High-density SNP genotyping to define beta-globin locus haplotypes

Abstract

Five major beta-globin locus haplotypes have been established in individuals with sickle cell disease (SCD) from the Benin, Bantu, Senegal, Cameroon, and Arab-Indian populations. Historically, beta-haplotypes were established using restriction fragment length polymorphism (RFLP) analysis across the beta-locus, which consists of five functional beta-like globin genes located on chromosome 11. Previous attempts to correlate these haplotypes as robust predictors of clinical phenotypes observed in SCD have not been successful. We speculate that the coverage and distribution of the RFLP sites located proximal to or within the globin genes are not sufficiently dense to accurately reflect the complexity of this region. To test our hypothesis, we performed RFLP analysis and high-density single nucleotide polymorphism (SNP) genotyping across the beta-locus using DNA samples from healthy African Americans with either normal hemoglobin A (HbAA) or individuals with homozygous SS (HbSS) disease. Using the genotyping data from 88 SNPs and Haploview analysis, we generated a greater number of haplotypes than that observed with RFLP analysis alone. Furthermore, a unique pattern of long-range linkage disequilibrium between the locus control region and the beta-like globin genes was observed in the HbSS group. Interestingly, we observed multiple SNPs within the HindIII restriction site located in the Ggamma-globin intervening sequence II which produced the same RFLP pattern. These findings illustrated the inability of RFLP analysis to decipher the complexity of sequence variations that impacts genomic structure in this region. Our data suggest that high-density SNP mapping may be required to accurately define beta-haplotypes that correlate with the different clinical phenotypes observed in SCD.

Fig. 1. β-haplotypes established using RFLP genotypes

A) Schematic diagram showing the distributions of 88 SNPs (red dot) on the custom SNP-chip and five RFLP sites (yellow bar) that were analyzed. Each globin gene is indicated by a colored box and ψβ-globin is shown by a white box. The β^S mutation (rs334) is shown as a green circle. Abbreviations: LCR, locus control region; HS, DNase I hypersensitive site. B) The linkage disequilibrium (LD) patterns and haplotypes established by Haploview analysis for the RFLP genotypes are shown for the HbAA subjects. Pair-wise computation was performed for the SNPs which are shown on each side of the boxes. The degree of LD is defined by value of D′ and LOD (the logarithm of the likelihood odds ratio), which is a measure of the confidence of D′ values. The red boxes indicate strong LD (LOD >2, D′ = 1), white boxes no LD (LOD < 2, D′ < 1), and pink (LOD = 2, D′ <1), and blue (LOD < 2, D′ =1) boxes indicate intermediated LD. The D′=1 unless indicated in the boxes where D′ is multiplied by 100. SNPs with strong LD were defined into haplotype blocks (black triangle) and the size of the region in LD is shown in parentheses. The inferred haplotypes and frequency observed are shown in the lower corner; htSNPs are indicated by a gray triangle beneath the RFLP number. Symbols: “+” = cut with the specific restriction enzyme used for analysis; “-“= no cut. C) The LD patterns and haplotypes for the RFLP genotypes established by Haploview analysis are shown for the HbSS group. The symbols are the same as defined in Panel B.

Fig. 2. Haploview-inferred haplotypes using SNP-chip genotypes

A) A genotype heat map was established for the HbAA subjects. Genotypes produced using the SNP-chip that satisfied the criteria detailed in Materials and Methods are shown. The rows represent the SNPs analyzed and the columns represent each DNA sample tested. SNPs are numbered according to that defined in Table 1; the β^S SNP (rs334) is number 73 and is highlighted in green. At each SNP position, a blue box represents wild-type homozygous genotypes, a yellow box heterozygous and the red box homozygous mutant genotypes. White boxes indicate genotype data is missing for the SNP indicated. B) Genotype heat map for the HbSS subjects. The color code is the same as described in Panel A. C) Haploview software was used to infer haplotypes using SNP-chip genotype data for the HbAA subjects. The numbers in the gray shaded area represent the SNPs listed in Table 1. The inferred haplotypes in each haplotype block and linkage between blocks are shown. Nine haplotype blocks were defined (numbers above the gray shaded area). The lines between haplotype blocks indicate the linkage frequency with a thick line for > 10% and a thin line for a frequency from 1-10%. Haplotype-tagging SNPs (htSNPs) are indicated underneath the SNP numbers by a triangle (▼). Haplotype frequency within each haplotype block is shown next to each haplotype. The number between two blocks is the Hedrick's multi-allelic D′; D′ >= 0.8 is indicative of strong LD between the blocks. D) Inferred haplotypes and linkages between five haplotype blocks (numbers above the gray shaded area) for the HbSS subjects. The methods and symbols are the same as described in Panel C.

Fig. 2. Haploview-inferred haplotypes using SNP-chip genotypes

A) A genotype heat map was established for the HbAA subjects. Genotypes produced using the SNP-chip that satisfied the criteria detailed in Materials and Methods are shown. The rows represent the SNPs analyzed and the columns represent each DNA sample tested. SNPs are numbered according to that defined in Table 1; the β^S SNP (rs334) is number 73 and is highlighted in green. At each SNP position, a blue box represents wild-type homozygous genotypes, a yellow box heterozygous and the red box homozygous mutant genotypes. White boxes indicate genotype data is missing for the SNP indicated. B) Genotype heat map for the HbSS subjects. The color code is the same as described in Panel A. C) Haploview software was used to infer haplotypes using SNP-chip genotype data for the HbAA subjects. The numbers in the gray shaded area represent the SNPs listed in Table 1. The inferred haplotypes in each haplotype block and linkage between blocks are shown. Nine haplotype blocks were defined (numbers above the gray shaded area). The lines between haplotype blocks indicate the linkage frequency with a thick line for > 10% and a thin line for a frequency from 1-10%. Haplotype-tagging SNPs (htSNPs) are indicated underneath the SNP numbers by a triangle (▼). Haplotype frequency within each haplotype block is shown next to each haplotype. The number between two blocks is the Hedrick's multi-allelic D′; D′ >= 0.8 is indicative of strong LD between the blocks. D) Inferred haplotypes and linkages between five haplotype blocks (numbers above the gray shaded area) for the HbSS subjects. The methods and symbols are the same as described in Panel C.

Fig. 3. Correlation of HindIII digestion with direct sequencing of the Gγ-IVSII region

The PCR products generated for RFLP analysis was used for direct sequencing analysis. The HindIII restriction site AAGCTT and position of three nucleotides according to the HBB record (U01317) are shown at the top of the figure. Each row represents data for individual DNA samples; each nucleotide position and genotype is shown for both alleles based on the sequencing results. For RFLP patterns the positive symbol (+) refers to HindIII digestion, while the negative symbol (-) no cut.

Fig. 4. Linkage Disequilibrium and haplotype block patterns across the β-locus

A) Shown are the LD patterns and haplotype blocks established by Haploview analysis with the SNP-chip data for the HbAA group. The numbers on top represent the SNPs used for the analysis. Haplotype blocks are shown by inverted triangles. Within each haplotype block, the distance of the region covered is indicated in kilo-bases (kb). The color code is the same as defined in the legend for Fig. 1B. B) The LD patterns and haplotype blocks established by the Haploview analysis with the SNP-chip genotype data for the HbSS group are shown. The symbols are the same as defined in Panel A