StemCell Therapy

The molecular genetics research interests are in human population genetics, anthropological genetics, immunogenetics, and the genetics of complex diseases. Ultimate goals surround elucidating questions of human variation, the evolutionary history of genes within populations and how these gene histories are involved in the etiology of complex diseases. While the laboratory's research goals have shared consequences for all humanity, specific interests focus on populations of African ancestry.

Operational Objectives:

1. Develop a SNP database for mapping functional mutations linked to diseases common in African peoples.

2. Utilization of evolutionary history of candidate genes to identify polymorphisms that are associated with diseases.

3. Exploit the linkage disquilibrium generated by admixture in the African American population for gene mapping.

CURRENT RESEARCH PROJECTS

The biological transition of enslaved Africans-to-African Americans is marked by the transition of environmental stresses from Africa to those in the Americas, and to a lesser extent, by The incorporation of non-African genes into the African American gene pool. The transition from the various African environments of origin to the diverse American environments is far from insignificant. The American environment imposed new selective pressures on the Africans. These selective pressures may have favored certain genes while eliminating others. This evolutionary hypothesis has been a controversial explanation for the high incidence of diseases such as hypertension in African Americans. Thus, African American biology has been significantly shaped by periods of intermixture creating high heterogeneity, and selective pressures emanating from the unique and particularly adverse social, economic, and political conditions in the US. All of these factors might contribute to the high incidence of diseases with a significant genetic component such as type 2 diabetes, asthma, hereditary cancer (prostate, breast and lung), and hypertension in African Americans.

Prostate cancer is the most common solid malignancy among men in the United States. African American men have the highest incidence of prostate cancer compared to other ethnic groups. This cohort also appears to present more commonly at an advanced stage with aggressive histology and increased cancer-related mortality. Thus, there is a critical need to explore the etiologic pathways (genetic and environmental factors) that contribute to this disparity. In on of our projects "Genes, environment and prostate cancer in populations of African descent" we seek to understand the relative contribution of allelic variations of candidate genes and environmental factors to determine an individuals risk of prostate cancer. The work is geared towards the African American population, for whom genomic studies are limited. African Americans share a common genetic background with West Africans yet vastly different environments. Comparative genetic and epidemiological research on the two populations reveal potential risk factors. This project will provide a better understanding of gene-gene (epistasis), and gene-environment effects on prostate cancer. At research sites in Washington, DC, Chicago, Illinois, and Benin City, Nigeria the goals of the project are to (1) recruit a well characterized cohort of 1200 cases and controls and collect blood for biochemical and molecular assays, along with diet and other environmental information; (2) use state of the art DHPLC technology to provide a formal evaluation of single nucleotide polymorphism (SNP) variation in 22 candidate genes for prostate cancer (androgen associated genes, apoptosis related genes, and diet related genes); (3) construct a web-based database of the SNPs discovered; (4) determine if haplotypic variation in candidate genes accounts for phenotypic variation in prostate cancer, prostate specific antigen (PSA) levels, and disease progression; and (5) assess whether gene-gene and gene-environment interactions exist by examining if prostate cancer risk is modified after stratification of genetic and/or environmental factors. This is the first study which examines SNP markers within the proposed candidate genes, diet, and other environmental variables in clinically evaluated African and African Americans and which evaluates their relative interactions and contribution, if any, to prostate cancer.

In another project, "Haplotype analyses of X chromosome variants: population genetics and implications for prostate cancer" the goals are to (1) provide a formal evaluation of X chromosome variation and linkage disequilibrium in the African American population, (2) determine the relationship of microsatellite alleles (CAG and GGN repeats) within the androgen receptor with the risk for prostate cancer and (3) exploit the evolutionary history of X chromosome haplotypes in order to determine if differences in X chromosome haplotypes account for phenotypic variation in prostate cancer and prostate specific antigen (PSA) levels.

While the molecular genetic research has shared consequences for all humanity, our specific interests focus on populations of African ancestry. Other areas of immediate interest are molecular evolutionary genetics, and biological anthropology. In another project, "the genetics of human pigmentation," we seek to understand the relative contribution of allelic variations of candidate genes responsible for variation in human pigmentation. Pigmentation is a classic anthropological trait that has been studied objectively using reflectance spectroscopy for over 50 years. Skin pigmentation is likely the trait that shows the largest degree of variability among human populations. That there are such dramatic differences in the levels of skin pigmentation among human populations is almost definite evidence for the action of natural selection. The identification of the genes that determine normal within-population variation in pigmentation and differences between populations is the first essential step in the elucidation of the molecular history of human pigmentation. The goals of this project are to (1) develop a database and sample collection that will allow for the delineation of the genes that determine pigmentation, and (2) genotype these individuals for a number of candidate genes to identify those which determine natural variation in pigmentation.

