M. A. Province

Familial resemblance for VO2max in the sedentary state: the HERITAGE family study

This study investigates the familial resemblance of maximal oxygen uptake (VO2max) based on data from 86 nuclear families of Caucasian descent participating in the HERITAGE Family Study. In the current study, VO2max was measured twice on a cycle ergometer in 429 sedentary individuals (170 parents and 259 of their offspring), aged between 16 and 65 yr. The VO2max was adjusted by regression procedures for the effects of 1) age and sex; 2) age, sex, and body mass; and 3) age, sex, body mass, fat mass, and fat-free mass, as determined by underwater weighing. Evidence for significant familial resemblance was observed for each of the three VO2max phenotypes. Spouse, sibling, and parent-offspring correlations were significant, suggesting that both genetic and environmental factors contribute to the familial resemblance for VO2max. Maximal heritability estimates were at least 50%, a value inflated to an undetermined degree by nongenetic factors. The hypothesis of maternal inheritance, with the fathers contribution being environmental, was also found to fit the data with estimates of maternal heritability, potentially associated in part with mitochondrial inheritance, reaching about 30%. These results suggest that genetic and nongenetic factors as well as maternal influences contribute to the familial aggregation of VO2max in sedentary individuals.

Sequence Variants in the Sulfonylurea Receptor (SUR) Gene Are Associated With NIDDM in Caucasians

NIDDM is a common heterogeneous disorder, the genetic basis of which has yet to be determined. The sulfonylurea receptor (SUR) gene, now known to encode an integral component of the pancreatic β-cell ATP-sensitive potassium channel, IKATP, was investigated as a logical candidate for this disorder. The two nucleotide-binding fold (NBF) regions of SUR are known to be critical for normal glucose regulation of insulin secretion. Thus, singlestrand conformational polymorphism analysis was used to find sequence changes in the two NBF regions of the SUR gene in 35 NIDDM patients. Eight variants were found; and three were evaluated in two Northern European white populations (Utah and the U.K.): 1) a missense mutation in exon 7 (S1370A) was found with equal frequency in patients (n = 223) and control subjects (n = 322); 2) an ACC→ACṮ silent variant in exon 22 (T761T) was more common in patients than in control subjects (allele frequencies 0.07 vs. 0.02, P = 0.0008, odds ratio (OR) 3.01, 95% CI 1.54–5.87); and 3) an intronic t→c change located at position –3 of the exon 24 splice acceptor site was also more common in patients than in control subjects (0.62 vs. 0.46, P < 0.0001, OR 1.91, 95% Cl 1.50–2.44). The combined genotypes of exon 22 C/T or T/T and intron 24 –3c/–3c occurred in 8.9% of patients and 0.5% of control subjects (P < 0.0001, OR 21.5,95% CI 2.91–159.6). These results suggest that defects at the SUR locus may be a major contributor to the inherited basis of NIDDM in Northern European Caucasians.

Common Variants in the 5′ Region of the Leptin Gene Are Associated with Body Mass Index in Men from the National Heart, Lung, and Blood Institute Family Heart Study

Linkage of body mass index (BMI) to a broad region of chromosome 7q22-35 has been reported in multiple studies. We previously published a multipoint LOD score of 4.9 at D7S1804 for BMI from the National Heart, Lung, and Blood Institute Family Heart Study. Leptin (LEP), the human homolog of the mouse obesity (ob) gene, is positioned near the linkage peak and is the most prominent candidate gene in this region. Interest in LEP as a susceptibility gene for human obesity has led to numerous linkage and association studies, but the results of these studies are still controversial. In the present study, we employed family-based tests of association with both a quantitative measure of BMI adjusted for age and sex and a dichotomously defined obesity trait. We genotyped 29 single-nucleotide polymorphisms (SNPs) spanning 240 kb around the LEP gene in the 82 extended pedigrees with the strongest evidence for linkage. When the programs TRANSMIT and FBAT were used, a number of SNPs showed association in men but not women, for both the quantitative and qualitative trait definitions (P<.05). Five SNPs (H1328084, H1328083, H1328082, H1328081, and H1328080) positioned 2 kb beyond the previously defined promoter region showed strong association in single-marker and multiple-marker haplotype analysis. This five-marker haplotype (frequency 49% in this sample) is overtransmitted to obese offspring (P=.00005). All five of these SNPs are predicted to modify transcription-factor binding sites. This may indicate new functional variants in an extended promoter region of LEP.

Genetic analysis of glucose tolerance in inbred mouse strains. Evidence for polygenic control.

