Disruption of sonic hedgehog signaling in Ellis-van Creveld dwarfism confers protection against bipolar affective disorder.

Ellis-van Creveld syndrome, an autosomal recessively inherited chondrodysplastic dwarfism, is frequent among Old Order Amish of Pennsylvania. Decades of longitudinal research on bipolar affective disorder (BPAD) revealed cosegregation of high numbers of EvC and Bipolar I (BPI) cases in several large Amish families descending from the same pioneer. Despite the high prevalence of both disorders in these families, no EvC individual has ever been reported with BPI. The proximity of the EVC gene to our previously reported chromosome 4p16 BPAD locus with protective alleles, coupled with detailed clinical observations that EvC and BPI do not occur in the same individuals, led us to hypothesize that the genetic defect causing EvC in the Amish confers protection from BPI. This hypothesis is supported by a significant negative association of these two disorders when contrasted with absence of disease (P=0.029, Fisher's exact test, two-sided, verified by permutation to estimate the null distribution of the test statistic). As homozygous Amish EVC mutations causing EvC dwarfism do so by disrupting sonic hedgehog (Shh) signaling, our data implicate Shh signaling in the underlying pathophysiology of BPAD. Understanding how disrupted Shh signaling protects against BPI could uncover variants in the Shh pathway that cause or increase risk for this and related mood disorders.

Some capabilities for model-based and model-free linkage analysis using the program package S.A.G.E. (Statistical Analysis for Genetic Epidemiology).

For both model-free and model-based linkage analysis the S.A.G.E. (Statistical Analysis for Genetic Epidemiology) program package has some unique capabilities in analyzing both continuous traits and binary traits with variable age of onset. Here we highlight model-based linkage analysis of a quantitative trait (plasma dopamine ß hydroxylase) that is known to be largely determined by monogenic inheritance, using a prior segregation analysis to produce the best fitting model for the trait. For a binary trait with variable age of onset (schizophrenia), we illustrate how using age of onset information to obtain a quantitative susceptibility trait leads to more statistically significant linkage signals, suggesting better power.

Finding genes underlying human disease.

With the surge in genome-wide association studies (GWAS), many have asked the question 'Are linkage studies dead?' In this article, we survey the approaches used in mapping human disease genes, reviewing the analysis strategies that preceded and laid the groundwork for GWAS. We note that earlier approaches are still useful and the development of new methodology is warranted.

Using linkage analysis to identify quantitative trait loci for sleep apnea in relationship to body mass index.

To understand the genetics of sleep apnea, we evaluated the relationship between the apnea hypopnea index (AHI) and body mass index (BMI) through linkage analysis to identify genetic loci that may influence AHI and BMI jointly and AHI independent of BMI. Haseman-Elston sibling regression was conducted on AHI, AHI adjusted for BMI and BMI in African-American and European-American pedigrees. A comparison of the magnitude of linkage peaks was used to assess the relationship between AHI and BMI. In EAs, the strongest evidence for linkage to AHI was on 6q23-25 and 10q24-q25, both decreasing after BMI adjustment, suggesting loci with pleiotropic effects. Also, a promising area of linkage to AHI but not BMI was observed on 6p11-q11 near the orexin-2 receptor, suggesting BMI independent pathways. In AAs the strongest evidence of linkage for AHI after adjusting for BMI was on chromosome 8p21.3 with linkage increasing after BMI adjustment and on 8q24.1 with linkage decreasing after BMI adjustment. Novel linkage peaks were also observed in AAs to both BMI and AHI on chromosome 13 near the serotonin-2a receptor. These analyses suggest genetic loci for sleep apnea that operate both independently of BMI and through BMI-related pathways.

The HIV-1 protease substitution K55R: a protease-inhibitor-associated substitution involved in restoring viral replication.

OBJECTIVES: The identification and in vitro characterization of novel protease mutations strongly associated with known protease resistance mutations. METHODS: The association between pairs of protease amino acid substitutions was identified using a database of protease sequences derived from protease inhibitor-experienced patients (n = 803). In vitro characterization included drug susceptibility and viral replication studies performed on recombinant viruses harbouring site-directed mutations. RESULTS: The K55R mutation, which is not a natural polymorphism, was identified to be strongly associated with protease mutations M46I/L and to a lesser extent L24I, I54V and V82A/T/S/F. In vitro characterization of the K55R substitution indicated a primary role for this substitution in increasing replicative capacity in the presence of specific protease mutations. CONCLUSIONS: The K55R mutation is a secondary drug resistance mutation that can improve viral replication capacity in the presence of other primary protease mutations.

