Mary J. Maliarik

HLA-DRB1*1101: A Significant Risk Factor for Sarcoidosis in Blacks and Whites

Sarcoidosis is a granulomatous disorder of unknown etiology, associated with an accumulation of CD4+ T cells and a TH1 immune response. Since previous studies of HLA associations with sarcoidosis were limited by serologic or low-resolution molecular identification, we performed high-resolution typing for the HLA-DPB1, HLA-DQB1, HLA-DRB1, and HLA-DRB3 loci and the presence of the DRB4 or DRB5 locus, to define HLA class II associations with sarcoidosis. A Case Control Etiologic Study of Sarcoidosis (ACCESS) enrolled biopsy-confirmed cases (736 total) from 10 centers in the United States. Seven hundred six (706) controls were case matched for age, race, sex, and geographic area. We studied the first 474 ACCESS patients and case-matched controls. The HLA-DRB1 alleles were differentially distributed between cases and controls (P<.0001). The HLA-DRB1*1101 allele was associated (P<.01) with sarcoidosis in blacks and whites and had a population attributable risk of 16% in blacks and 9% in whites. HLA-DRB1-F(47) was the amino acid residue most associated with sarcoidosis and independently associated with sarcoidosis in whites. The HLA-DPB1 locus also contributed to susceptibility for sarcoidosis and, in contrast to chronic beryllium disease, a non-E(69)-containing allele, HLA-DPB1*0101, conveyed most of the risk. Although significant differences were observed in the distribution of HLA class II alleles between blacks and whites, only HLA-DRB1*1501 was differentially associated with sarcoidosis (P<.003). In addition to being susceptibility markers, HLA class II alleles may be markers for different phenotypes of sarcoidosis (DRB1*0401 for eye in blacks and whites, DRB3 for bone marrow in blacks, and DPB1*0101 for hypercalcemia in whites). These studies confirm a genetic predisposition for sarcoidosis and present evidence for the allelic variation at the HLA-DRB1 locus as a major contributor.

The BTNL2 Gene and Sarcoidosis Susceptibility in African Americans and Whites

The BTNL2 gene is a member of the B7 receptor family that probably functions as a T-cell costimulatory molecule. It resides in the class II major histocompatibility complex (MHC) region of chromosome 6p and has recently been associated with sarcoidosis susceptibility in a white German population. We sought to replicate the BTNL2 association in an African American family-based study population (n=219 nuclear families) and two case-control populations--one African American (n=295 pairs) and one white (n=366 pairs). Ten SNPs were detected within a 490-bp region spanning exon/intron 5 of BTNL2. Haplotype variation within this region was significantly associated with sarcoidosis in all three study populations but more so in whites (P=.0006) than in the African American case-control (P=.02) or family-based (P=.03) samples. The previously reported BTNL2 SNP with the strongest sarcoidosis association, rs2076530, was also the SNP with the strongest association in our white population (P<.0001). The A allele of rs2076530 results in a premature exon-splice site and increases risk for sarcoidosis (odds ratio=2.03; 95% confidence interval 1.32-3.12). Although rs2076530 was not associated with sarcoidosis in either African American sample, a three-locus haplotype that included rs2076530 was associated with sarcoidosis across all three study samples. Multivariable logistic regression analyses showed that BTNL2 effects are independent of human leukocyte antigen class II genes in whites but may interact antagonistically in African Americans. Our results underscore the complexity of genetic risk for sarcoidosis emanating from the MHC region.

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.

Heterogeneity of familial risk in sarcoidosis.

Familial clustering of sarcoidosis and the higher prevalence and clinical severity of sarcoidosis in African Americans suggests etiologic heterogeneity. To test for heterogeneity in familial risk of sarcoidosis, we studied 3,395 siblings and parents of 558 index cases (361 African American, 197 Caucasian) diagnosed at Henry Ford Hospital between 1951 and 1994. Using the age‐ and sex‐specific cumulative incidence of sarcoidosis in our sample, we found a statistically significant heterogeneity in familial risk of disease (P < .001). To determine if this was due to a greater risk of sarcoidosis in African Americans, we recalculated disease probabilities using age‐, sex‐, and race‐specific disease cumulative incidence and found the same amount of heterogeneity in familial risk (P < .001). Index cases (n = 69) from high‐risk families were more likely to be African American (odds ratio [OR] = 3.24; 95% confidence interval (CI) = 1.71−6.14) and to have an offspring or second‐degree relative affected (OR = 6.21; 95% CI = 2.86−13.45).