Mutation analyses of BRCA1 and BRCA2. We are analyzing the breast cancer predisposing genes, BRCA1 and BRCA2, for germline mutations in African American families at high-risk for hereditary breast cancer. Patients are considered high-risk if they have a family history of the disease, early onset breast cancer, bilateral breast cancer, breast and ovarian cancer, or a male affected with breast cancer. The entire BRCA1 and BRCA2 coding and flanking intron regions are being examined for mutation detection. In preliminary studies of BRCA1 using the technique of single strand conformation polymorphism, we identified 11 different germline mutations/ variations in 7 patients from 45 high-risk families. Two pathogenic, protein-truncating mutations were detected in exon 11. A ten base pair tandem duplication, 943ins10, was present in a woman with breast and ovarian cancer whose first-degree relatives had prostate cancer. A four base pair deletion, 3450del4, was detected in a breast cancer patient with five cases of breast cancer in the family; two of the proband's sisters with breast cancer also carried the same mutation. Four amino acid substitutions (Lys1183Arg, Leu1564Pro, Gln1785His, and Glu1794Asp) and four nucleotide substitutions in intron 22 (IVS22+78 C/A, IVS22+67 T/C, IVS22+8 T/A and IVS22+7 T/C) were observed in patients and not in control subjects. One early onset breast cancer patient carried five distinct BRCA1 variations, two amino acid substitutions and three substitutions in intron 22. An amino acid substitution in exon 11, Ser1140Gly, was identified in 3 different unrelated patients and in 6 of 92 control samples. The latter probably represents a benign polymorphism. BRCA1 and BRCA2 analyses for the detection of mutations are ongoing.

Genetic variation in asthma. Asthma families collected by HU investigators were part of the Collaborative Study on the Genetics of Asthma (CSGA) genome-wide search for asthma susceptibility loci in ethnically diverse populations. Asthma is an inflammatory airways disease associated with intermittent respiratory symptoms, bronchial hyper-responsiveness (BHR) and reversible airflow obstruction and is phenotypically heterogeneous. Patterns of clustering and segregation analyses in asthma families have suggested a genetic component to asthma. Previous studies reported linkage of BHR and atopy to chromosomes 5q, 6p, 11q, 14q, and 12q. One genome-wide search in atopic sib pairs had been reported, however, only 12% of their subjects had asthma. The CSGA conducted a genome-wide search in 140 families with > or = 2 asthmatic sibs, from three different populations and reported evidence for linkage to six novel regions: 5p15 (P = 0.0008) and 17p11.1-q11.2 (P = 0.0015) in African Americans; 11p15 (P = 0.0089) and 19q13 (P = 0.0013) in Caucasians; 2q33 (P = 0.0005) and 21q21 (P = 0.0040) in Hispanics. Evidence for linkage was also detected in five regions previously reported to be linked to asthma-associated phenotypes: 5q23-31 (P = 0.0187), 6p21.3-23 (P = 0.0129), 12q14-24.2 (P = 0.0042), 13q21.3-qter (P = 0.0014), and 14q11.2-13 (P = 0.0062) in Caucasians and 12q14-24.2 (P = 0.0260) in Hispanics.