Genome-wide linkage scan for nicotine dependence in European Americans and its converging results with African Americans in the Mid-South Tobacco Family sample.

Previously, we reported a genome-wide scan for nicotine dependence (ND) in the African American (AA) sample of the Mid-South Tobacco Family (MSTF) cohort. In this study, we conducted a genome-wide scan in 629 individuals representing 200 nuclear families of European American (EA) origin of the MSTF cohort with the goals of identifying vulnerability loci for ND in the EAs and determining converging regions across the ethnic groups. We examined 385 autosomal microsatellite markers for ND, which was assessed by smoking quantity (SQ), the Heaviness of Smoking Index (HSI) and the Fagerström test for ND (FTND). After performing linkage analyses using various methods implemented in the GENEHUNTER and SAGE programs, we found eight regions on chromosomes 2, 4, 9-12, 17 and 18 that met the criteria for suggestive linkage to at least one ND measure in the EA sample. Of these, the region on chromosome 4 at 43 cM showed suggestive linkage to indexed SQ, the HSI and the FTND, and the region on chromosome 9 at 24 cM showed suggestive linkage to the HSI and the FTND. To increase detection power, we analyzed a combined AA and EA sample using age, gender and ethnicity as covariates and found that the region on chromosome 12 near marker D12S372 showed significant linkage to SQ. Additionally, we found six regions on chromosomes 9-11, 13 and 18 that showed suggestive linkage to at least one ND measure in the combined sample. When we compared the linkage peaks detected for ND among the two samples and a combined sample, we found that four regions on chromosomes 9 (two regions), 11 and 18 overlapped. On the other hand, we identified five regions on chromosomes 2, 4, 10, 12 and 17 that showed linkage to ND only in the EA sample, and two regions on chromosomes 10 and 13 that showed linkage to ND only in the AA sample. For those linkages identified in only one sample, we found that the combined analysis of AA plus EA samples actually decreased the linkage signal. This indicates that some chromosomal regions may be more homogenous than others across the ethnic samples. All regions except for the one on chromosome 12 have been detected at nominally significant levels in other studies, providing independent replication of ND loci in different populations.

Genetic linkage analysis of sarcoidosis phenotypes: the sarcoidosis genetic analysis (SAGA) study.

The sarcoidosis genetic analysis (SAGA) study previously identified eight chromosomal regions with suggestive evidence for linkage to sarcoidosis susceptibility in African-American sib pairs. Since the clinical course of sarcoidosis is variable and likely under genetic control, we used the affected relative pair portion of the SAGA sample (n=344 pairs) to perform multipoint linkage analyses with covariates based on pulmonary and organ involvement phenotypes. Chest radiographic resolution was the pulmonary phenotype with the highest LOD (logarithm of the backward odds, or likelihood ratio) score of 5.11 at D1S3720 on chromosome 1p36 (P=4 x 10(-5)). In general, higher LOD scores were attained for covariates that modeled clustered organ system involvement rather than individual organ systems, with the cardiac/renal group having the highest LOD score of 6.65 at chromosome 18q22 (P=2 x 10(-5)). The highest LOD scores for the other three organ involvement groups of liver/spleen/bone marrow, neuro/lymph and ocular/skin/joint were 3.72 at 10p11 (P=0.0004), 5.16 at 7p22 (P=4 x 10(-5)) and 2.93 at 10q26 (P=0.001), respectively. Most of the phenotype linkages did not overlap with the regions previously found linked to susceptibility. Our results suggest that genes influencing clinical presentation of sarcoidosis in African Americans are likely to be different from those that underlie disease susceptibility.

Regression-based multivariate linkage analysis with an application to blood pressure and body mass index.

