Determination of the allelic frequency in Smith-Lemli-Opitz syndrome by analysis of massively parallel sequencing data sets.

Data from massively parallel sequencing or 'Next Generation Sequencing' of the human exome has reached a critical mass in both public and private databases, in that these collections now allow researchers to critically evaluate population genetics in a manner that was not feasible a decade ago. The ability to determine pathogenic allele frequencies by evaluation of the full coding sequences and not merely a single nucleotide polymorphism (SNP) or series of SNPs will lead to more accurate estimations of incidence. For demonstrative purposes, we analyzed the causative gene for the disorder Smith-Lemli-Opitz Syndrome (SLOS), the 7-dehydrocholesterol reductase (DHCR7) gene and determined both the carrier frequency for DHCR7 mutations, and predicted an expected incidence of the disorder. Estimations of the incidence of SLOS have ranged widely from 1:10,000 to 1:70,000 while the carrier frequency has been reported as high as 1 in 30. Using four exome data sets with a total of 17,836 chromosomes, we ascertained a carrier frequency of pathogenic DHRC7 mutations of 1.01%, and predict a SLOS disease incidence of 1/39,215 conceptions. This approach highlights yet another valuable aspect of the exome sequencing databases, to inform clinical and health policy decisions related to genetic counseling, prenatal testing and newborn screening.

A cooperative interaction between LPHN3 and 11q doubles the risk for ADHD.

In previous studies of a genetic isolate, we identified significant linkage of attention deficit hyperactivity disorder (ADHD) to 4q, 5q, 8q, 11q and 17p. The existence of unique large size families linked to multiple regions, and the fact that these families came from an isolated population, we hypothesized that two-locus interaction contributions to ADHD were plausible. Several analytical models converged to show significant interaction between 4q and 11q (P<1 × 10(-8)) and 11q and 17p (P<1 × 10(-6)). As we have identified that common variants of the LPHN3 gene were responsible for the 4q linkage signal, we focused on 4q-11q interaction to determine that single-nucleotide polymorphisms (SNPs) harbored in the LPHN3 gene interact with SNPs spanning the 11q region that contains DRD2 and NCAM1 genes, to double the risk of developing ADHD. This interaction not only explains genetic effects much better than taking each of these loci effects by separated but also differences in brain metabolism as depicted by proton magnetic resonance spectroscopy data and pharmacogenetic response to stimulant medication. These findings not only add information about how high order genetic interactions might be implicated in conferring susceptibility to develop ADHD but also show that future studies of the effects of genetic interactions on ADHD clinical information will help to shape predictive models of individual outcome.

A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication.

Attention-Deficit/Hyperactivity Disorder (ADHD) has a very high heritability (0.8), suggesting that about 80% of phenotypic variance is due to genetic factors. We used the integration of statistical and functional approaches to discover a novel gene that contributes to ADHD. For our statistical approach, we started with a linkage study based on large multigenerational families in a population isolate, followed by fine mapping of targeted regions using a family-based design. Family- and population-based association studies in five samples from disparate regions of the world were used for replication. Brain imaging studies were performed to evaluate gene function. The linkage study discovered a genome region harbored in the Latrophilin 3 gene (LPHN3). In the world-wide samples (total n=6360, with 2627 ADHD cases and 2531 controls) statistical association of LPHN3 and ADHD was confirmed. Functional studies revealed that LPHN3 variants are expressed in key brain regions related to attention and activity, affect metabolism in neural circuits implicated in ADHD, and are associated with response to stimulant medication. Linkage and replicated association of ADHD with a novel non-candidate gene (LPHN3) provide new insights into the genetics, neurobiology, and treatment of ADHD.

Recruitment strategies and comparison of prostate cancer-specific clinical data on African-American and Caucasian males with and without family history.

Prostate cancer is the most common cancer in men in the United States. This is a complex disease with high heterogeneity and the exact causes are unknown in population-specific samples. Family history is a primary risk factor irrespective of race. Identifying prostate cancer families with multiple affected cancer cases is challenging. Herein we document recruitment techniques and present prostate cancer clinical factors described in a cohort of African Americans and Caucasians with or without a strong family history. A total of 521 prostate cancer patients (241 African Americans and 280 Caucasians) were identified using a novel cooperative methodology involving a combination of treating physicians and tumor registries. Higher prostate-specific antigen (PSA, P=0.0269) was found in familial cases as compared to sporadic cases in African-American men. In addition, PSA values for familial cases were higher (P=0.0093) in African-American as compared to Caucasian men. No differences were detected in Gleason score values in either race, regardless of family history. These findings remained the same after adjustment was made for age at diagnosis. In conclusion, methodologies for cohort acquisition, and clinical characteristics, are described for men with and without a family history of prostate cancer using both Caucasian and African-American populations.

