Imputation of class I and II HLA loci using high-density SNPs from ImmunoChip and their associations with Kawasaki disease in family-based study.

Kawasaki disease (KD) is the leading cause of acquired heart disease in children in most developed countries including the United States. The etiology of KD is not known; however, epidemiological and immunological data suggest infectious or immune-related factors in the manifestation of the disease. Further, KD has several hereditary features that strongly suggest a genetic component to disease pathogenesis. Human leucocyte antigen (HLA) loci have also been reported to be associated with KD, but results have been inconsistent, in part, because of small study samples and varying linkage disequilibrium (LD) patterns observed across different ethnic groups. To maximize the informativeness of single nucleotide polymorphism (SNP) genotypes in the major histocompatibility (MHC) region, we imputed classical HLA I (A, B, C) and HLA II (DRB1, DQA1, DQB1) alleles using SNP2HLA method from genotypes of 6700 SNPs within the extended MHC region contained in the ImmunoChip among 112 White patients with KD and their biological parents from North America and tested their association with KD susceptibility using the transmission disequilibrium test. Mendelian consistency in the trios suggested high accuracy and reliability of the imputed alleles (class I = 97.5%, class II = 96.6%). While several SNPs in the MHC region were individually associated with KD susceptibility, we report over-transmission of HLA-C*15 (z = +2.19, P = 0.03) and under-transmission of HLA-B*44 (z = -2.49, P = 0.01) alleles from parents to patients with KD. HLA-B*44 has been associated with KD in other smaller studies, and both HLA-C*15 and HLA-B*44 have biological mechanisms that could potentially be involved in KD pathogenesis. Overall, inferring HLA loci within the same ethnic group, using family-based information is a powerful approach. However, studies with larger sample sizes are warranted to evaluate the correlations of the strength and directions between the SNPs in MHC region and the imputed HLA alleles with KD.

SNP screening of central MHC-identified HLA-DMB as a candidate susceptibility gene for HIV-related Kaposi's sarcoma.

The major histocompatibility complex (MHC) region on chromosome 6p21.3 is suspected to host susceptibility loci for HIV-related Kaposi's sarcoma (HIV-KS). A nested case-control study in the Multicenter AIDS Cohort Study was designed to conduct fine genetic association mapping across central MHC. Individuals co-infected with HIV-1 and human herpes virus-8 who later developed KS were defined as cases (n=354) and were matched 1:1 with co-infected KS-free controls. We report data for new independent MHC class II and III susceptibility loci. In particular, class II HLA-DMB emerged as a strong candidate, with the intronic variant rs6902982 A>G associated with a fourfold increase of risk (odds ratio (OR)=4.09; 95% confidence interval (CI)=1.90-8.80; P=0.0003). A striking multiplicative effect on the estimated risk was associated with further carriage of two non-synonymous variants, rs1800453 A>G (Asp697Gly) and rs4148880 A>G (Ile393Val), in the linked TAP1 gene (OR=10.5; 95% CI=2.54-43.6; P=0.0012). The class III susceptibility variant is moderately associated with HIV-KS and lies within a 120-kb-long haplotype (OR=1.52; 95% CI=1.01-2.28; P=0.047) formed by rs7029 A>G (GPANK1 3' untranslated region), rs1065356 G>A (LY6G6C), rs3749953 A>G (MSH5-SAPCD1 read through) and rs707926 G>A (VARS). Our data suggest that antigen processing by MHC class II molecules is a target pathway in the pathogenesis of HIV-KS.

RYR3 gene polymorphisms and cardiovascular disease outcomes in the context of antihypertensive treatment.

Nearly one-third of adults in the United States have hypertension, which is associated with increased cardiovascular disease (CVD) morbidity and mortality. The goal of antihypertensive pharmacogenetic research is to enhance understanding of drug response based on the interaction of individual genetic architecture and antihypertensive therapy to improve blood pressure control and ultimately prevent CVD outcomes. In the context of the Genetics of Hypertension Associated Treatment study and using a case-only design, we examined whether single-nucleotide polymorphisms in RYR3 interact with four classes of antihypertensive drugs, particularly the calcium channel blocker amlodipine versus other classes, to modify the risk of coronary heart disease (CHD; fatal CHD and non-fatal myocardial infarction combined) and heart failure (HF) in high-risk hypertensive individuals. RYR3 mediates the mobilization of stored Ca(+2) in cardiac and skeletal muscle to initiate muscle contraction. There was suggestive evidence of pharmacogenetic effects on HF, the strongest of which was for rs877087, with the smallest P-value=0.0005 for the codominant model when comparing amlodipine versus all other treatments. There were no pharmacogenetic effects observed for CHD. The findings reported here for the case-only analysis of the antihypertensive pharmacogenetic effect of RYR3 among 3058 CHD cases and 1940 HF cases show that a hypertensive patient's genetic profile may help predict which medication(s) might better lower CVD risk.

