Juleen A. Cavanaugh

International Collaboration Provides Convincing Linkage Replication in Complex Disease through Analysis of a Large Pooled Data Set: Crohn Disease and Chromosome 16

Numerous familial, non-Mendelian (i.e., complex) diseases have been screened by linkage analysis for regions harboring susceptibility genes. Except for rare, high-penetrance syndromes showing Mendelian inheritance, such as BRCA1 and BRCA2, most attempts have failed to produce replicable linkage findings. For example, in multiple sclerosis and other complex diseases, there have been many reports of significant linkage, followed by numerous failures to replicate. In inflammatory bowel disease (IBD), linkage to two regions has elsewhere been reported at genomewide significance levels: the pericentromeric region on chromosome 16 (IBD1) and chromosome 12q (IBD2). As with other complex diseases, the subsequent support for these localizations has been variable. In this article, we report the results of an international collaborative effort to investigate these putative localization by pooling of data sets that do not individually provide convincing evidence for linkage to these regions. Our results, generated by the genotyping and analysis of 12 microsatellite markers in 613 families, provide unequivocal replication of linkage for a common human disease: a Crohn disease susceptibility locus on chromosome 16 (maximum LOD score 5.79). Despite failure to replicate the previous evidence for linkage on chromosome 12, the results described herein indicate the need to further investigate the potential role of this locus in susceptibility to ulcerative colitis. This report provides a convincing example of the collaborative approach necessary to obtain the sample numbers required to achieve statistical power in studies of complex human traits.

Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodium-dependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B0. We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G → A and 0.001 for 718C → T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder.

Analysis of Australian Crohn's disease pedigrees refines the localization for susceptibility to inflammatory bowel disease on chromosome 16.

A number of localizations for the putative susceptibility gene(s) have been identified for both Crohns disease and ulcerative colitis. In a genome wide scan, Hugot et al. (1996) identified a region on chromosome 16 which appeared to be responsible for the inheritance of inflammatory bowel disease in a small proportion of families. Subsequent work has suggested that this localization is important for susceptibility to Crohns disease rather than ulcerative colitis (Ohmen et al. 1996; Parkes et al. 1996). We investigated the contribution of this localization to the inheritance of inflammatory bowel disease in 54 multiplex Australian families, and confirmed its importance in a significant proportion of Crohns disease families; we further refined the localization to a region near to D16S409, obtaining a maximum LOD score of 6.3 between D16S409 and D16S753.

Prevalence of CARD15/NOD2 mutations in Caucasian healthy people

BACKGROUND:Crohns disease (CD) has been associated with CARD15/NOD2 mutations in Caucasians. The R702W, G908R, and 1007fs mutations represent 82% of the mutated chromosomes. The relative risk of developing CD in homozygous or compound heterozygous people has been estimated as between 10 and 40 times that of the general population. This high risk may support the opinion that CARD15/NOD2 variants are strong CD risk factors at the individual and population levels.SUBJECTS AND METHODS:The allele and genotype frequencies were calculated for the R702W, G908R, and 1007fs mutations in 3,575 Caucasian healthy controls recruited by 15 groups distributed on three continents. Geographic homogeneity was tested and the observed proportion of double mutants was compared with the expected value using χ2 tests.RESULTS:The allele frequencies of the R702W, G908R, and 1007fs mutations were 4.3% (3.6–4.9), 1.2% (0.8–1.6), and 2.3% (1.8–2.8), respectively, with large geographic fluctuations of the G908R, 1007fs, and wild-type alleles (P < 0.0001). At the population level, no simple relationship was observed between mutation frequencies and the disease incidences in the studied populations. At the individual level, no significant deficit of double-dose mutation carriers among healthy controls was found, providing strong evidence that the penetrances of the most at-risk genotypes are low.CONCLUSION:Altogether, these data confirm that CARD15/NOD2 acts in interaction with other unknown risk cofactors.

CARD15/NOD2 risk alleles in the development of Crohn's disease in the Australian population.

We have previously reported strong evidence for linkage between IBD1 and Crohns disease (CD) in Australian Crohns disease families. Three risk alleles for Crohns disease, (Arg702Trp (C/T), Gly908Arg (G/C) and 980fs981 (‐/C), were recently identified in the CARD15/NOD2 gene on chromosome 16, implicating this as the IBD1 locus. Using a novel diagnostic PCR‐RFLP, we have examined the frequency of these alleles in 205 multiplex IBD families, 107 sporadic Crohns disease cases and 409 normal individuals. We demonstrate that the three risk alleles are more frequent in Crohns disease, than in controls, with allelic frequencies of 0.11, 0.02 and 0.07 respectively. Heterozygosity for individual variants conferred a three‐fold increase in risk for Crohns disease while substantially higher risks were associated with being homozygous or compound heterozygous. Despite a significantly lower population allele frequency for the frameshift mutation than reported by other groups, we see a similar contribution by this allele to the risk of developing Crohns disease. While the three risk alleles influence susceptibility to Crohns disease in Australia, we show that these alleles do not fully explain the linkage evidence and suggest that there are very likely additional IBD1 susceptibility alleles yet to be described in Australian CD at the NOD2 locus. We also show a second linkage peak in Australian CD that provides some support for a second disease susceptibility locus on chromosome 16.

Characterisation of a highly sensitive troponin I assay and its application to a cardio-healthy population.