Population isolates in South Tyrol and their value for genetic dissection of complex diseases.

The study of genetic isolates is a promising approach for the study of complex genetic traits. The small and constant population size, lack of migration, and multiple relationships between individuals in the isolate population could reduce the genetic diversity, and lead to increased levels of linkage disequilibrium (LD). We studied the extent of LD on Xq13 in six population isolates from South Tyrol in the Eastern Italian Alps. We found different levels of LD in our study samples, probably reflecting their degrees of isolation and their demographic histories. The highest values were obtained in Val Gardena (ranking among the highest levels of LD in Europe) and in Stelvio, which qualified as a microisolate according to historical information, and biodemographic and genealogical criteria. Phylogenetic analysis revealed that the two Ladin-speaking populations are genetically distant from each other, and from their German-speaking neighbours, and are characterized by a smaller effective population size than the neighbouring valleys. These peculiar characteristics suggest that South Tyrol could be a unique resource for the study of complex diseases, showing all the characteristics of isolated populations with the advantage of including, in a fairly homogeneous environment, two genetically differentiated sub-populations. This could allow investigators to gain an insight into the contribution of genetic heterogeneity in complex diseases.

The value of molecular haplotypes in a family-based linkage study.

Novel methods that could improve the power of conventional methods of gene discovery for complex diseases should be investigated. In a simulation study, we aimed to investigate the value of molecular haplotypes in the context of a family-based linkage study. The term "haplotype" (or "haploid genotype") refers to syntenic alleles inherited on a single chromosome, and we use the term "molecular haplotype" to refer to haplotypes that have been determined directly by use of a molecular technique such as long-range allele-specific polymerase chain reaction. In our study, we simulated genotype and phenotype data and then compared the powers of analyzing these data under the assumptions that various levels of information from molecular haplotypes were available. (This information was available because of the simulation procedure.) Several conclusions can be drawn. First, as expected, when genetic homogeneity is expected or when marker data are complete, it is not efficient to generate molecular haplotyping information. However, with levels of heterogeneity and missing data patterns typical of complex diseases, we observed a 23%-77% relative increase in the power to detect linkage in the presence of heterogeneity with heterogeneity LOD scores >3.0 when all individuals are molecularly haplotyped (compared with the power when only standard genotypes are used). Furthermore, our simulations indicate that most of the increase in power can be achieved by molecularly haplotyping a single individual in each family, thereby making molecular haplotyping a valuable strategy for increasing the power of gene mapping studies of complex diseases. Maximization of power, given an existing family set, can be particularly important for late-onset, often-fatal diseases such as cancer, for which informative families are difficult to collect.

A genetic model for determining MSH2 and MLH1 carrier probabilities based on family history and tumor microsatellite instability.

Mutation-predicting models can be useful when deciding on the genetic testing of individuals at risk and in determining the cost effectiveness of screening strategies at the population level. The aim of this study was to evaluate the performance of a newly developed genetic model that incorporates tumor microsatellite instability (MSI) information, called the AIFEG model, and in predicting the presence of mutations in MSH2 and MLH1 in probands with suspected hereditary non-polyposis colorectal cancer. The AIFEG model is based on published estimates of mutation frequencies and cancer penetrances in carriers and non-carriers and employs the program MLINK of the FASTLINK package to calculate the proband's carrier probability. Model performance is evaluated in a series of 219 families screened for mutations in both MSH2 and MLH1, in which 68 disease-causing mutations were identified. Predictions are first obtained using family history only and then converted into posterior probabilities using information on MSI. This improves predictions substantially. Using a probability threshold of 10% for mutation analysis, the AIFEG model applied to our series has 100% sensitivity and 71% specificity.

A major lung cancer susceptibility locus maps to chromosome 6q23-25.