To determine genetic factors involved in diabetes susceptibility in inbred strains of mice, we initially evaluated differences in fed plasma glucose and insulin concentrations among six strains (AKR/J, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J). There was considerable variation in fed plasma glucose concentration, with C3H/HeJ mice the most glucose tolerant (174 ± 7 mg/dl) and C57BL/6J mice the least glucose tolerant (252 ± 7 mg/dl, P < .0001 vs. C3H/HeJ mice). Glycosylated hemoglobin of C57BL/6J mice (4.0 ± 0.06%) was also higher than that of C3H/HeJ mice (3.52 ± 0.06%, P < .0001). The fed plasma insulin concentration did not differ between these two strains. Glucose tolerance was further evaluated in overnight-fasted C3H/HeJ and C57BL/6J mice by an intraperitoneal glucose tolerance test (IPGTT). Although fasting plasma glucose did not differ, the most remarkable difference in plasma glucose during IPGTT between C57BL/6J and C3H/HeJ mice was noted at 30 min (489 ± 29 vs. 227 ± 20 mg/dl, P < .001). To determine the number of genes involved in the phenotypic difference in glucose tolerance, C57BL/6J males were crossed with C3H/HeJ females (F1, C3H/HeJ × C57BL/6J), and the F1 hybrid females were backcrossed with C57BL/6J males (backcrossed, F1 × C57BL/6J). Plasma glucose after 30 min on IPGTT was 219 ± 8 (n = 21), 456 ± 18 (n = 23), and 292 ± 13 (n = 23) mg/dl for C3H/HeJ, C57BL/6J, and F1 mice, respectively (P < .001 for all comparisons). The range of glucose levels on IPGTT of the backcrossed mice was 250–500 mg/dl. If a single gene were determining the difference in glucose tolerance in C57BL/6J mice, analysis of the plasma glucose level of individual backcrossed mice would show segregation into two classes, one like the F1 hybrid and the other like the inbred parent. This segregation was not observed; therefore, we conclude that modification of glucose tolerance in these two inbred strains is under polygenic control. Plasma insulin levels after 15 min on IPGTT for C57BL/6J mice were lower than those for C3H/HeJ mice (1.0 ± 0.09 vs. 1.3 ± 0.09 ng/ml, n = 6, P < .001). On the other hand, insulin levels after 30 min on IPGTT for C3H/HeJ (0.9 ± 0.08 ng/ml, n = 21), C57BL/6J (1.05 ± 0.1 ng/ml, n = 23), F1 (1.05 ± 0.1 ng/ml, n = 23), and backcrossed mice (1.0 ± 0.1 ng/ml, n = 23) were not different from each other. A positive correlation between plasma glucose and insulin concentrations was observed in C3H/HeJ (r = .42, P < .05) and F1 (r = .46, P < .05) mice, whereas a negative correlation was seen in C57BL/6J mice (r = −.45, P < .05). This suggests that glucose-stimulated insulin secretion may be significantly impaired in C57BL/6J mice, indicating one of the possible mechanisms for the phenotypic expression of glucose intolerance.

Associations of candidate loci angiotensinogen and angiotensin-converting enzyme with severe hypertension: The NHLBI Family Heart Study.

PURPOSE In studies conducted in several different populations, the M235T substitution in the angiotensinogen (AGT) locus has been associated with hypertension. METHODS A case-control study was initiated in an attempt to replicate this finding. Persons with hypertension, age- and sex-matched normotensive controls, and randomly sampled individuals were probands from the Family Heart Study of the National Heart, Lung, and Blood Institute. Subjects were recruited from the Atherosclerosis Risk in Communities study (ARIC) in North Carolina and Minneapolis, MN, and from the Framingham Heart Study in Massachusetts. Genotypes were determined for the M235T substitution in the AGT locus and for the insertion/deletion polymorphism in the angiotensin-converting enzyme (ACE) locus. Simple association tests as well as logistic regression analyses were performed. RESULTS The association of AGT-T235 with hypertension was replicated in the Framingham sample (odds ratio, 1.60; 95% confidence interval, 1.11-2.30), but not in the ARIC white or black subjects. However, logistic regression analysis suggested a significant association of AGT with hypertension in both the ARIC white and Framingham samples when the effects of body mass index, triglycerides, and the presence of significant coronary heart disease were controlled. These analyses further suggested that, in the ARIC data, the relationship with the AGT locus is stronger in women than men and that there may be interaction (epistasis) between homozygotes for T235 and ACE-DD in the Framingham data. While the small sample size precluded logistic regression analysis, the frequency of the T235 allele in the black random sample was much higher than in the comparable white sample. CONCLUSIONS These results are compatible with the presence of a genetic risk factor for hypertension in or near the angiotensinogen locus.

