Multiple common variants for celiac disease influencing immune gene expression.

We performed a second-generation genome-wide association study of 4,533 individuals with celiac disease (cases) and 10,750 control subjects. We genotyped 113 selected SNPs with P(GWAS) < 10(-4) and 18 SNPs from 14 known loci in a further 4,918 cases and 5,684 controls. Variants from 13 new regions reached genome-wide significance (P(combined) < 5 x 10(-8)); most contain genes with immune functions (BACH2, CCR4, CD80, CIITA-SOCS1-CLEC16A, ICOSLG and ZMIZ1), with ETS1, RUNX3, THEMIS and TNFRSF14 having key roles in thymic T-cell selection. There was evidence to suggest associations for a further 13 regions. In an expression quantitative trait meta-analysis of 1,469 whole blood samples, 20 of 38 (52.6%) tested loci had celiac risk variants correlated (P < 0.0028, FDR 5%) with cis gene expression.

A two-tier approach to mutation detection in the COL4A5 gene for Alport syndrome.

About 85% of Alport syndrome is an X-linked semi-dominant condition caused by mutations in the collagen gene, COL4A5. The large size and high GC content of this gene have presented diagnostic laboratories with problems in identifying mutations with greater than about a 50% success rate since the gene was first cloned 16 years ago. An RNA based approach is adopted here for a first pass mutation scanning coupled with more traditional exon-by-exon screening to increase the rate of mutation identification. Twenty-one mutations were identified in twenty-five patients with clear Alport syndrome including four gross deletions, two deep intronic mutations, three frameshifts, three splice site mutations, eight missense mutations and one inframe deletion.

Gene conversion and evolution of Xq28 duplicons involved in recurring inversions causing severe hemophilia A.

Inversions breaking the 1041 bp int1h-1 or the 9.5-kb int22h-1 sequence of the F8 gene cause hemophilia A in 1/30,000 males. These inversions are due to homologous recombination between the above sequences and their inverted copies on the same DNA molecule, respectively, int1h-2 and int22h-2 or int22h-3. We find that (1) int1h and int22h duplicated more than 25 million years ago; (2) the identity of the copies (>99%) of these sequences in humans and other primates is due to gene conversion; (3) gene conversion is most frequent in the internal regions of int22h; (4) breakpoints of int22h-related inversions also tend to involve the internal regions of int22h; (5) sequence variations in a sample of human X chromosomes defined eight haplotypes of int22h-1 and 27 of int22h-2 plus int22h-3; (6) the latter two sequences, which lie, respectively, 500 and 600 kb telomeric to int22h-1 are five-fold more identical when in cis than when in trans, thus suggesting that gene conversion may be predominantly intrachromosomal; (7) int1h, int22h, and flanking sequences evolved at a rate of about 0.1% substitutions per million years during the divergence between humans and other primates, except for int1h during the human-chimpanzee divergence, when its rate of evolution was significantly lower. This is reminiscent of the slower evolution of palindrome arms in the male specific regions of the Y chromosome and we propose, as an explanation, that intrachromosomal gene conversion and cosegregation of the duplicated regions favors retention of the ancestral sequence and thus reduces the evolution rate.

Unusual deep intronic mutations in the COL4A5 gene cause X linked Alport syndrome.

The X-linked form of Alport syndrome is caused by mutations in the COL4A5 gene in Xq22. This large multiexonic gene has, in the past, been difficult to screen, with several studies detecting only about 50% of mutations. We report three novel intronic mutations that may, in part, explain this poor success rate and demonstrate that single base changes deep within introns can, and do, cause disease: one mutation creates a new donor splice site within an intron resulting in the inclusion of a novel in-frame cryptic exon; a second mutation results in a new exon splice enhancer sequence (ESE) that promotes splicing of a cryptic exon containing a stop codon; a third patient exhibits exon skipping as a result of a base substitution within the polypyrimidine tract that precedes the acceptor splice site. All three cases would have been missed using an exon-by-exon DNA screening approach.

Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A.

The messenger RNA (mRNA) from 5 of 69 patients with severe hemophilia A did not support amplification of complementary DNA containing the first few exons of the factor VIII (F8) gene but supported amplification of mRNA containing exon 1 of F8 plus exons of the VBP1 gene. This chimeric mRNA signals an inversion breaking intron 1 of the F8 gene. Using an inversion patient, one deleted for F8 exons 1 to 6, and cosmids mapped 70 to 100 kb telomeric of the F8 gene, this study shows that this break strictly affects a sequence (int1h-1) repeated (int1h-2) about 140 kb more telomerically, between the C6.1A and VBP1 genes. The 1041-base pair repeats differ at a single nucleotide (although int1h-2 also showed one polymorphism) and are in opposite orientation. The results demonstrate that they cause inversions by intrachromosome or intrachromatid homologous recombination. The genomic structure of the inversion region shows that transcription traverses intergenic spaces to produce the 2 chimeric mRNAs containing the F8 sequences and characteristic of the inversion. This observation prompts the suggestion that nature may use such extended transcription to test whether the addition of novel domains from neighboring genes creates desirable new genes. A rapid polymerase chain reaction test was developed for the inversion in both patients and carriers. This has identified 10 inversions, affecting F8 genes with 5 different haplotypes for the BclI, introns 13 and 22 VNTR polymorphism, among 209 unrelated families with severe hemophilia A. This indicates a prevalence of 4.8% and frequent recurrence of the inversion. This should result in absence of F8, and one inversion patient is known to have inhibitors. (Blood. 2002;99:168-174)