A sensitive denaturing gradient-Gel electrophoresis assay reveals a high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region.

A population study of heteroplasmy in the hypervariable region 1 (HV1) portion of the human mtDNA control region was performed. Blood samples from 253 randomly chosen individuals were examined using a sensitive denaturing gradient-gel electrophoresis (DGGE) system. This method is capable of detecting heteroplasmic proportions as low as 1% and virtually all heteroplasmy where the minor component is > or = 5%. Heteroplasmy was observed in 35 individuals (13.8%; 95% confidence interval [CI] 9.6-18.0). Of these individuals, 33 were heteroplasmic at one nucleotide position, whereas 2 were heteroplasmic at two different positions (a condition known as "triplasmy"). Although heteroplasmy occurred at a total of 16 different positions throughout HV1, it was most frequently observed at positions 16093 (n=13) and 16129 (n=6). In addition, the majority of heteroplasmic variants occurred at low proportions and could not be detected by direct sequencing of PCR products. This study indicates that low-level heteroplasmy in HV1 is relatively common and that it occurs at a broad spectrum of sites. Our results corroborate those of other recent reports indicating that heteroplasmy in the control region is more common than was previously believed-a finding that is of potential importance to evolutionary studies and forensic applications that are based on mtDNA variation.

Segregation analysis of two-locus models regulating apolipoprotein-A1 levels.

For quantitative traits associated with risk to complex diseases, such as heart disease, single major locus models are likely to be too simplistic. Currently, researchers have begun to use oligogenic models of inheritance, but the resolving power of these models remains to be determined. As the major apoprotein of high density lipoprotein (HDL), apolipoprotein A1 (apo-A1) is generally accepted as a protective factor for coronary artery disease. Although familial aggregation of apo-A1 levels has been reported, the mode of inheritance of apo-A1 remains ill defined. In the present study, we conducted a segregation analysis comparing a series of one-locus and two-locus univariate models for apo-A1 levels in a sample of 137 families ascertained through probands undergoing elective, diagnostic coronary angiography. A two-locus Mendelian model fit these data significantly better than any one-locus model. The incorporation of the second major locus into the model of inheritance leads to a significant improvement in the fit, and a significant decrease of the residual heritability. The best-fitting model included two loci with a reciprocal pattern of epistasis generating 4 distinct genotypic distributions. Taken together, these two major loci account for 58% of the variance of adjusted apo-A1 levels. This demonstration of a second major locus controlling apo-A1 levels may explain the equivocal results obtained from previous studies. This two-locus model may be more powerful for linkage analysis to map one or both of these quantitative trait loci.

Linkage of the apo CIII microsatellite with isolated low high-density lipoprotein cholesterol.

To evaluate whether a highly polymorphic microsatellite region within intron 3 of the apolipoprotein (apo) CIII gene is linked to the isolated low HDL-C phenotype, we studied eight unrelated probands (mean HDL-C = 10+/-5 mg/dl) and 157 biological family members. After PCR amplification of genomic DNA and denaturing polyacrylamide gel electrophoresis, 26 alleles were identified in this microsatellite including 9 alleles heretofore unreported. Quantitative sib-pair linkage analysis demonstrated strong evidence of linkage between the isolated low HDL-C phenotype and the apo CIII microsatellite region (P = 0.007). The microsatellite was also linked to apo AI (P = 0.001), the primary apolipoprotein of HDL-C. Therefore, this highly polymorphic microsatellite region is a potentially important marker in the genetic evaluation of the isolated low HDL-C phenotype.

A comprehensive analysis of complex traits in problem 2A.

We used descriptive analysis to investigate the relationship between affection status and five quantitative traits (Q1-Q5) in Problem 2A and results suggested the five quantitative traits fall into two groups. The first group comprised three strongly correlated traits, Q1-Q3, which underlie affection status, and the second group comprised Q4 and Q5, which are not directly related to affection status. Segregation and linkage analyses of traits Q1-Q3 and affection status from the first replicate detected one of the major loci for Q1 (MG1) linked to marker 14 on chromosome 5 (D5G14). Because our segregation analysis failed to show evidence of a major locus effect on Q2, and we overlooked the interaction between MG3 and sex, we did not detect either MG2 or MG3. Using Haseman-Elston sib-pair analysis [Haseman and Elston, 1972], we also examined the statistical power of Q1 and type I error rate (using the environmental factor as an index), for the remaining 199 replicates in the context of a genome screen.

Segregation analysis of HDL3-C levels in families of patients undergoing coronary arteriography at an early age.