Multivariate linkage analysis has been suggested for the analysis of correlated traits, such as blood pressure (BP) and body mass index (BMI), because it may offer greater power and provide clearer results than univariate analyses. Currently, the most commonly used multivariate linkage methods are extensions of the univariate variance component model. One concern about those methods is their inherent sensitivity to the assumption of multivariate normality which cannot be easily guaranteed in practice. Another problem possibly related to all multivariate linkage analysis methods is the difficulty in interpreting nominal p-values, because the asymptotic distribution of the test statistic has not been well characterized. Here we propose a regression-based multivariate linkage method in which a robust score statistic is used to detect linkage. The p-value of the statistic is evaluated by a simple and rapid simulation procedure. Theoretically, this method can be used for any number and type of traits and for general pedigree data. We apply this approach to a genome linkage analysis of blood pressure and body mass index data from the Beaver Dam Eye Study.

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families.

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.

In vitro development of resistance to human immunodeficiency virus protease inhibitor GW640385.

Development of in vitro resistance to GW640385, a new human immunodeficiency virus type 1 protease inhibitor, was studied. Variants characterized included one with <4-fold resistance and amino acid substitutions Q58E/A71V (protease) and P452K (Gag) and one with >50-fold resistance and amino acid substitutions L10F/G16E/E21K/A28S/M46I/F53L/A71V (protease) and L449F/P453T (Gag). The A28S substitution substantially reduced replication capacity.

Genome-wide search for sarcoidosis susceptibility genes in African Americans.

Sarcoidosis, a systemic granulomatous disease of unknown etiology, likely results from an environmental insult in a genetically susceptible host. In the US, African Americans are more commonly affected with sarcoidosis and suffer greater morbidity than Caucasians. We searched for sarcoidosis susceptibility loci by conducting a genome-wide, sib pair multipoint linkage analysis in 229 African-American families ascertained through two or more sibs with a history of sarcoidosis. Using the Haseman-Elston regression technique, linkage peaks with P-values less than 0.05 were identified on chromosomes 1p22, 2p25, 5p15-13, 5q11, 5q35, 9q34, 11p15 and 20q13 with the most prominent peak at D5S2500 on chromosome 5q11 (P=0.0005). We found agreement for linkage with the previously reported genome scan of a German population at chromosomes 1p and 9q. Based on the multiple suggestive regions for linkage found in our study population, it is likely that more than one gene influences sarcoidosis susceptibility in African Americans. Fine mapping of the linked regions, particularly on chromosome 5q, should help to refine linkage signals and guide further sarcoidosis candidate gene investigation.

Linkage of serum leptin levels in families with sleep apnea.

OBJECTIVE: To identify regions on the genome linked to plasma leptin levels. DESIGN: Full genome scan with 402 microsatellite markers, spaced approximately 10 cM apart. Data were analyzed using the Haseman-Elston regression linkage analysis. SUBJECTS: A total of 160 sibling pairs from 59 predominantly African American, obese families recruited to participate in a genetic-epidemiological study of obstructive sleep apnea. MEASUREMENTS: Serum leptin levels adjusted for age, sex, race and body mass index (BMI). RESULTS: Suggestive linkage peaks were observed on chromosomes 2 (P=0.00170; marker D2S1384), 3 (P=0.00007; marker D3S3034), 4 (P=0.00020; marker D4S1652) and 21 (P=0.00053; marker D21s1411). CONCLUSION: The peak on chromosome 3 is near the gene for glycogensynthase kinase 2 beta, an important factor in glucose homeostasis. Linkage was generally stronger after BMI adjustment, suggesting the potential influence of a number of metabolic pathways on leptin levels other than those that directly determine obesity levels. The evidence of linkage for leptin levels is consistent with prior linkage analyses for cholesterol, hypertension and other metabolic phenotypes.

A meta-analytic investigation of linkage and association of common leptin receptor (LEPR) polymorphisms with body mass index and waist circumference.

METHODS: We analyzed data pooled from nine studies on the human leptin receptor (LEPR) gene for the association of three alleles (K109R, Q223R and K656N) of LEPR with body mass index (BMI; kg/m(2)) and waist circumference (WC). A total of 3263 related and unrelated subjects from diverse ethnic backgrounds including African-American, Caucasian, Danish, Finnish, French Canadian and Nigerian were studied. We tested effects of individual alleles, joint effects of alleles at multiple loci, epistatic effects among alleles at different loci, effect modification by age, sex, diabetes and ethnicity, and pleiotropic genotype effects on BMI and WC. RESULTS: We found that none of the effects were significant at the 0.05 level. Heterogeneity tests showed that the variations of the non-significant effects are within the range of sampling variation. CONCLUSIONS: We conclude that, although certain genotypic effects could be population-specific, there was no statistically compelling evidence that any of the three LEPR alleles is associated with BMI or WC in the overall population.