Dermatophagoides pteronyssinus (Der p) is one of the most frequently implicated allergens in atopic diseases. Although HLA could play an important role in the development of the IgE response to the Der p allergens, genetic regulation by non-HLA genes influences certain HLA-associated IgE responses to complex allergens. To clarify genetic control for the expression of Der p-specific IgE responsiveness, a genome-wide search was conducted for genes influencing Der p-specific IgE antibody levels by using 45 Caucasian and 53 African American families ascertained as part of the Collaborative Study on the Genetics of Asthma (CSGA). Specific IgE antibody levels to the Der p crude allergen and to the purified allergens Der p 1 and Der p 2 were measured. Multipoint, nonparametric linkage analysis of 370 polymorphic markers was performed with the GENEHUNTER program. The best evidence of genes controlling specific IgE response to Der p was obtained in 2 novel regions: chromosomes 2q21-q23 (P = .0033 for Caucasian subjects) and 8p23-p21 (P = .0011 for African American subjects). Three regions previously proposed as candidate regions for atopy, total IgE, or asthma also showed evidence for linkage to Der p- specific IgE responsiveness: 6p21 (P = .0064) and 13q32-q34 (P = 0.0064) in Caucasian subjects and 5q23-q33 (P = 0.0071) in African American subjects. No single locus generated overwhelming evidence for linkage in terms of established criteria and guidelines for a genome-wide screening, which supports previous assertions of a heterogeneous etiology for Der p-specific IgE responsiveness. Two novel regions, 2q21-q23 and 8p23-p21, that were identified in this study merit additional study. In addition genome-wide screening was conducted for genes influencing Dermatophagoides pteronyssinus-specific IgE responsiveness as a part of the Collaborative Study on the Genetics of Asthma (CSGA). Evidence for linkage was found in some regions, including chromosomes 5131-q33 and 11q13 in African American families. Plans are underway to initiate an international study of the genetics of asthma in collaboration with medical scientists in Ghana and investigators at the NHGC. These investigations will target regions where associations with specific IgE responses have been indicated in African Americans.

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DEVELOPING PROJECTS

Characterization of African American Ancestral HLA Haplotypes in West Africa.

An important area of investigation at the NHGC is the inclusion of evolutionary history of genes as a diagnostic probe in tracing the history of disease in a population.

This project builds upon the foundation of research on the genetics of complex diseases common in African Americans already established with the NHGRI in partnership with the NIH Office of Research on Minority Health. More specifically, it would build upon African American Diabetes mellitus (AADM) an international human gnome research initiative to map genes for type 2 diabetes in ancestral populations of African Americans. Because of the overlap in clinical phenotype of some subsets of types 1 and 2 diabetes, the rationale for this study is that characterization of HLA class II haplotypes in the west African study population may assist in refining the clinical phenotype of a subset of type 2 diabetes patients.

The association of HLA class II genes with susceptibility to type 1 diabetes is well documented in many populations. In African Americans type 1 diabetes patients, unique HLA class II polymorphisms have been instructive in determining risk assessment of closely linked HLA loci. We have reported the association of a unique HLA-DR3 haplotype in African Americans that appears to be associated with resistance to type 1 diabetes. The higher frequency of this haplotype among controls raises questions about the frequency of this haplotype in west African ancestral populations of African Americans.

The long range goal of research at the NHGC is to improve the health status of African Americans through research on human DNA sequence variation and to apply the knowledge gained to better understand the biomedical significance of gene-based differences already known to exist among populations in the immune response to organ transplants; sensitivity to drugs; influence of environment on health, and susceptibility to complex diseases, such as cancer and diabetes.

The research goals of the molecular genetics component are predicated upon the two broad hypotheses of population variation in DNA polymorphic markers used to map genes and the correlation of population-based variation in DNA polymorphic markers with disease.Studies of human leukocyte antigen (HLA) polymorphisms and other genetic polymorphic systems have consistently shown greater genetic variability in African populations. The biomedical implications of population-based variation in HLA genes are seen in association in the arena of clinical transplantation, where decisions regarding the distribution of limited donor organs must be informed by science and balanced by the ethical concerns of the larger society.

The goal of this study is to define HLA alleles and haplotypes in the study population and determine whether allele and haplotype frequencies in diabetics differ from controls. If a difference is found, the implication of HLA associations with the clinical phenotype of type 2 diabetes will be investigated. The study of HLA haplotypes in west African ancestral populations of African Americans will help identify HLA polymorphisms that are common in this population.Since HLA has been associated with a variety of autoimmune diseases, the results of this study should not only be useful in the analysis of HLA haplotypes in type 2 diabetes, but also informative for population-based HLA evolutionary studies.

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Linkage disequilibrium (LD) in African Americans.

Linkage disequilibrium is a population genetic phenomenon that has been useful for gene mapping efforts. LD can usually be found in populations for genes that are tightly (close genetic distance) linked, and can be generated by mutation, selection, or admixture of populations with different allele frequencies. Generally, disequilibrium is dependent on population size, time (generations), and distance between genetic markers. Normally, the greater the distance between markers, the faster the decay of disequilibrium. The nonrandom association of alleles at different genetic loci can be measured by a variety of linkage disequilibrium measures.

Within the African American population one would expect to find short genomic areas of tight LD, a legacy of this population's roots in the antiquity of African human history, together with large areas of LD, a legacy of more recent admixture with Europeans and Native Americans. Assessment of the level of genetic variation and LD in the African American population is important for several reasons. It will allow us to better understand the mechanisms responsible for the creation and maintenance of LD over genomic regions.