HDL cholesterol (HDL-C) level is a risk factor for coronary heart disease. Studies have shown a strong genetic influence on HDL-C levels in addition to environmental influences, but no definite major gene control has been demonstrated. Since HDL subfractions may better reflect the actions of distinct metabolic alterations than total HDL2 we tested the hypothesis that the amount of cholesterol in the denser HDL3 subfraction (HDL3-C) is under the control of a major gene. The study population included 676 family members of 116 probands who underwent coronary arteriography at an early age (men < or = 50 and women < or = 60 years). HDL3-C level was measured by using enzymatic methods after preparative ultracentrifugation at a density of 1.125 g/mL. HDL3-C was adjusted for age, gender, alcohol consumption, and smoking, which combined accounted for 3% of its variance. Segregation analysis was conducted on adjusted HDL3-C by using regressive models. The familial correlations for HDL3-C levels were spouse .03 +/- .08, parent-offspring .14 +/- .05, and sibling .24 +/- .05. The data strongly supported a codominant mendelian model, with the common allele coding for lower HDL3-C levels and the rarer allele (frequency, 25%) coding for higher HDL3-C levels. This major gene explained 34% of the variation in HDL3-C levels and 9% of the variation in total HDL-C levels. These results suggest that HDL3-C levels exhibit clearer genetic control than total HDL-C and may therefore be a useful target for further genetic studies.

A locus for cerebral cavernous malformations maps to chromosome 7q in two families.

Cavernous malformations (angiomas) affecting the central nervous system and retina can be inherited in autosomal dominant pattern (OMIM 116860). These vascular lesions may remain clinically silent or lead to a number of neurological symptoms including seizure, intracranial hemorrhage, focal neurological deficit, and migraine. We have mapped a gene for this disorder in two families, one of Italian-American origin and one of Mexican-American origin, to markers on proximal 7q, with a combined maximum lod score of 3.92 (theta of zero) with marker D7S479. Haplotype analysis of these families places the locus between markers D7S502 proximally and D7S515 distally, an interval of approximately 41 cM. The location distinguishes this disorder from an autosomal dominant vascular malformation syndrome where lesions are primarily cutaneous and that maps to 9p21.

Prospective study of occupational asthma to laboratory animal allergens: stability of airway responsiveness to methacholine challenge for one year.

The stability of airway hyperresponsiveness was studied in a group of 178 young adults working with laboratory animals. At the time of their entry into the study, 132 of 178 subjects (74%) had less than 20% response to the inhalation of 25 mg/ml methacholine, whereas 26 (15%) had a methacholine dose causing a 20% fall in forced expiratory volume in 1 second after fewer than 80 breath units. The distribution of methacholine responsiveness did not differ at 6 months and 1 year; 155 of 178 volunteers (90.4%) responded during the repeated challenges to doses within one dilution of their results at entry. One hundred forty-one subjects were consistently unreactive during the year, and 17 were consistently reactive. Approximately equal numbers gained and lost reactivity. Those with consistently positive responses to methacholine were more likely to have skin test reactivity and chest symptoms. The presence of consistent chest symptoms was loosely associated with consistent methacholine responsiveness; 55% of those with consistent hyperresponsive airways had symptoms, and 24% of those who consistently had symptoms had hyperresponsive airways. We concluded that the methacholine response is relatively stable during the course of a year in laboratory animal workers who remain at their jobs and that the presence of a positive skin test response to laboratory animals or of chest symptoms does not change the pattern of stable responsiveness.

Two-locus approach of segregation and linkage analysis in the study of complex traits.

A two-locus segregation and linkage-analysis approach was used to characterize the genetic control of a complex trait (Q1) and to localize the genes that have detectable effects. The results suggested that a two-locus Mendelian model fit the data significantly better than a one-locus model. The linkage results based on the most parsimonious two-locus model revealed linkage of Q1 to two areas (MG2 and MG3), while there was less evidence for linkage using one-locus models. Results also suggested that the subphenotypes (Q2 and Q3) provided useful information for further analysis of Q1 using two-locus models.

Genetic epidemiologic study of hearing loss in an adult population.

Previous population studies of hearing loss have been limited to children with moderate to profound impairment, and have reported that heritability accounts for at least 50% of congenital or early-onset cases. The present study was designed to assess genetic factors associated with late-onset hearing impairment in an adult population. A brief family history and audiologic questionnaire was sent to approximately 11,200 members of the consumer organization, Self Help for the Hard of Hearing, Inc., and 4,039 questionnaires were returned. All respondents reported having at least one previous audiologic exam. Reported data were verified against audiograms when available. Regardless of the reported causes, 49% of early-onset cases (< or = 20 years of age) had one or two parent(s) with some form of hearing loss compared with 62% in later-onset cases. As expected, mean age at onset was substantially younger for cases with positive family histories than cases with negative family histories. Results from nuclear segregation analysis showed that fully recessive and dominant models failed to explain the early- or late-onset hearing loss data. In this nationwide survey, the large proportion of cases with positive family histories clearly indicates the importance of genetic factors in adult-onset forms of hearing loss. Comparison with younger-onset cases will permit further delineation of differences in inheritance patterns. This study should identify more homogeneous groups of adult-onset families for further genetic study, and provide empiric information for use in genetic counselling.