Lung cancer is a major cause of death in the United States and other countries. The risk of lung cancer is greatly increased by cigarette smoking and by certain occupational exposures, but familial factors also clearly play a major role. To identify susceptibility genes for familial lung cancer, we conducted a genomewide linkage analysis of 52 extended pedigrees ascertained through probands with lung cancer who had several first-degree relatives with the same disease. Multipoint linkage analysis, under a simple autosomal dominant model, of all 52 families with three or more individuals affected by lung, throat, or laryngeal cancer, yielded a maximum heterogeneity LOD score (HLOD) of 2.79 at 155 cM on chromosome 6q (marker D6S2436). A subset of 38 pedigrees with four or more affected individuals yielded a multipoint HLOD of 3.47 at 155 cM. Analysis of a further subset of 23 multigenerational pedigrees with five or more affected individuals yielded a multipoint HLOD score of 4.26 at the same position. The 14 families with only three affected relatives yielded negative LOD scores in this region. A predivided samples test for heterogeneity comparing the LOD scores from the 23 multigenerational families with those from the remaining families was significant (P=.007). The 1-HLOD multipoint support interval from the multigenerational families extends from C6S1848 at 146 cM to 164 cM near D6S1035, overlapping a genomic region that is deleted in sporadic lung cancers as well as numerous other cancer types. Parametric linkage and variance-components analysis that incorporated effects of age and personal smoking also supported linkage in this region, but with somewhat diminished support. These results localize a major susceptibility locus influencing lung cancer risk to 6q23-25.

Attention-deficit/hyperactivity disorder (ADHD): feasibility of linkage analysis in a genetic isolate using extended and multigenerational pedigrees.

Segregation analyses converge in explaining the predisposition to attention-deficit/hyperactivity disorder (ADHD) as the consequence of a major gene and exclude purely environmental or cultural transmission. As a result of the ADHD phenotype restrictions, collection of extended families or design of linkage studies using families has been extremely difficult and thus currently linkage studies have been performed using only concordant or discordant sib-pairs rather than large families. On the other hand, intergenerational studies are represented by the transmission disequilibrium test (TDT) using trios. We collected pedigree data on ADHD from the Paisa community from Antioquia, Colombia, a genetic isolate. The goal of this study was to genetically map a putative gene predisposing to ADHD in a set of 27 multigenerational Paisa families. Here we present the results of a power simulation using SIMLINK to detect linkage of ADHD. ADHD was assumed to be a dichotomous trait with incomplete penetrance and a phenocopy rate of 3% in males and 0.2% in females. We simulated cosegregation of the trait and a marker locus in our pedigrees. We assumed Hardy-Weinberg and linkage equilibrium, equally frequent marker alleles and evaluated power at several recombination fractions between the trait and marker loci. Also, the ADHD trait was assumed to be genetically heterogeneous and different functions of age-dependent penetrance were simulated. We found exceptionally good power to detect linkage (expected LOD > 14 if theta is 0.1 or less), and that the presence of heterogeneity up to 50% does not affect substantially the projected LOD scores even for a theta recombination value of 0.05 (eLOD > 5.87). Having now obtained blood samples and confirmatory interviews in five families (representing 20% of the projected number of families), we performed a new analysis. The expected mean LOD in these five families reached values close to 10 and remained invariant when heterogeneity and different penetrance models were considered. We discuss the relative benefits of using extended and multigenerational families for genetic mapping studies as opposed to using nuclear families, affected sib pairs or sporadic cases which require the collection of over 1000 analytical units to get the same power exhibited by the small number of pedigrees described here.

Effects of misspecification of allele frequencies on the power of Haseman-Elston sib-pair linkage method for quantitative traits.

It is well known that the Haseman-Elston (H-E) sib-pair linkage method does not assume that the genetic model underlying the trait phenotype is known without error, although this assumption is made for marker loci. However, misspecification of allele frequencies at the marker locus decreases power when some or all parental genotypes are unknown. In this study, the power of the H-E sib-pair method was compared for different types of traits when some or all parental data were missing and allele frequencies at the marker loci were misspecified. Data were generated for a quantitative trait and marker loci in nuclear families using G.A.S.P. (V3.3). Three types of traits were simulated with two equifrequent alleles with a random environmental effect (10%, 30%, and 50%). The simulated data were analyzed using (i) one of the parent's marker data, and (ii) no parental marker data, with both correct and incorrect marker allele frequencies. This test is found to be robust in most of the situations considered except for a slight decrease in power when sample size is small and when the marker locus is not very polymorphic.

Autosomal dominant inheritance of prostate cancer: a confirmatory study.