Abstract Background: Abbott Diagnostics have developed a new highly sensitive troponin I (hs-TnI) assay. We have assessed its analytical characteristics and applied the assay to a population of apparently cardio-healthy persons. Methods: We assessed imprecision, bias compared to the previous generation assay, matrix effects, and interferences and applied the assay to an apparently healthy population, deriving the 99th percentile limit of the distribution of values in reference populations for men and women separately. Results: The dynamic range of the assay was ranged from 0.5–50,000 ng/L (pg/mL). The 10% CV was at a concentration of 3.9 ng/L, and the 20% CV was at a concentration of 1.8 ng/L. The new and current version of the TnI assay were highly correlated [slope: 0.98 (95%CI:0.88–1.07), y-intercept:1.20 (95%CI:-2.35–4.75) r2=0.99]. The 99th percentile limit of the distribution of values in a reference population was different for males and females: for males 14.0 ng/L and for females 11.1 ng/L and at these concentrations the assay CV was 5.0%. TnI was detectable in nearly all patient samples from the healthy reference population (98.6%). Conclusions: This new hs-TnI assay is able to measure to an order of magnitude lower than the current generation TnI assay from the same manufacturer. With TnI being detectable in nearly all apparently healthy subject samples this suggests that TnI presence does not always indicate cardiomyocyte necrosis.

ATG16L1 T300A Shows Strong Associations With Disease Subgroups in a Large Australian IBD Population: Further Support for Significant Disease Heterogeneity

OBJECTIVES:Crohns disease (CD) and ulcerative colitis (UC) are the two most common forms of inflammatory bowel disease (IBD), representing a significant health-care burden. A variant in the autophagy gene ATG16L1 (T300A) has been newly identified as a CD susceptibility locus by genome-wide association. Our aim was to assess the contribution of T300A in determining disease susceptibility and phenotype in two independent Australian IBD cohorts and explore the relationship between T300A and known CD risk factors (NOD2[ nucleotide-binding oligomerization domain containing 2] status and smoking).METHODS:In total, 669 CD and 543 UC cases, and 1,244 controls (study 1), 154 CD cases and 420 controls (study 2), and 702 unaffected parents from both groups were genotyped. We conducted case–control and family association analyses, and investigated relationships between T300A and disease subgroups and between NOD2 status and cigarette smoking (CD only).RESULTS:The strong association between CD and T300A was confirmed (P < 0.001), with a two-fold increase in disease risk associated with the GG genotype (odds ratio [OR] 1.96, 95% confidence interval [CI] 1.49–2.58), while ileal CD risk was almost three-fold (OR 2.73, CI 1.87–4.0). ATG16L1 and NOD2 were found to contribute independently to CD risk. A greater than seven-fold increased CD risk was observed for current smokers with a GG genotype (vs nonsmoking AA genotype; P < 0.001, OR 7.65, CI 4.21–13.91). A significant inverse association was found between T300A and UC (P= 0.002). This was strongest for patients with extensive, severe disease.CONCLUSIONS:We confirm the strong association between T300A and CD, specifically ileal subphenotype, and also report the first strong association of this variant with UC.

Iminoglycinuria and hyperglycinuria are discrete human phenotypes resulting from complex mutations in proline and glycine transporters

Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes.

The molecular basis of neutral aminoacidurias

Recent success in the molecular cloning and identification of apical neutral amino acid transporters has shed a new light on inherited neutral amino acidurias, such as Hartnup disorder and Iminoglycinuria. Hartnup disorder is caused by mutations in the neutral amino acid transporter B0 AT1 (SLC6A19). The transporter is found in kidney and intestine, where it is involved in the resorption of all neutral amino acids. The molecular defect underlying Iminoglycinuria has not yet been identified. However, two transporters, the proton amino acid transporter PAT1 (SLC36A1) and the IMINO transporter (SLC6A20) appear to play key roles in the resorption of glycine and proline. A model is presented, involving all three transporters that can explain the phenotypic variability of iminoglycinuria.

The IBD international genetics consortium provides further evidence for linkage to IBD4 and shows gene‐environment interaction

Background and Aims: The inflammatory bowel diseases (IBDs) Crohns disease (CD) and ulcerative colitis are complex disorders with an important genetic determinant. One gene associated with CD has been identified: NOD2/CARD15. Two independent genome‐wide scans found significant evidence (logarithm of odds [LOD] 3.6) and suggestive evidence (LOD 2.8) for linkage on locus 14q11‐12, also known as the IBD4 locus. To further characterize this locus, we assessed gene‐environment interaction (IBD4 × smoking) and phenotypic heterogeneity in a large cohort of IBD‐affected sibling pairs as part of an ongoing international collaborative effort. Patients and Methods: A total of 733 IBD families, comprising 892 affected sibling pairs, were genotyped for microsatellites D14S261, D14S283, D14S972, and D14S275, spanning the IBD4 locus. Information on gender, ethnicity, age at onset, smoking at diagnosis, extraintestinal manifestations, and disease location was available. Results: A significant distortion in the mean allele sharing (MAS) between affected siblings was observed for CD patients only at each of the four markers (54.6%, 52.8%, 50.4%, and 53.3%, respectively). Maximum linkage for CD was observed at marker D14S261 (multipoint nonparametric linkage score 2.36; P ≤ 0.01; MAS 54.6%). MAS was higher in CD families in which all siblings or at least one sibling smoked compared with nonsmoking CD families (MAS, 58.90%, 57.50%, and 52.80%, respectively). Conclusions: The IBD International Genetics Consortium replicated the IBD4 locus on chromosome 14q for CD and also showed evidence for a gene‐environment interaction at this locus. Further studies are needed to explore the mechanism by which smoking influences IBD4.