Glucokinase Gene is Genetic Marker for NIDDM in American Blacks

Glucokinase (ATP:d-glucose-6-phosphotransferase), expressed exclusively in liver and pancreatic islet β-cells, catalyzes the first step of glycolysis and acts as glucose sensor and metabolic signal generator in these tissues. The enzyme plays a key role in glucose homeostasis and as such is an excellent candidate for inherited defects predisposing to non-insulin-dependent diabetes mellitus (NIDDM). A compound-imperfect dinucleotide (CA)n repeat element was found ∼10-kb 3′ of the human glucokinase gene on chromosome 7p, which revealed polymorphism with alleles differing in size by 2–15 nucleotides in unrelated individuals. A polymerase chain reaction assay was developed, and genomic DNA from 275 biologically unrelated American black individuals was typed for glucokinase alleles. The differences in allelic frequencies between individuals with NIDDM and nondiabetic individuals were compared. After typing 112 diabetic and 163 nondiabetic subjects, we found five different-sized alleles, with Z defined as the most common allele, Z + 2, Z + 4, Z + 10, and Z − 15. The Z allele was more common in nondiabetic subjects than in diabetic patients (60.4 vs. 49.6%, P = 0.012). The Z + 4 allele was more common in diabetic patients than in nondiabetic subjects (20.1 vs. 12.0%, P = 0.009). After adjusting for age, sex, and body mass index, the Z + 4 allele continued to have a positive association with NIDDM (P = 0.0018), and the Z allele had a negative association with NIDDM (P = 0.0334). The Z + 4 allele, transmitted as an autosomal dominant trait, appeared to be the most significant one at this locus. No difference was found in the clinical characteristics between the diabetic patients with or without the Z + 4 allele, after adjusting for multiple comparisons. These results indicate that the dinucleotide (CA)n repeat polymorphism at the glucokinase locus is a genetic marker for NIDDM in American blacks. We estimate that the presence of at least one Z + 4 allele increases the odds of NIDDM in this racial group by 2.85 times for the same age, sex, and body mass index combination.

Associations between candidate loci angiotensin-converting enzyme and angiotensinogen with coronary heart disease and myocardial infarction: The NHLBI family heart study☆

Angiotensin-converting enzyme (ACE) and angiotensinogen (AGT) are major components of the renin-angiotensin systems. An association between myocardial infarction (MI) and the ACE DD genotype of the insertion/deletion (ID) polymorphism in intron 16 of the ACE gene has been reported. However, other similarly designed studies have not found such an association. Angiotensin II, the product of AGT, has a direct effect on vascular tone; and a variant in the AGT gene has been found to be associated with MI in the Japanese. This case-control study was initiated to investigate whether the ACEI/D and AGT M235T polymorphisms are associated with an increased risk for coronary heart disease (CHD) and MI. Our study groups were composed of participants in the National Heart Lung Blood Institute (NHLBI) Family Heart Study (FHS) selected from three population-based studies: two Atherosclerosis Risk in Communities (ARIC) centers (Forsyth County, NC, and Minneapolis, MN), and the Framingham Heart Study. In multivariate analysis within ARIC Caucasians, a significant positive association was found between CHD (controls = 230, cases = 232) and the AGT TT genotype (P = 0.022; OR = 1.84, 1.09-3.10 95% CI). When we restricted the analysis to a low-risk group for CHD (controls = 70, cases = 35) an interaction between the ACE DD and AGT TT genotypes was significant (P = 0.025; OR = 5.02 1.22-20.6 95% CI). After further subsetting low-risk cases to those with a definite MI (controls = 74, cases = 16), we found that the associations with the ACE DD genotype was also significant (P = 0.013, OR = 3.94, 1.28-12.2 95% CI). Comparable tests in the Framingham sample failed to support an association of these markers with CHD. In conclusion, within selected groups the ACE D and AGT 235T alleles are statistically associated with CHD and MI, and there is a synergistic interaction between the two alleles. These results and those from previous studies together suggest that the association of these two loci is neither strong nor consistent and involves a complex interaction among risk factors and genotypes.