Fine physical and transcript mapping of a 1.8 Mb region spanning the locus for childhood acute lymphoblastic leukemia on chromosome 12p12. 3.

Rearrangements of the short arm of chromosome 12 are frequently observed in hematological disorders. Previous studies of loss of heterozygosity identified a small genetic interval on chromosome 12p12.3 that is frequently deleted in childhood acute lymphoblastic leukemia (ALL). Two genes, ETV6/TEL and p27/KIP1, are located in this interval. Evidence has accumulated that an as-yet unidentified tumor suppressor gene is closely linked to these. To facilitate the identification of candidate genes, a long-range high-resolution restriction map of the ALL locus was constructed using a contig of YAC clones. Several marker loci, including 11 STS, three newly developed YAC end-based STS, six EST, and seven genes were unambiguously positioned in the new map. The map covers 1.8Mb and extends from the distal salivary proline-rich protein gene cluster to the proximal p27/KIP1 gene. The data confirmed the order tel-D12S358-p27/KIP1-cen and excluded p27/KIP1 as a candidate tumor suppressor gene. The critical region delimited by D12S89 and D12S358 is a 750kb CpG-island rich region that includes the 240kb TEL/ETV6 gene as well as CLAPS3 (clathrin-adaptor small chain 3). The new map provides a molecular framework for the identification of novel genes and transcriptional units in the ALL interval.

Childhood acute lymphoblastic leukemia: is there a tumor suppressor gene in chromosome 12p12.3?

Cytogenetic deletions on the short arm of chromosome 12 are common, recurring alterations found in a wide range of hematological neoplasias, including childhood acute lymphoblastic leukemia (ALL), the most frequent pediatric malignancy. This loss of genetic material suggests the presence of a tumor suppressor gene playing an important role in growth regulation or in the differentiation of hematopoietic stem cells. In order to substantiate this hypothesis and determine the chromosomal location of this putative gene, we and others have applied a deletion mapping strategy based on the detection of loss of heterozygosity (LOH) at specific genomic loci in leukemic cells. Hemizygous deletions at chromosome 12p12.3 were observed in childhood B cell precursor ALL, and proved to be one of the most frequent genetic alterations seen in this disease. The shortest region of overlapping deletions (SRO) was delimited by the markers D12S89 (distal) and D12S358 (proximal), separated by a genetic interval of approximately 3 cM. LOH in the same region in other hematological diseases, as well as in a variety of solid tumors, suggests either the presence of several tumor suppressor genes or the existence of a single gene with a wide range of activity. This genetic interval contains two known genes: TEL/ETV6, an ets-like transcription factor, and the cyclin-dependant kinase inhibitor, p27/kip1. Accumulating evidence suggests that an as yet unidentified tumor suppressor gene is closely linked to these two genes. Long-range restriction mapping of the SRO region allowed the construction of a approximately 750 kb physical map containing 4 known genes, 7 STSs, 3 chromosome 12 ESTs and 8 CpG islands. The construction of a 12p12.3 framework map is a crucial step towards the identification of candidate genes and should provide a valuable tool for the characterization of transcriptional units.

Isochores and CpG islands in YAC contigs in human Xq26.1-qter.

GC levels were assessed at 37 loci across 30 Mb of Xq26.1-qter, a region physically mapped in overlapping yeast artificial chromosome clones. In 8 Mb of R band Xq26, GC is relatively high (up to 44%) in the proximal 4 Mb and relatively low (40-41%) in the distal 4 Mb. Consistently low GC values (38-41%) are observed in G band Xq27. In contrast, further toward the telomere in Xq28, the GC level rises progressively to reach 52% at 2 to 4 Mb from the end of the chromosome; this region is delimited by low GC loci. Across these regions of Xq, the content of rare-cutter restriction enzyme sites containing CpG, including "CpG islands" in the most completely mapped Xq26-27.1 region, is correlated with GC level. Isochore mapping can thus provide one index of putative gene content across mapped regions.

Compositional mapping of the human dystrophin-encoding gene.

