 women with RPL history and a control group of 106 unrelated healthy women, who have given birth to at least one child conceived in natural way, were genotyped by a PCR based restriction fragment length polymorphism assay. Results. There was no significant difference in IL6 -174G/C or ESR1 -397C/T genotype and allele frequencies between the case and control groups. Combined genotype distribution analysis showed significantly (p < 0.05) lower frequency of individuals homozygous for both IL6 -174G and ESR1 -397C alleles in case group (0.026) comparing to control (0.094). Conclusions. Genotype comprising IL6 -174G and ESR1 -397C alleles in homozygous state may be considered as a genetic marker of successful pregnancy maintenance during gestation early stages.

The polymorphisms of XRCC1 gene and susceptibility to pulmonary cancer

To explore the relationship between the polymorphisms of DNA repair gene XRCC1 and susceptibility to pulmonary cancer. A case-control study of 209 lung cancer patients and 256 control subjects was conducted to investigate the role of XRCC1 gene in lung cancer. Genotyping was performed using PCR based restriction fragment length polymorphism (PCR-RFLP) technique. The frequency (19.1%) of XRCC1-194 Trp/Trp in case group was significantly higher than that (10.9%) in control group (P < 0.05), OR for lung cancer was 2.215 (95% CI: 1.276-3.845). The frequency (6.7%) of XRCC1-280 His/His in case group was significantly higher than that (4.3%) in control group (P < 0.05), OR for lung cancer was 2.46 (95% CI: 1.141-5.304). There was no significant difference for XRCC1-399 Gln/Gln genotype between the two groups. Interaction analysis of gene polymorphisms and environment factors indicated that there was interactions between XRCC1-194 Trp/Trp and XRCC1-280 His/His genotypes and smoking. The risks of lung cancer in smokers with XRCC1-194 Arg/Trp+Trp/Trp and XRCC1-280 His/His+Arg/His were 4.889 (95% CI: 2.828-8.452) and 6.281(95% CI: 3.572-11.046), respectively. These findings supported the hypothesis that the interaction of polymorphisms of XRCC1-194 Trp/Trp, XRCC1-280 His/His with smoking resulted in the increased risk of lung cancer, and the polymorphisms of XRCC and smoking could play an role in development of lung cancer.

An OGG1 polymorphism is associated with mitochondrial DNA content in pesticide-exposed fruit growers

Exposure to pesticides has the capacity to cause mitochondrial dysfunction. An increase mitochondrial DNA (mtDNA) content has also been suggested to relate with DNA damaging agent. In mitochondria, the manganese superoxide dismutase (MnSOD) is a scavenger of reactive oxygen species, and the 8-oxoguanine DNA glycosylase (OGG1) is the major DNA glycosylase for the repair of 8-oxoG lesions. However, the alteration of mtDNA content elicited by pesticide exposure in people with genetic variations in MnSOD or OGG1 has not been investigated. In this study, the mitochondrial to nuclear DNA ratio was quantified in the peripheral blood of 120 fruit growers who experienced pesticide exposure and 106 unexposed controls by real-time quantitative polymerase chain reaction (real-time qPCR). Questionnaires were administered to obtain demographic data and occupational history. The MnSOD and OGG1 genotypes were identified by the PCR based restriction fragment length polymorphism analysis. After adjusting for confounding effects, multiple regression model revealed that subjects experiencing high or low pesticide exposure had a greater mtDNA content than that of controls. The OGG1 Ser-Ser genotype was also associated with an increased mtDNA content. No association between MnSOD genotype and mtDNA content was revealed. Thus, subjects experiencing pesticide exposure had greater mtDNA content and the OGG1 genotype may modulate mtDNA content in pesticide-exposed fruit growers.

Association of beta 2 -adrenergic receptor gene polymorphisms and nocturnal asthma in Saudi patients

BACKGROUND AND OBJECTIVES:Two polymorphisms of beta2-adrenergic receptor (β2-AR) gene, namely the substitution from arginine (Arg) to glycine (Gly) at codon 16 and from glutamine (Gln) to glutamic (Glu) at codon 27, are linked with functional changes in the β2-AR in the respiratory system even though they are not deemed to be susceptibility genes for asthma per se. The objective of this study was to investigate this association in a subset of asthmatic patients, namely those with nocturnal asthma.METHODS:The β2-AR gene polymorphisms at codon 16 and 27 were assessed in 40 patients clinically diagnosed with nocturnal asthma and 96 normal controls. Genomic DNA was obtained from whole blood and genotyping was carried out by a PCR based restriction fragment length polymorphism technique.RESULTS:There was a statistically significant difference in genotype frequencies at codon 16 (Arg/Gly) between nocturnal asthmatic patients and normal control subjects (P < 0.05). However, there was no statistically significant difference in allele frequencies between the two groups. In addition, there was a significant association between Arg16-Gly genotype with nocturnal asthma compared to homozygous Gly16 (codominant model P = 0.0033, OR = 3.69: 95% CI: 1.49-9.12). However, there were no statistically significant differences in genotype and allele frequencies at codon 27 (Gln/Glu) between the normal control and nocturnal asthmatic groups (χ2 = 1.81, P = 0.41). The results also indicate that linkage disequilibrium existed between the β2-AR codon 16 and β2-AR codon 27 polymorphism (|D´| = 0.577). The data for all haplotypes did not show a statistically significant association.CONCLUSION:We present the genotype and allele frequencies of β2-AR gene polymorphisms in normal Saudi subjects and nocturnal asthmatic patients. There was a significant difference in genotype frequencies at codon 16 (Arg/Gly). However, our study indicates a poor association of individual single nuceotide polymorphisms with nocturnal asthma.