OBJECTIVES: To confirm, in a study of a large, independent cohort of families with prostate cancer, the findings of three segregation analyses that have suggested the existence of an inherited form of prostate cancer with an autosomal dominant inheritance mode. METHODS: Between January 1991 and December 1993, 1199 pedigrees were ascertained through single, unrelated, prostate cancer probands who presented for radical prostatectomy at the Division of Urologic Surgery, Washington University Medical Center in St. Louis, Missouri. Maximum likelihood segregation analysis was used to test specifically for mendelian inheritance of prostate cancer. RESULTS: Segregation analyses revealed that the familial aggregation of prostate cancer can be best explained by the autosomal dominant inheritance of a rare (q = 0.0037) high-risk allele. According to the best-fitting autosomal dominant model, 97% of all carriers will be affected by 85 years of age compared with 10% of noncarriers. Furthermore, the autosomal dominant model predicts that the high-risk allele accounts for a large proportion (65%) of all patients diagnosed with prostate cancer before 56 years of age. However, of all prostate cancer cases, a relatively small proportion is inherited (8% by 85 years old). CONCLUSIONS: These results are in agreement with earlier reports of segregation analyses of prostate cancer and strengthen the evidence that prostate cancer is inherited in a mendelian fashion within a subset of families.

Comparison of novel and existing methods for detection of linkage disequilibrium using parent-child trios in the GAW12 genetic isolate simulated data.

A novel method for joint detection of association caused by linkage disequilibrium (LD) and estimation of both recombination fraction and linkage disequilibrium parameters was compared to several existing implementations of the transmission/disequilibrium test (TDT) and modifications of the TDT in the simulated genetic isolate data from Genetic Analysis Workshop 12. The first completely genotyped trio of affected child and parents was selected from each family in each replicate so that the TDT tests are valid tests of linkage and association, rather than being only valid as tests for linkage. In general, power to detect LD using the genome-wide scan markers was inadequate in the individual replicate samples, but the power was better when analyzing several SNP markers in candidate gene 1.

Linkage heterogeneity for the IBD1 locus in Crohn's disease pedigrees by disease onset and severity.

BACKGROUND & AIMS: There is evidence for the IBD1 Crohn's disease (CD) susceptibility locus on chromosome 16 in several but not all populations studied. Genetic and phenotypic heterogeneity may underlie ability to replicate IBD1. We determined if age and severity stratification could identify a clinical subgroup at risk for IBD1. METHODS: Linkage analysis at microsatellites spanning chromosome 16 was performed in 2 groups of CD pedigrees: group 1, 57 pedigrees with at least one affected relative classified as having "severe" disease, by history of surgical resection or immunomodulator therapy, and with disease diagnosed before age 22; and group 2, 33 pedigrees with no history of early-onset, severe CD. RESULTS: Group 1 pedigrees demonstrated genomewide significant linkage evidence for the IBD1 locus (nonparametric multipoint logarithm of the odds [Mlod], 3.84; P = 1.3 x 10(-5)) with linkage evidence greater than all 90 pedigrees (Mlod, 2.12; P = 9.0 x 10(-4)). Group 2 pedigrees had near zero nonparametric 2-point and Mlod scores for the IBD1 region. Heterogeneity between groups 1 and 2 was significant (P = 0.002). CONCLUSIONS: Presence of early-onset, more severe CD identifies pedigrees at high risk for IBD1. These pedigrees will have more power to refine the IBD1 locus and identify the causative gene.

Analysis of HPC1, HPCX, and PCaP in Icelandic hereditary prostate cancer.

Putative prostate cancer susceptibility loci have recently been identified by genetic linkage analysis on chromosomes 1q24-25 (HPC1). 1q44.243 (PCaP), and Xq27-28 (HPCX). In order to estimate the genetic linkage in Icelandic prostate cancer families, we genotyped 241 samples from 87 families with eleven markers in the HPC1 region, six markers at PCaP, and eight at HPCX. Concurrently, we assessed allelic imbalance at the HPC1 and PCaP loci in selected tumors from the patients. For each of the candidate regions, the combined parametric and non-parametric LOD scores were strongly negative. Evidence for linkage allowing for genetic heterogeneity was also insignificant for all the regions. The results were negative irrespective of whether calculations were performed for the whole material or for a selected set of early age at onset families. The prevalence of allelic imbalance was relatively low in both the HPC1 (0%-9%) and PCaP (5%-20%) regions and was not elevated in tumors from positively linked families. Our studies indicate that the putative cancer susceptibility genes at chromosomes 1q24-25, 1q44.2-43, and Xq27-28 are unlikely to contribute significantly to hereditary prostate cancer in Iceland and that selective loss of the HPC1 and PCaP loci is a relatively rare somatic event in prostate cancers.