Pooling analysis of genetic data: the association of leptin receptor (LEPR) polymorphisms with variables related to human adiposity.

Analysis of raw pooled data from distinct studies of a single question generates a single statistical conclusion with greater power and precision than conventional metaanalysis based on within-study estimates. However, conducting analyses with pooled genetic data, in particular, is a daunting task that raises important statistical issues. In the process of analyzing data pooled from nine studies on the human leptin receptor (LEPR) gene for the association of three alleles (K109R, Q223R, and K656N) of LEPR with body mass index (BMI; kilograms divided by the square of the height in meters) and waist circumference (WC), we encountered the following methodological challenges: data on relatives, missing data, multivariate analysis, multiallele analysis at multiple loci, heterogeneity, and epistasis. We propose herein statistical methods and procedures to deal with such issues. With a total of 3263 related and unrelated subjects from diverse ethnic backgrounds such as African-American, Caucasian, Danish, Finnish, French-Canadian, and Nigerian, we tested effects of individual alleles; joint effects of alleles at multiple loci; epistatic effects among alleles at different loci; effect modification by age, sex, diabetes, and ethnicity; and pleiotropic genotype effects on BMI and WC. The statistical methodologies were applied, before and after multiple imputation of missing observations, to pooled data as well as to individual data sets for estimates from each study, the latter leading to a metaanalysis. The results from the metaanalysis and the pooling analysis showed that none of the effects were significant at the 0.05 level of significance. Heterogeneity tests showed that the variations of the nonsignificant effects are within the range of sampling variation. Although certain genotypic effects could be population specific, there was no statistically compelling evidence that any of the three LEPR alleles is associated with BMI or waist circumference in the general population.

Segregation analyses of asthma and respiratory allergy: the Humboldt family study.

We performed segregation analyses of asthma and respiratory allergy based on data from 309 nuclear families comprising 1,053 individuals living in the town of Humboldt, Saskatchewan, in 1993, using the REGD program of the S.A.G.E. program package. For adults, information on asthma and history of respiratory allergy was provided by the subjects themselves, and for children by their parents. When asthma was considered as the trait in segregation analysis, models of no major effect, with or without familial effects, were rejected, but they were not rejected after adjusting for history of respiratory allergy. The major gene hypothesis was not rejected before adjusting for history of respiratory allergy. When respiratory allergy was analyzed as the trait, both major gene and multifactorial models fitted the data well, regardless of whether there was adjustment for asthma or not. Other covariates adjusted for in the segregation analyses were age, sex, number of household smokers, current smoking, number of household members, generation, and house type. The data suggest that a major gene related to respiratory allergy may explain the familial aggregation of asthma.

Linkage analysis of alpha 1-antitrypsin deficiency: lessons for complex diseases.

OBJECTIVES: Severe alpha 1-antitrypsin (A1AT) deficiency is the one proven genetic risk factor for chronic obstructive pulmonary disease (COPD). Familial aggregation has been demonstrated for COPD among individuals who do not have A1AT deficiency, but linkage analysis of COPD has not been reported. To investigate the optimal phenotype definitions and analytical methods for the linkage analysis of COPD, we examined a set of 28 A1AT- deficient families containing 155 individuals. We have used the protease inhibitor (PI) type as a genetic marker rather than a disease gene, and we have performed linkage analysis between PI type and serum A1AT level and spirometry-related phenotypes. METHODS: Linkage analysis was performed on the quantitative phenotypes forced expiratory volume at 1 s (FEV(1) as % predicted), the ratio of FEV(1) to forced vital capacity (FEV(1)/FVC as % predicted), and serum A1AT level using the variance component approach in SOLAR, the generalized estimating equation approach in RELPAL, and the model-based classical lod score method in LINKAGE. Linkage analysis with qualitative A1AT and spirometry phenotypes was performed using a model-based method (LINKAGE) and a model-free method (GENEHUNTER). Adjustments for smoking effects were investigated under each method. RESULTS: All of the methods demonstrated linkage of PI type to serum A1AT level. Interestingly, however, the other quantitative phenotypes provided only weak evidence for linkage of PI type to lung disease. Better evidence for linkage of lung disease to PI type was found using a moderate or a mild threshold for the definition of airflow obstruction. CONCLUSIONS: For linkage analysis of spirometry phenotypes in A1AT deficiency, qualitative phenotypes provided stronger evidence for linkage than quantitative phenotypes. Possible contributors to the stronger evidence for linkage to qualitative spirometry phenotypes include the ascertainment scheme and the nonnormality of the pulmonary function data in PI Z subjects. This study provides guidelines for studies of the genetics of COPD unrelated to A1AT deficiency.