GENETICS OF SARCOIDOSIS

Sarcoidosis likely results from an interplay of environmental and genetic factors. Despite a recent large multicenter study, A Case-Control Etiologic Study of Sarcoidosis (ACCESS), no single causative environmental agent has been identified. Family clustering and differences in racial incidence of sarcoidosis support an inherited susceptibility to sarcoidosis. Siblings of patients with sarcoidosis have about a fivefold increased risk of developing sarcoidosis. Certain human leukocyte antigen (HLA) alleles have been consistently associated with sarcoidosis susceptibility. Furthermore, HLA-DRB1*0301/DQB1*0201 has been associated with good prognosis in Löfgrens syndrome. Many candidate genes studied based on their potential function in immunopathogenesis have been evaluated in case-control studies, but few have been consistently associated with sarcoidosis across populations. Two genome scans have been reported in sarcoidosis. One in African Americans reporting linkage to chromosome 5 and the other in German families reporting linkage to chromosome 6. Follow-up studies on chromosome 6 identified the BTNL2 gene, a B7 family costimulatory molecule to be associated with sarcoidosis. Advances in genotyping and statistical analysis are helping to elucidate the genetics of sarcoidosis.

TGF-β1 Variants in Chronic Beryllium Disease and Sarcoidosis

Evidence suggests a genetic predisposition to chronic beryllium disease (CBD) and sarcoidosis, which are clinically and pathologically similar granulomatous lung diseases. TGF-β1, a cytokine involved in mediating the fibrotic/Th1 response, has several genetic variants which might predispose individuals to these lung diseases. We examined whether certain TGF-β1 variants and haplotypes are found at higher rates in CBD and sarcoidosis cases compared with controls and are associated with disease severity indicators for both diseases. Using DNA from sarcoidosis cases/controls from A Case Control Etiologic Study of Sarcoidosis Group (ACCESS) and CBD cases/controls, TGF-β1 variants were analyzed by sequence-specific primer PCR. No significant differences were found between cases and controls for either disease in the TGF-β1 variants or haplotypes. The −509C and codon 10T were significantly associated with disease severity indicators in both CBD and sarcoidosis. Haplotypes that included the −509C and codon 10T were also associated with more severe disease, whereas one or more copies of the haplotype containing the −509T and codon 10C was protective against severe disease for both sarcoidosis and CBD. These studies suggest that the −509C and codon 10T, implicated in lower levels of TGF-β1 protein production, are shared susceptibility factors associated with more severe granulomatous disease in sarcoidosis and CBD. This association may be due to lack of down-regulation by TGF-β1, although future studies will be needed to correlate TGF-β1 protein levels with known TGF-β1 genotypes and assess whether there is a shared mechanisms for TGF-β1 in these two granulomatous diseases.

Blood Lead Level and Risk of Asthma

Asthma and lead poisoning are prevalent among urban children in the United States. Lead exposure may be associated with excessive production of immunoglobulin E, possibly increasing asthma risk and contributing to racial disparities. The objective of this study was to examine racial differences in the association of blood lead level (BLL) to risk of developing asthma. We established and followed a cohort prospectively to determine asthma onset, using patient encounters and drug claims obtained from hospital databases. Participants were managed care enrollees with BLL measured and documented at 1–3 years of age. We used multiple variable analysis techniques to determine the relationship of BLL to period prevalent and incident asthma. Of the 4,634 children screened for lead from 1995 through 1998, 69.5% were African American, 50.5% were male, and mean age was 1.2 years. Among African Americans, BLL ≥5 and BLL ≥10 μg/dL were not associated with asthma. The association of BLL ≥5 μg/dL with asthma among Caucasians was slightly elevated, but not significant [adjusted hazard ratio (adjHR) = 1.4; 95% confidence interval (CI), 0.7–2.9; p = 0.40]. Despite the small number of Caucasians with high BLL, the adjHR increased to 2.7 (95% CI, 0.9–8.1; p = 0.09) when more stringent criteria for asthma were used. When compared with Caucasians with BLL < 5 μg/dL, African Americans were at a significantly increased risk of asthma regardless of BLL (adjHR = 1.4–3.0). We conclude that an effect of BLL on risk of asthma for African Americans was not observed. These results demonstrate the need for further exploration of the complex interrelationships between race, asthma phenotype, genetic susceptibilities, and socioenvironmental exposures, including lead.