Segregation analysis of esophageal cancer in a moderately high-incidence area of northern China.

In order to explore the mode of inheritance of esophageal cancer in a moderately high-incidence area of northern China, we conducted a pedigree survey on 225 patients affected by esophageal cancer in Yangquan, Shanxi Province. Segregation analysis was performed using the REGTL program of S.A.G.E. The results showed that Mendelian autosomal recessive inheritance of a major gene that influences susceptibility to esophageal cancer provided the best fit to the data. In the best-fitting recessive model, the frequency of the disease allele was.2039. There was a significant sex effect on susceptibility to the disease. The maximum cumulative probability of esophageal cancer among males with the AA genotype was 100%, but, among females, it was 63.5%. The mean age at onset for both men and women was 62 years. The age-dependent penetrances for males with the AA genotype by the ages of 60 and 80 years were 41.6% and 95.2%, respectively, whereas, for females, they were 26.4% and 60.5%, respectively. Incorporating environmental risk factors-such as cigarette smoking, pipe smoking, alcohol drinking, eating hot food, and eating pickled vegetables-into the models did not provide significant improvement of the fit of the models to these data. The results suggest a major locus underlying susceptibility to esophageal cancer with sex-specific penetrance.

Effects of misspecification of allele frequencies on the type I error rate of model-free linkage analysis.

Linkage analyses of simulated quantitative trait data were performed using the Haseman-Elston (H-E) sib pair regression test to investigate the effects of inaccurate allele frequency estimates on the type I error rates of this test. Computer simulations generating a quantitative trait in nuclear families were performed using GASP [1]. Assuming no linkage, several data sets were simulated; they differed in marker allele numbers and frequencies, number of sib pairs and number of sibships. Each set of simulated data was analyzed using (1) all parental marker data, (2) half of the parental marker data, and (3) no parental marker data, using both correct and incorrect allele frequencies in the latter 2 cases. The H-E sib pair linkage method was found to be robust to misspecification of marker allele frequencies regardless of the number of alleles.

The transmission/disequilibrium test for linkage on the X chromosome.

The transmission/disequilibrium test (TDT), which detects linkage between a marker and disease loci in the presence of linkage disequilibrium, was introduced by Spielman et al. The original TDT requires families in which the genotypes are known for both parents and for at least one affected offspring, and this limits its applicability to diseases with late onset. The sib-TDT, or S-TDT, which utilizes families with affected and unaffected siblings, was introduced as an alternative method, by Spielman and Ewens, and the TDT and S-TDT can be combined in an overall test (i.e., a combined-TDT, or C-TDT). The TDT statistics described so far are for autosomal chromosomes. We have extended these TDT methods to test for linkage between X-linked markers and diseases that affect either males only or both sexes. For diseases of late onset, when parental genotypes are often unavailable, the X-linkage C-TDT may allow for more power than is provided by the X-linkage TDT alone.

Genetic epidemiology of breast cancer: segregation analysis of 389 Icelandic pedigrees.

A genetic epidemiologic investigation of breast cancer involving 389 breast cancer pedigrees including information on 14,721 individuals from the Icelandic population-based cancer registry is presented. Probands were women born in or after 1920 and reported to have breast cancer in the cancer registry. The average age of the 389 probands was 45.5 years (SD 8.92). Segregation analyses was performed evaluating residual maternal effects, a dichotomous cohort effect, and assuming the age at diagnosis followed a logistic distribution after log-transformation. Familial aggregation could be best explained by the inheritance of a high-risk allele leading to early onset breast cancer among the homozygotes, which represent approximately 2.6% of the population. A Mendelian codominant model was selected as the best fitting model, with an estimated age at diagnosis of 51.8 years among these high-risk homozygotes, 64.0 years for heterozygotes and 76.3 years for the low-risk genotype. The predicted cumulative risk for homozygote carriers of the high-risk allele is 32.2% by age 60, compared to 16.4% for heterozygotes and 5.0% for non-carriers of the same age. These predicted age profiles in the current study complement recent reports from Iceland of a majority of BRCA2 mutation carriers being diagnosed with breast cancer below the age of 50 years, and 60 years being the mean age at diagnosis for non-carriers. This model also predicted a high background risk of breast cancer for women in this population (estimated susceptibility gamma = 0.44 +/- 0.08). This implies that if carriers and non-carriers did not die of competing causes, the estimated risk of being diagnosed with breast cancer by age 80 years irrespective of carrier status is 11.4%.