The genomes of warm-blooded vertebrates are mosaics of long DNA segments (> 300 kb, on the average), the isochores, homogeneous in GC levels, which belong to a small number of compositional families. In the present work, the human dystrophin-encoding gene, spanning more than 2.3 Mb in Giemsa band Xp21 (on the short arm of the X chromosome), was analyzed in its isochore organization by hybridizing cDNA probes, corresponding to eight contiguous segments of the coding sequence, on compositional fractions from human DNA. Five DNA regions of uniform (+/- 0.5%) GC content, separated by compositional discontinuities of about 2% GC, were found, so providing the first high-resolution compositional map obtained for a human genome locus and the first direct estimate of isochore size (360 kb to more than 770 kb, in the locus under consideration). One of the isochores contains 71% and another one 21% of deletion breakpoints found in patients suffering from Duchenne's and Becker's muscular dystrophies.

Compositional properties of telomeric regions from human chromosomes.

We have investigated the GC levels of third codon position of genes localized in G- (Giemsa), R-(reverse) and T-(telomeric) bands of human metaphase chromosomes, as well as the hybridization of telomeric probes on fractionated human DNA. The first set of results shows much higher GC levels for genes localized in T-bands than in G- or R-bands (the latter being higher than the former). The second set of data shows that telomeric probes corresponding to T-bands hybridize on the GC-richest family (H3) of isochores, whereas telomeric probes corresponding to R-bands hybridize on GC-rich families H1 and H2; in agreement with these findings, the telomeric repeat common to all chromosomes hybridized on isochore families H1, H2 and H3.

CpG islands: features and distribution in the genomes of vertebrates.

We have investigated the distribution of unmethylated CpG islands in vertebrate genomes fractionated according to their base composition. Genomes from warm-blooded vertebrates (man, mouse and chicken) are characterized by abundant CpG islands, whose frequency increases in DNA fractions of increasing % of guanine + cytosine; % G + C (GC), in parallel with the distribution of genes and CpG doublets. Small, yet significant, differences in the distribution of CpG islands were found in the three genomes. In contrast, genomes from cold-blooded vertebrates (two reptiles, one amphibian, and two fishes) were characterized by an extreme scarcity or absence of CpG islands (detected in these experiments as HpaII tiny fragments or HTF). CpG islands associated with homologous genes from cold- and warm-blooded vertebrates were then compared by analyzing CpG frequencies, GC levels, HpaII sites, rare-cutter sites and G/C boxes (GGGGCGGGGC and closely related motifs) in sequences available in gene banks. Small, yet significant, differences were again detected among the CpG islands associated with homologous genes from warm-blooded vertebrates, in that CpG islands associated with mouse or rat genes often showed low CpG and/or GC levels, as well as low numbers of HpaII sites, rare-cutter sites and G/C boxes, compared to homologous human genes; more rarely, CpG islands were just absent. As far as cold-blooded vertebrates were concerned, a number of genes showed CpG islands, which exhibited a much lower frequency of CpG doublets than that found in CpG islands of warm-blooded vertebrates, but still approached the statistically expected frequency; none of the other features of CpG islands associated with genes from warm-blooded vertebrates were present. Other genes did not show any associated CpG islands, unlike their homologues from warm-blooded vertebrates.

CpG islands, genes and isochores in the genomes of vertebrates.

We have shown that human genes associated with CpG islands increase in number as they increase in % of guanine + cytosine (GC) levels, and that most genes associated with CpG islands are located in the GC-richest compartment of the human genome. This is an independent confirmation of the concentration gradient of CpG islands (detected as HpaII tiny fragments, or HTF) which was demonstrated in the genome of warm-blooded vertebrates [Aïssani and Bernardi, Gene 106 (1991) 173-183]. We then reassessed the location of CpG islands using the data currently available and confirmed that CpG islands are most frequently located in the 5'-flanking sequences of genes and that they overlap genes to variable extents. We have shown that such extents increase with the increasing GC levels of genes, the GC-richest genes being completely included in CpG islands. Under such circumstances, we have investigated the properties of the 'extragenic' CpG islands located in the 5'-flanking segments of homologous genes from both warm- and cold-blooded vertebrates. We have confirmed that, in cold-blooded vertebrates, CpG islands are often absent; when present, they have lower GC and CpG levels; the latter attain, however, statistically expected values. Finally, we have shown that CpG doublets increase with the increasing GC of exons, introns and intergenic sequences (including 'extragenic' CpG islands) in the genomes from both warm- and cold-blooded vertebrates. The correlations found are the same for both classes of vertebrates, and are similar for exons, introns and intergenic sequences (including 'extragenic' CpG islands). The findings just outlined indicate that the origin and evolution of CpG islands in the vertebrate genome are associated with compositional transitions (GC increases) in genes and isochores.