Polymorphisms of aquaporin5 gene in chronic obstructive pulmonary disease in a Chinese population.

Chronic obstructive pulmonary disease (COPD) is a major cause of chronic morbidity and mortality. It is influenced by both environmental and genetic factors. Aquaporin5 plays a critical role in the maintenance of normal lung water homeostasis. We investigated whether polymorphisms in the gene aquaporin5 had any bearing on individual susceptibility to the development of COPD. 332 COPD patients and 373 unrelated, age-matched healthy people were recruited for the study. All participants were Chinese Han people. We designed denaturing high performance liquid chromatography (DHPLC) to detect SNP in exons1, 2, 3 of AQP5 and DNA sequencing to confirm it. The allele +2254A>G (rs3736309) in intron3 and +3088A>G (Thr-Thr, rs41308104) in exon4 from NCBI dbSNP were genotyped by PCR-based restriction fragment length polymorphism. We found no SNPs in exons1-4 of the AQP5 gene in our study population. However, the genotype frequency of +2254A>G SNP in intron3 was significantly different in cases and controls. The frequencies of AA AG GG in cases were 45.2%, 40.7%, and 14.1%, while in controls they were 34.5%, 51.6%, and 13.9% (chi2 = 9.899, P = 0.007), respectively. Higher OR for COPD was seen for persons with +2254AA genotype against +2254AG genotype (OR = 2.73; 95%CI = 1.88-3.97). Carriers of the variant allele +2254G had a lower risk of COPD than homozygous wild type carriers (OR = 0.44; 95%CI = 0.307-0.631). The +2254A to G variant in intron 3 of AQP5 was associated with a decreased risk of COPD in a Chinese population.

PCR Based RFLP Genotyping of Bovine Lymphocyte Antigen DRB3.2 in Iranian Holstein Population

Major Histocompatibility Complex (MHC) class II locus DRB3 was investigated by PCR based restriction fragment length polymorphism (PCR-RFLP) assay. A total of 262 Holstein cows participating in the national recording system were sampled from 10 herds. A two-step polymerase chain reaction was carried out in order to amplify a 284 base-pair fragment of exon 2 of the target gene. Second PCR products were treated with three restriction endonucleas enzymes RsaI, BstYI and HaeIII. Digested fragments were analyzed by polyacrylamid gel electrophoresis. Twenty-eight BoLA-DRB3 alleles were identified. Identified alleles are: BoLA-DRB3.2 *3, *6, *7, *8, *9, *10, *11, *12, *13, *14, *15, *16,20, *21, *22, *23, *24, *25, *26, *27, *28, *32, *36, *37, *40, *51, *iaa and *ibb. The BoLA-DRB3.2*40 allele that was observed in this study has not been reported previously. The calculated frequencies were as follows: 2.29, 1.34, 0.19, 14.5, 0.38, 3.05, 12.21, 1.34, 2.29, 1.34, 2.48, 9.16, 0.95, 0.77, 6.68, 9.16, 17.94, 1.15, 0.57, 1.15, 0.95, 0.57, 0.38, 1.91, 0.38, 5.73, 0.19 and 0.95% respectively. The six most frequently observed alleles (BoLA-DRB3.2 *8, *11, *16, *22, *23 and *24) accounted for 69.65% of the alleles in these 10 herds. The results of this study confirm the allelic distribution of six most frequent alleles in Holstein population's worldwide.

Relationship between polymorphisms of DNA repair gene XRCC1 and susceptibility to radiation injury

To explore the relationship between polymorphisms of DNA repair gene XRCC1 and susceptibility to radiation injury. In 1:1 case-control study, 113 abnormal chromosome workers exposed to ionizing radiation were selected as cases and 113 normal chromosome as controls who matched with case for sex, age (+/- 5 years), nation, type of work, the same or more but in 2 years work length and the same similar levels of the cumulative exposure radiation dose. Genotypes were analysed using PCR based restriction fragment length polymorphism techniques. The frequency of XRCC1 26304TT allele in case group (18.58%) was significantly higher than that in control group (7.08%), with OR for radiation damage being 3.47 (95% CI 1.43 - 8.44, P < 0.05). No association was observed between XRCC1 G27466A and G28152A and susceptibility to radiation injury. The mutation of XRCC1 C26304T is related with the susceptibility to radiation injury. The polymorphisms of XRCC1 G27466A and G28152A are not found to have association with abnormal chromosomes.