This better understanding will aid in the mapping of genes responsible for complex diseases. We expect to observe a diverse pattern of LD among the African American chromosomes when compared to other populations. While the pattern observed among African Americans is not restricted to the population, it is observed at higher frequency than others with diffferent populatioin histories. African American chromosomes with ancestry in West Africa should exhibit closely linked disequilibrium while chromosomes with ultimate ancestry from Europe will reveal broader regions of disequilibrium. What this study will do is assess patterns and level of LD among chromosomal regions within the African American population.

Significance of the African American population for gene mapping

As stated above, LD can be generated by admixture between divergent populations. Thus, a genetic consequence of the unique population history of African Americans is increased LD. We caution that much of the disequilibrium may not actually be due to genetic linkage, but are artifacts of divergent allele frequencies in the parental populations. However, it is expected that linked loci will also show significant disequilibrium in the African American population. The analysis of LD between marker and disease loci has proven to be a powerful tool for positional cloning of disease genes.

When a disease or trait manifests variation between populations, admixed populations provide a population based approach to evaluate the relative importance of genetic factors. A variety of statistical genetic methods for disease studies exploit the LD created by admixture. These include the Transmission Disequilibrium Test (TDT) and Mapping by Admixture Linkage Disequilibrium (MALD). An important assumption of many of these methods is that the ancestry of alleles at each locus be assigned to one of the two founding populations. The assignment of alleles to parent populations is problematic at times, however as more informative genetic markers are found and more individuals and populations sampled, the statistical power to assign alleles increases.

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RESOURCES

The Molecular Genetics Laboratory in the National Human Genome Center is newly renovated and is located on the 6th floor of the Howard University Cancer Center. This facility is approximately 7,500 square feet. There are two large laboratories (~1500 sq. ft. each), a DNA sequencing and genotyping room (~800 sq. ft.), two cold rooms, dark room, and a walk-in freezer. The laboratory space is equipped with benches, tables, sinks, distilled water, fume hoods and separate areas for tissue culture, PCR, and radioisotope use.

Four Pentium III NT Workstations (400-500 mHz) and four Power Macintosh G4's provide the computational hardware for the Molecular Genetics laboratory. The eight computers are networked together via the Genome Center NT server with the 5 computers operating three ABI 377 DNA sequencers and two DNA Wave Machines in addition to the computers used by the Genetic Epidemiology and Statistical Genetics units. The molecular genetics laboratory contains all the standard equipment necessary for large-scale, high throughput molecular analysis of DNA variation. These items include centrifuges, waterbaths, gel electrophoresis apparatus, pipettes, glassware, balances, etc. The laboratory also has two Transgenomics DNA Wave machines for SNP detection using dHPLC. The genotyping room contains three ABI 377 automated sequencers, ten Perkin Elmer 9700 thermocyclers, and the PSQ 96 Pyrosequencing platform for SNP genotyping.

Molecular genetics laboratory space on the 5th floor of the cancer center, contains two ABI 373 automated sequencers. The immunogenetics core research laboratory, also on the 5thfloor of the cancer center, provides approximately 800 sq ft of additional laboratory space for molecular genetics work.

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CORE SERVICES

The Molecular Genetics Laboratory will utilize current SNP technologies to:

1) identify and characterize DNA sequence variation in the NHGC African American population resource,

2) generate databases for locating functional mutations in candidate genes involved in the biology and pathophysiology of complex diseases common in African Americans and other populations in the African Diaspora,

3) develop a database of allele and haplotype frequencies for a reference panel of SNP variants in the NHGC population resource. This will include a set of candidate genes for complex diseases common in African Americans,

Prostate Cancer

Breast cancer

Asthma

Type 2 diabetes

Hypertension

HIV aids

4) Use coalescence models to construct phylogenies of the candidate genes in order to evaluate the evolutionary history of the genes in various populations. Construct haplotype phylogenies for a reference set of DNA loci/markers representative of various types of polymorphic systems found in the genome. This will include but is not limited to the following:

Single nucleotide polymorphisms (SNPs)

Microsatellites (mono, di, tri, and tetra nucleotide repeats)

Minisatellites (variable number of tandem repeats/VNTRs)

Nucleotide insertions and deletions

Alu repeats

MOLECULAR GENETICS UNIT GROUP PICTURE

06-Jan-2008

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See the article here:
Howard University National Human Genome Center

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