The compositional properties of human genes.

The present work represents the first attempt to study in greater detail previously proposed compositional correlations in genomes, based on a body of additional data relating to gene localizations as well as to extended flanking sequences extracted from gene banks. We have investigated the correlations that exist between (1) the GC levels of exons of human genes, and (2) the GC levels of either intergenic sequences or introns associated with the genes under consideration. In both cases, linear relationships with slopes close to unity were found. The similarity of the linear relationships indicates similar GC levels in intergenic sequences and introns located in the same isochores. Moreover, both intergenic sequences and introns showed GC levels 5-10% lower than the corresponding exons. The above findings considerably strengthen the previously drawn conclusion that coding and noncoding sequences (both inter- and intragenic) from the same isochores of the human genome are compositionally correlated. In addition, we find linear correlations between the GC levels of codon positions and of the intergenic sequences or introns associated with the corresponding genes, as well as among the GC levels of codon positions of genes.

Correlations between the compositional properties of human genes, codon usage, and amino acid composition of proteins.

We have analyzed the correlation that exists between the GC levels of third and first or second codon position for about 1400 human coding sequences. The linear relationship that was found indicates that the large differences in GC level of third codon positions of human genes are paralleled by smaller differences in GC levels of first and second codon positions. Whereas third codon position differences correspond to very large differences in codon usage within the human genome, the first and second codon position differences correspond to smaller, yet very remarkable, differences in the amino acid composition of encoded proteins. Because GC levels of codon positions are linearly correlated with the GC levels of the isochores harboring the corresponding genes, both codon usage and amino acid composition are different for proteins encoded by genes located in isochores of different GC levels. Furthermore, we have also shown that a linear relationship with a unit slope and a correlation coefficient of 0.77 exists between GC levels of introns and exons from the 238 human genes currently available for this analysis. Introns are, however, about 5% lower in GC, on average, than exons from the same genes.

The distribution of genes in the human genome.

Previous investigations on the human genome determined: (i) the base compositions (GC levels) and the relative amounts of its isochore families; (ii) the compositional correlations (i.e., the correlations between GC levels) between third codon positions of a set of genes and the DNA fractions in which the genes were localized; and (iii) the compositional correlations between (a) third and first + second codon positions, as well as that between (b) introns and exons from the set of 'localized genes' and from all the coding sequences and genes (genomic sequences of exons + introns) available in gene banks. Here, we have shown that the correlations (iii, a and b) for 'localized genes' and genes from the bank are in full agreement, indicating that the former set is representative of the latter. We have then used the data (i) and the correlation (ii) to estimate the distribution of genes in isochore families. We have found that 34% of the genes are located in the GC-poor isochores (which represent 62% of the genome), 38% in the GC-rich isochores (31% of the genome) and 28% in the GC-richest isochores (3% of the genome). There is, therefore, a compositional gradient of gene concentration in the human genome. The gene density in the GC-richest 3% of the genome is about eight times higher than in the GC-rich 31%, and about 16 times higher than in the GC-poorest 62%.

A compositional map of human chromosome 21.

GC-poor and GC-rich isochores, the long (greater than 300 kb) compositionally homogeneous DNA segments that form the genome of warm-blooded vertebrates, are located in G- and R-bands respectively of metaphase chromosomes. The precise correspondence between GC-rich isochores and R-band structure is still, however, an open problem, because GC-rich isochores are compositionally heterogeneous and only represent one-third of the genome, with the GC-richest family (which is by far the highest in gene concentration) corresponding to less than 5% of the genome. In order to clarify this issue and, more generally, to correlate DNA composition and chromosomal structure in an unequivocal way, we have developed a new approach, compositional mapping. This consists of assessing the base composition over 0.2-0.3 Mb (megabase) regions surrounding landmarks that were previously localized on the physical map. Compositional mapping was applied here to the long arm of human chromosome 21, using 53 probes that had already been used in physical mapping. The results obtained provide a direct demonstration that the DNA stretches of G-bands essentially correspond to GC-poor isochores, and that R-band DNA is characterized by a compositional heterogeneity that is much more striking than expected, in that it comprises isochores covering the full spectrum of GC levels. GC-poor isochores of R-bands may, however, correspond to 'thin' G-bands, as visualized at high resolution, leaving GC-rich and very GC-rich isochores as the real components of (high-resolution) R-band DNA.(ABSTRACT TRUNCATED AT 250 WORDS)