Richard Bagnall

Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn's disease susceptibility.

A genome-wide association scan in individuals with Crohns disease by the Wellcome Trust Case Control Consortium detected strong association at four novel loci. We tested 37 SNPs from these and other loci for association in an independent case-control sample. We obtained replication for the autophagy-inducing IRGM gene on chromosome 5q33.1 (replication P = 6.6 × 10−4, combined P = 2.1 × 10−10) and for nine other loci, including NKX2-3, PTPN2 and gene deserts on chromosomes 1q and 5p13.

Int22h‐related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection

Summary.  Background: Intrachromosomal, homologous recombination of the duplicon int22h‐1 with int22h‐2 or int22h‐3 causes inversions accounting for 45% of severe hemophilia A, hence the belief that int22h‐2 and int22h‐3 are in opposite orientation to int22h‐1. However, inversions involving int22h‐2 are five times rarer than those involving its virtually identical copy: int22h‐3. Recent sequencing has indicated that int22h‐2 and int22h‐3 form the internal part of the arms of an imperfect palindrome so that int22h‐2, in the centromeric arm, has the same orientation as int22h‐1 and, upon recombination with int22h‐1, should produce deletions and duplications but not inversions. Aim: This work aims to provide rapid tests for all the mutations that can result from recombinations between the int22h sequences and to investigate whether int22h‐2‐related inversions causing hemophilia A arise in chromosomes, where the arms of the palindrome have recombined so that int22h‐2 and int22h‐3 swap places and orientation. Patients/methods: Twenty patients with int22h‐related inversions were examined together with a control and inversion carriers using reverse transcription‐polymerase chain reaction (RT‐PCR), long‐range PCR and sequencing. Results and conclusions: Analysis of mRNA in patients and a control provided evidence confirming the palindromic arrangement of int22h‐2 and int22h‐3 and the proposed inversion polymorphism that allows int22h‐2 to be in the telomeric arm of the palindrome and in opposite orientation to int22h‐1. New long‐range PCR reactions were used to develop a single tube test that detects and discriminates inversions involving int22h‐2 or int22h‐3 and a two‐tube test that can distinguish inversions, deletions, and duplications due to recombination between int22h sequences.

Haemophilia A mutations in the UK: results of screening one-third of the population.

One‐third of the UK haemophilia A population was screened to establish a national database of mutations and pedigrees and advance knowledge of the disease. The following mutations were found: 131 intron 22‐ and 13 intron1‐breaking inversions; 11 gross deletions and an insertion; 65 frameshifts; three in‐frame deletions and one insertion; 46 nonsense; 30 intronic mutations affecting splice sites and four generating new sites; 469 non‐synonymous mutations due to 203 different base substitutions of which four affected, and nine were predicted to affect, splicing; three promoter mutations; two synonymous exon 14 mutations possibly affecting splicing; two VWF mutations. Of the above mutations, 176 are not listed in the Haemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS). Four gross deletions arose by non‐homologous end‐joining; we detected unexpected splicing in some mutations; substitution of amino acids conserved for less than 90 million years are rare; the risk of developing inhibitors for patients with nonsense mutations is greater when the stop codon is in the 3′ half of the mRNA; changes likely to generate splice sites causing frameshifts are over‐represented among non‐synonymous mutations associated with inhibitors; our data and those in HAMSTeRS enabled the size of the spectrum of specific mutations causing the disease to be estimated and to determine how much of it is known.

Polymorphism and hemophilia A causing inversions in distal Xq28: a complex picture.

Two varieties of an inversion breaking the factor VIII gene (F8) and causing half of all severe hemophilia A have previously been described. These were thought to involve the intron 22 sequence: int22h-1 [1] interacting with either the proximal (int22h-2), or the distal (int22h-3) of two extragenic and telomeric copies. However, new sequence data show that only int22h-3 should be involved in these inversions [2]. We explain this discrepancy and note that the new sequence data predict a confounding complication for mutation detection in some families with hemophilia A. In 1993, it was shown that int22h-3 is in inverse orientation to int22h-1 and the same was thought to be true for int22h-2 because BclI blots indicated that both int22h-2 and int22h-3 recombined with int22h-1 to cause inversions [3]. By contrast, the new sequence of the human X chromosome [2] shows that int22h-1 is in the same orientation as int22h-2 so that their interaction should lead to deletion and duplication rather than inversion. These data show that int22h-2 and int22h-3 form the arms of an imperfect palindrome separated by a 65 kb

Creation of a novel donor splice site in intron 1 of the factor VIII gene leads to activation of a 191 bp cryptic exon in two haemophilia A patients

We have constructed a confidential U.K. database of haemophilia A mutations and pedigrees by characterizing the gene defect of one index patient in each U.K. family. Mutations were identified by screening all coding regions of the factor VIII (FVIII) mRNA, using solid‐phase fluorescent chemical cleavage of mismatch and examining additional non‐coding regions of the gene. Here we report two haemophilia A patients (UK 114 FVIII:C 2% and UK 243 FVIII:C < 1%) with an abnormal FVIII mRNA due to an A to G point mutation, 1.4 kb downstream from exon 1 in the FVIII gene. This mutation creates a new donor splice site in intron 1 and leads to insertion of a 191 bp novel exon in the mRNA. Haplotype analysis suggests that the mutation may have originated in a common ancestor of the two patients, who further illustrate how mRNA analysis allows higher efficiency of haemophilia A mutation detection, because their mutation would not have been identified by direct analysis of the factor VIII gene.

Novel isoforms of the CARD8 ( TUCAN ) gene evade a nonsense mutation

CARD8 (TUCAN) is implicated in the regulation of apoptosis and inflammation, and is a positional and functional candidate gene for inflammatory bowel disease (IBD). Recent investigations have reported conflicting results of association between a CARD8 nonsynonymous SNP, rs2043211, and IBD. SNP rs2043211 results in an A>T transversion in the CARD8 template strand, which introduces a stop codon polymorphism (Cys10Stop), and genotyping of the Cys10Stop variant revealed that 9% of the control population was homozygous for the ‘Stop’ allele. The effect of the Stop allele on mRNA and protein expression of the two known isoforms of this gene was investigated. IBD patients homozygous for the Stop allele showed somewhat reduced expression of CARD8 mRNA, but, contrary to expectation, expressed a 48 kDa protein isoform. A search of the EST database and reverse transcription-PCR analysis revealed a novel coding exon and three novel CARD8 mRNA isoforms that are conserved in primates. The isoforms of CARD8 differ in their N-termini, resulting in diverse predicted molecular weights (47, 48, 51, 54 and 60 kDa) and multiple outcomes for the variant including Cys10Stop, Cys34Stop, Phe52Ile and Phe102Ile; one isoform may arise through transcription and translation initiated downstream of rs2043211 to yield a novel protein isoform of ∼47 kDa. The multiple isoforms and differing consequences for a predicted stop codon polymorphism underline the importance of detailed analysis of the effects of proposed functional variants on gene expression.