Ethnicity and Human Genetic Linkage Maps

Human genetic linkage maps are based on rates of recombination across the genome. These rates in humans vary by the sex of the parent from whom alleles are inherited, by chromosomal position, and by genomic features, such as GC content and repeat density. We have examined--for the first time, to our knowledge--racial/ethnic differences in genetic maps of humans. We constructed genetic maps based on 353 microsatellite markers in four racial/ethnic groups: whites, African Americans, Mexican Americans, and East Asians (Chinese and Japanese). These maps were generated using 9,291 subjects from 2,900 nuclear families who participated in the National Heart, Lung, and Blood Institute-funded Family Blood Pressure Program, the largest sample used for map construction to date. Although the maps for the different groups are generally similar, we did find regional and genomewide differences across ethnic groups, including a longer genomewide map for African Americans than for other populations. Some of this variation was explained by genotyping artifacts--namely, null alleles (i.e., alleles with null phenotypes) at a number of loci--and by ethnic differences in null-allele frequencies. In particular, null alleles appear to be the likely explanation for the excess map length in African Americans. We also found that nonrandom missing data biases map results. However, we found regions on chromosome 8p and telomeric segments with significant ethnic differences and a suggestive interval on chromosome 12q that were not due to genotype artifacts. The difference on chromosome 8p is likely due to a polymorphic inversion in the region. The results of our investigation have implications for inferences of possible genetic influences on human recombination as well as for future linkage studies, especially those involving populations of nonwhite ethnicity.

Linkage between a muscle-specific CK gene marker and VO2max in the HERITAGE Family Study.

PURPOSE We have reported a significant association between VO2max in the sedentary state and its response (delta VO2max) to an endurance training program with a muscle-specific creatine kinase (CKMM) gene polymorphism. The purpose of this study was to test the hypothesis of genetic linkage between the same CKMM marker and VO2max in the sedentary state as well as delta VO2max. METHODS Sib-pair linkage analysis was performed on 277 full sib-pairs from 98 Caucasian nuclear families of the HERITAGE Family Study. VO2max was measured during cycle ergometry tests before and after 20 wk of endurance training. The CKMM polymorphism was detected by the polymerase chain reaction and digestion with the Ncol restriction enzyme. RESULTS Frequencies for the rare (1170 base pairs) and common (985 + 185 base pairs) alleles were 0.32 and 0.68, respectively. No significant linkage (t = -0.02, P = 0.49) was detected between the CKMM marker and the age and sex adjusted VO2max (mL x kg(-1) x min(-1)) in the sedentary state. However, after adjustment of delta VO2max for the effects of age, sex, initial VO2max, and body mass, evidence for linkage between the CKMM locus and delta VO2max was suggestive (P = 0.04). CONCLUSION The present results provide further support for the notion that the CKMM gene, or some gene in close linkage disequilibrium with it, may contribute to individual differences in the VO2max response to endurance training.

A genetic marker at the glucokinase gene locus for Type 2 (non-insulin-dependent) diabetes mellitus in Mauritian Creoles

SummaryThe prevalence of Type 2 (non-insulin-dependent) diabetes mellitus is high in Mauritius, a multiethnic island nation in the southwestern Indian Ocean. Evaluation of candidate genes in the different ethnic groups represents a means of assessing the genetic component. As glucokinase is known to be a key regulator of glucose homeostasis in liver and pancreatic Beta-cells, the human gene was isolated and a dinucleotide repeat (CA)n marker was identified at this locus. A polymerase chain reaction assay was developed, and alleles differing in size were observed in individuals, according to the number of repeats in the amplified fragment. Eighty-five Creoles and 63 Indians of known glucose tolerance status were typed by amplification of genomic DNA for this dinucleotide (CA)n repeat marker. Four different alleles were observed including Z, the most common allele, and Z+2, Z+4, and Z+10, which differed from Z by 2, 4, and 10 nucleotides respectively. In Mauritian Creoles, the frequency of the Z+2 allele was greater in Type 2 diabetic subjects than in control subjects (23.8 % vs 8.9 %, p=0.008), and the frequency of the Z allele was lower in Type 2 diabetic subjects (60% vs 75.6%, p=0.03). Analysis with univariate logistic regression models indicated that the Z+2 allele had the highest odds ratio, 3.08 (95% confidence interval 1.14–8.35, p=0.0416), among the other risk factors (age, sex, body mass index, and waist/hip ratio). The multivariate odds ratio for Type 2 diabetes was 2.88 (95% confidence interval 0.98–8.50, p=0.0551). In contrast, in the Mauritian Indian population, no differences were noted between the frequency of any glucokinase allele in the Type 2 diabetic and control groups. These data suggest that the Z+2 allele is an important risk factor for Type 2 diabetes in Mauritian Creoles, but not in Mauritian Indians, and also imply that the glucokinase gene may play a role in the pathogenesis of Type 2 diabetes in Mauritian Creoles. Further studies are needed to define the nature of this defect.