Sarcoidosis and granuloma genes: A family-based study in African-Americans

The evidence for a genetic component in the aetiology of sarcoidosis includes familial aggregation, associations with genetic polymorphisms, and linkage to the major histocompatibility complex class region on chromosome 6p. Unfortunately, the majority of genetic associations with sarcoidosis have not been consistently replicated. In the present study, using a family-based study design, which controls for population stratification, the authors attempted to replicate previously reported associations between sarcoidosis and three attractive candidate genes studied primarily in case-control samples. In 225 nuclear families, ascertained through African Americans with a history of sarcoidosis, no evidence was found for an association between sarcoidosis susceptibility and polymorphisms in the angiotensin converting enzyme, vitamin D receptor and tumour necrosis factor‐α genes. Further analyses of chronic and acute disease phenotypes failed to reveal any notable associations. Assuming an underlying inheritance model with an additive allelic effect on disease risk, the current study had ∼80–90% statistical power to detect a 3‐fold increased risk associated with the putative risk allele of the polymorphisms under study. The present authors conclude that in African-Americans, the angiotensin converting enzyme, vitamin D receptor, and tumour necrosis factor‐α genes are not significant risk factors for sarcoidosis susceptibility.

The influence of T cell receptor and cytokine genes on sarcoidosis susceptibility in African Americans

The pathogenesis of sarcoidosis, a multisystem granulomatous disorder, is mediated through immunoregulatory pathways. While sarcoidosis clusters in families, inherited risk factors remain undefined. In search of possible sarcoidosis susceptibility genes, we examined anonymous polymorphic genetic markers tightly linked to six different candidate gene regions on chromosomes 2q13, 5q31, 6p23-25, 7p14-15, 14q11 and 22q11. These candidate regions contain T cell receptor, interleukin (IL) and interferon regulatory factor (IRF) genes. Our study population consisted of 105 African-American sarcoidosis cases and 95 unrelated healthy controls. The allelic frequency distribution of two out of the six markers, IL-1 alpha marker (p = 0.010) on 2q13 and the F13A marker (p = 0.0006) on 6p23-25, was statistically significantly different in cases compared with controls. The two alleles most strongly associated with sarcoidosis were IL-1 alpha*137 (Odds Ratio (OR) = 2.60; 95% confidence interval (CI) = 1.36-4.98) and F13A*188 (OR = 2.42; 95% CI = 1.37-4.30). Individuals that had both of these alleles were at a six-fold increased risk for sarcoidosis (OR = 6.19; 95% CI = 2.54-15.10). Restricting the analysis to cases with at least one first or second-degree relative affected with sarcoidosis increased the OR to 15.38. IL-1 levels are elevated in sarcoidosis and the F13A marker is tightly linked to a gene that codes for a newly identified interferon regulatory factor protein (IRF-4), which is thought to play a role in T cell effector functions. Our results suggest genetic susceptibility to sarcoidosis may be conferred by more than one immune-related gene that act synergistically on disease risk.

The Distribution of Long Range Admixture Linkage Disequilibrium in an African-American Population

Objectives: To better understand the effect of admixture on long range linkage disequilibrium (LD), we characterized extended LD in gene-rich regions of an African-American population. Methods: Approximately 290 cM of chromosomes 1, 3, 6, 11–17, 20 and 22 were scanned using 109 polymorphic microsatellite markers spaced an average of 3 cM apart. Disequilibrium between loci (D′) was based on maximum-likelihood estimates of haplotype frequencies computed for 200 unrelated African Americans. Results: Mean D′ values were highest on chromosomes 6p23–p21.3 (D′ = 0.33) and 15p22.2–p25.3 (D′ = 0.34), and lowest on chromosome 12p11.2–q14 (D′ = 0.21). Overall, the variance in LD among chromosomes accounted for approximately two-thirds of the total LD variance. Of the 434 locus pairs spaced between 0.3 and 38.7 cM apart, there was no detectable correlation between LD and recombination distance and a weak negative correlation between LD and physical distance (rs = –0.12; p = 0.031). For the 192 intrachromosomal locus pairs where allele frequency data were available from the Centre d’Etude du Polymorphisme humain (CEPH), we found a statistically significant positive correlation between LD and the allelic frequency differences (δ) between the African-American study population and Caucasian reference CEPH population (rs = 0.53; p < 0.0001). The correlation between LD and both recombination and physical distance was markedly increased for locus pairs with high δ levels. Conclusions: Our results suggest that recent Caucasian admixture maintains a high level of long range LD in African Americans on a genomic scale, and selected markers with large African American/Caucasian δ levels may be useful in association studies.