Isolation of Campylobacter spp. from a pig slaughterhouse and analysis of cross-contamination

The purpose of this study was to establish the prevalence and possible contamination routes of Campylobacter spp. in a pig slaughterhouse. Swab samples were taken from the last part of rectum, from the carcasses surface before meat inspection and from slaughter line surface from 4 different pig herds during slaughtering. Identification of Campylobacter isolates was determined by the use of phase-contrast microscopy, hippurate hydrolysis, indoxyl acetate hydrolysis tests and PCR based restriction fragment length polymorphism method (PCR-RFLP). Pulsed-field gel electrophoresis (PFGE) typing using two macro-restriction enzymes SmaI and SalI was applied to in-slaughterhouse contamination analysis of pig carcasses. The study showed that 28 (63.6%) of the 44 samples collected at slaughterhouse were contaminated by Campylobacter spp. Up to 5 different colonies were obtained from each swab sample and a total of 120 different isolates were collected. 23.4% (28 of 120) isolates were identified as C. jejuni (19 from carcasses and 9 from slaughter line surfaces) and 76.6% (92 of 120) isolates as C. coli (28 from faeces, 47 from carcasses and 17 from slaughter line surfaces). The typing results showed identity between isolates from successive flocks, different carcasses, and places in the slaughterhouse in contact with carcasses. The results suggest that cross-contamination originated in the gastro-intestinal tract of the slaughtered pigs and that cross-contamination happened during the slaughter process.

Polymorphism of p16 INK4A and cyclin D1 in adenocarcinomas of the upper gastrointestinal tract

We investigated the prevalence of single nucleotide polymorphisms in the p16 gene (C540G) and the cyclin D1 gene (G870A), both known to regulate function in G1 arrest and therefore, may play an important role in carcinogenesis. Using PCR based restriction fragment length polymorphism and single strand conformational polymorphism, we determined single nucleotide exchanges in the p16 and cyclin D1 genes among 56 esophageal adenocarcinomas (ADC) arising in Barrett's esophagus, 95 cardiac gastric ADC, and in 191 distal gastric ADC. The allelic frequencies were compared to a control group of 253 healthy blood donors. The C/G genotype of p16 was identified in 10.4% of esophageal carcinomas, 13.3% of cardiac carcinomas, and in 14.1% of gastric carcinomas, compared to 17.4% in the healthy control group. All other cases showed the C/C wildtype, as no homozygous G/G nucleotide exchange was detected in the group of cancer patients or in the control group. In esophageal cancer, cyclin D1 G/G genotype was found 28.6%, A/G in 46.4%, and A/A in 25.0%. In cardiac carcinoma, frequency of cyclin D1 genotype was 27.4% for G/G, 57.9% for A/G, and 14.7% for AA. In distal gastric carcinoma, both homozygous genotypes (G/G and A/A) had a frequency of 15.2% each, while the heterozygous A/G genotype occurred in 69.6% of patients. The control group displayed 24.9% G/G, 53.8% A/G, and 21.3% A/A genotype. Our results show that frequencies of p16 or cyclin D1 polymorphisms in gastric and esophageal ADC do not differ significantly from the healthy control group. Therefore, these polymorphisms are unlikely to be associated with risk of ADC of the upper gastrointestinal tract.

Functional polymorphisms in cell death pathway genes FAS and FASL contribute to risk of lung cancer

Background: The FAS and FASL system plays a key role in regulating apoptotic cell death and corruption of this signalling pathway has been shown to participate in immune escape and...

Semiautomated typing of human papillomaviruses by restriction fragment length polymorphism analysis of fluorescence-labeled PCR fragments

A simplified version of a PCR-based reductional restriction fragment length polymorphism (rRFLP) approach for typing of human papillomaviruses (HPVs) is described previously [Wang et al., 1997]. It is achieved by the use of a biotin-labeled primer in PCR which, on restriction digestion and staining, is associated with only a single restriction fragment. In this report, we describe a further development of the rRFLP approach with the use of a fluorescence-labeled primer in PCR and fragment detection by laser scanning in an automatic sequencer. HPV typing is achieved by computer-assisted matching of the fluorescence-labeled rRFLP patterns with a database of rRFLP patterns of all known anogenital HPV types. On analysis of the typing of 133 HPV-positive cases using this procedure, 20 different HPV types were detected in exfoliated cervical cells in PAP smear samples derived from Taiwanese women. The results indicate the existence of a heterogeneous population of HPV types in Taiwan. Although most cases were associated with the more common HPV types, a significant fraction (about 20%) of the HPV types detected was related to the less common genotypes, which are often not included in commercial kits available for HPV typing. The results indicate the importance of covering as many HPV types as possible in clinical HPV genotyping protocols.

A new MspIPCR-RFLP in the human LDL receptor gene.

A new two-allele PCR-based MspI restriction fragment length polymorphism (RFLP) was revealed at the human LDL receptor locus. The polymorphic MspI restriction site is located in the 3'-untranslated region of exon 18 and caused by a T to C substitution at nucleotide 3960. This new MspI RFLP may be useful with other informative RFLPs to follow the segregation of the LDL receptor gene in families with hypercholesterolemia.