Two-locus and bivariate segregation analysis of HDL-C and apo AI.

Two-locus and bivariate segregation analyses of HDL-C and apo AI were carried out using the GAW8 Berkeley data set. For HDL-C, results are ambiguous. Both the mixed environmental model and the mixed Mendelian model fit the data equally well. For apo AI, however, univariate one-locus segregation analysis suggests a Mendelian major gene controlling its levels. Moreover, two-locus analysis suggests a second major gene is involved. Bivariate segregation analysis indicates these data cannot resolve the question as to whether a single Mendelian major gene with pleiotropic effects controls levels of both HDL-C and apo AI in humans.

A genetic model for control of hypertriglyceridemia and apolipoprotein B levels in the Johns Hopkins colony of St. Thomas Hospital rabbits.

The St. Thomas Hospital (STH) rabbit has been previously shown to have a Mendelian form of hypertriglyceridemia, accompanied by accelerated atherosclerosis, and these animals may serve as a useful model for human dyslipoproteinemia syndromes. Here we describe the establishment of a new colony of these STH animals, and present genetic analysis of triglyceride (TG) and apolipoprotein B (apoB) levels. Segregation analysis of TG in 39 STH animals and 24 controls gave evidence of Mendelian segregation for an allele leading to both elevated TG levels and increased variability in these levels. Predicted means from the most parsimonious model for the Johns Hopkins STH colony were quite similar to that seen in the original London colony, and this model accounted for 80% of the variation in TG seen in the sample. This hypertriglyceridemia locus indirectly influenced the mean apoB levels in these rabbits, and segregation analysis of mean apoB levels suggested a second locus controlling apoB levels. Analysis of residual apoB levels (adjusted for predicted effects of the hypertriglyceridemia locus) revealed clearer evidence for a second locus controlling mean apoB levels in this colony. Arguments for two distinct genetic mechanisms operating in these STH animals are presented.

Risk factors for increased airway responsiveness to methacholine challenge among laboratory animal workers.

As a first step in a prospective study of the incidence of asthma to laboratory animals, a group of 364 adults 18 to 48 yr of age who were beginning employment with laboratory animals were evaluated in terms of their past history, health status, allergy, and airway responsiveness to methacholine. At entry to the study, 269 had previous occupational contact with animals, 109 had chest symptoms in the previous year, 168 had a history of allergic symptoms to laboratory animals (any with asthmatic responses were systematically excluded), and 118 had positive immediate skin tests (29 had positive skin tests to laboratory animals). When defined as a PD20FEV1 of 80 breath units or less, 18.4% of these young adults had methacholine hyperresponsiveness (HRA). Significant risk factors for HRA were found to be younger age, female sex, lower educational level, a history of allergic symptoms to laboratory animals, and a history of chest symptoms. Positive skin tests to laboratory animals were present in 8% of workers; this was not a significant risk factor for HRA although positive skin tests to pollen and household allergens were. Previous work experience was a risk factor, especially among those with allergic symptoms, and a trend toward self-selection was suggested in that the rate of HRA was lowest in workers with more than 2 yr of experience or with two or more previous jobs with laboratory animals.

Genetic determination of high-density lipoprotein-cholesterol and apolipoprotein A-1 plasma levels in a family study of cardiac catheterization patients.

Plasma levels of two lipoprotein risk factors, high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-1 (apo A-1), have been shown to be negatively associated with the risk of developing coronary artery disease, and several reports have examined familial factors in HDL-C and apo A-1 levels. A number of studies suggest that shared genes influence familial resemblance of these lipoprotein levels far more than do shared environments. Possible mechanisms for the inheritance of these two risk factors (HDL-C and apo A-1 plasma levels) are explored using data from 390 individuals in 69 families ascertained through probands undergoing diagnostic cardiac catheterization. Segregation analysis was used to test a series of specific models of inheritance. Evidence for single-locus control of apo A-1 levels, with Mendelian transmission of a dominant allele leading to elevated apo A-1 levels, was seen in these families, although there was additional correlation among sibs present. This locus accounted for 48.6% and 37.2% of the total variation in apo A-1 levels in males and females, respectively. Similar evidence of segregation at a single locus controlling HDL-C levels was not seen in these families.