A tournament of linkage tests in complex inheritance.

The performance of some weakly parametric linkage tests in common use was compared on 200 replicates of oligogenic inheritance from Genetic Analysis Workshop 10. Each random sample for the quantitative trait was dichotomized at different thresholds and also selected through 2 affected sibs, generating 8 combinations of sample and variable. The variance component program SOLAR performed best with a continuous trait, even in selected samples, when the population mean was used. The sib-pair program SIBPAL2 was best in most other cases when the phenotype product, population mean, and empirical estimates of pair correlations were used. The BETA program that introduced phenotype products was slightly more powerful than maximum likelihood scores under the null hypothesis and approached but did not exceed SIBPAL2 under its optimal conditions. Type I errors generally exceeded expectations from a chi(2) test, but were conservative with respect to bounds on lods. All methods can be improved by use of the population mean, empirical correlations, logistic representation for affection status, and correct lods for samples that favour the null hypothesis. It remains uncertain whether all information can be extracted by weakly parametric methods and whether correction for ascertainment bias demands a strongly parametric model. Performance on a standard set of simulated data is indispensable for recognising optimal methods.

Sudden cardiac death, genes, and arrhythmogenesis : consideration of new population and mechanistic approaches from a national heart, lung, and blood institute workshop, part I.

Malignant ventricular arrhythmias are the leading mechanism of death in patients with acute and chronic cardiac pathologies. The extent to which inherited mutations and polymorphic variation in genes determining arrhythmogenic mechanisms affect these patients remains unknown, but based on recent population studies, this risk appears significant, deserving much greater investigation. This report summarizes a National Heart, Lung, and Blood Institute workshop that considered sources of genetic variation that may contribute to sudden cardiac death in common cardiac diseases. Evidence on arrhythmogenic mechanisms in recent population studies suggests a significant portion of the risk of sudden cardiac death in such broad populations may be unrelated to traditional risk factors for predisposing conditions such as atherosclerosis, hypertension, and diabetes and instead may involve unrecognized genetic and environmental interactions that influence arrhythmic susceptibility more directly. Additional population and genetic studies directed at discovering the sources of inherited molecular risk that are most directly linked to arrhythmia initiation and propagation, in addition to studies on previously well-described risk factors, would appear to have considerable potential for reducing premature cardiovascular mortality.

Electrocardiographic prediction of abnormal genotype in congenital long QT syndrome: experience in 101 related family members.

INTRODUCTION: Previous studies showed that diagnosing congenital long QT syndrome (LQTS) is difficult due to variable penetrance and genetic heterogeneity, especially when subjects from multiple families with diverse mutations are combined. We hypothesized that a combination of clinical and ECG techniques could identify gene carriers within a single family with congenital LQTS. METHODS AND RESULTS: One hundred one genotyped members of a family with LQTS, including 26 carriers of a HERG mutation, underwent history and ECG analysis. Forty-eight family members also underwent exercise testing with QT and T wave alternans (TWA) analysis and 24-hour Holter monitoring with QT and heart rate variability analysis. A logistic regression model, which included age, gender, QTc, and QTc by age, provided the best prediction of gene carrier status, although there was substantial overlap (78%) of QTc among subjects with and without the mutation. QTc was not helpful as a discriminator in children < or = 13 years. TWA (observed infrequently) did not add significantly to the model's ability to predict abnormal genotype. CONCLUSION: Even in this homogeneous LQTS population, the phenotype was so variable that clinical and detailed ECG analyses did not permit an accurate diagnosis of gene carrier status, especially in children. Sustained microvolt TWA was a specific (100%) but insensitive (18%) marker for LQTS. Its ability to predict risk of arrhythmia in this population remains to be determined. Genetic testing serves an essential role in screening for carriers of LQTS.