Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results

Abstract

Cystic fibrosis (CF) is a monogenic disease due to mutations in the CFTR gene. Yet, variability in CF disease presentation is presumed to be affected by modifier genes, such as those recently demonstrated for the pulmonary aspect. Here, we conduct a modifier gene study for meconium ileus (MI), an intestinal obstruction that occurs in 16–20% of CF newborns, providing linkage and association results from large family and case–control samples. Linkage analysis of modifier traits is different than linkage analysis of primary traits on which a sample was ascertained. Here, we articulate a source of confounding unique to modifier gene studies and provide an example of how one might overcome the confounding in the context of linkage studies. Our linkage analysis provided evidence of a MI locus on chromosome 12p13.3, which was segregating in up to 80% of MI families with at least one affected offspring (HLOD = 2.9). Fine mapping of the 12p13.3 region in a large case–control sample of pancreatic insufficient Canadian CF patients with and without MI pointed to the involvement of ADIPOR2 in MI (p = 0.002). This marker was substantially out of Hardy–Weinberg equilibrium in the cases only, and provided evidence of a cohort effect. The association with rs9300298 in the ADIPOR2 gene at the 12p13.3 locus was replicated in an independent sample of CF families. A protective locus, using the phenotype of no-MI, mapped to 4q13.3 (HLOD = 3.19), with substantial heterogeneity. A candidate gene in the region, SLC4A4, provided preliminary evidence of association (p = 0.002), warranting further follow-up studies. Our linkage approach was used to direct our fine-mapping studies, which uncovered two potential modifier genes worthy of follow-up.

Cystic Fibrosis Newborn Screening: Distribution of Blood Immunoreactive Trypsinogen Concentrations in Hypertrypsinemic Neonates

“…We identified 28 CF subjects with meconium ileus on a total of 120 CF (23.3%). Considering the fact that our center missed some cases of mecomium ileus because it was not notified to us, our results are supported by literature data showing that meconium ileus occurs in 16-20% of CF newborns (Dorfman et al 2009). One-hundred-and-sixty-six CF neonates were identified in the population, resulting in an incidence of 1:4,320, with an expected CF carrier incidence of 1:33.…”

TLR5 as an Anti-Inflammatory Target and Modifier Gene in Cystic Fibrosis

“…Specifically, to reduce genetic variability at the CFTR locus, we included only CF patients who had insufficiency of the exocrine pancreas (PI) and carried severe CFTR mutations on both alleles. For the purposes of this study, exocrine pancreatic function status was based on clinical status and CFTR mutations (29,41,42). PI status was assigned if both CFTR mutations were known to be associated with PI.…”

ADIPOR2 Polymorphisms in Cystic Fibrosis are Potential Modifiers of Clinical Severity

“…The genotyping for the 315 base deletions and the insertion of 134 bases was performed in multiplexed reactions using specific primers [ADIPOR2_315delF1 -5´-TGA CAG CCA CCA AGG AGA TTT GGA-3´ and ADIPOR2_315delR1 -5´-AGA CCA CGT CTT CTC CCT TCA ACA-3´;ADIPOR2_134delF1 -5´-AGC TTG ACA AAG ACA CTG CCT ACC-3´ and ADIPOR2_134delR1 -5´-AGC ATA ACC AGG TCC ATG TGG GAA-3´] in multiplexed PCR at standard cycling conditions (60°C for the annealing for 45 s, 1 min for the extension at 72°C). For the 315 base deletions the reaction had 991 bases (wild type) and 676 bases (deletion type); for the insertion of 134 bases, the reaction had 451 bases (wild type) and 585 bases (insertion type) [15].…”

“…The adiponectin receptor 2 (ADIPOR2) gene, located in the region 12p13.3, was studied in CF and associated with the presence of meconium ileus [15]. The ADIPOR2 gene encodes an integral membrane protein, the adiponectin receptor, which regulates the expression of a diabetic hormone secreted by adipocytes and the absence of which is associated with insulin resistance [16].…”

“…However, it is likely that the differential expression of the ADIPOR2 protein is associated with variations in the introns or promoter region of the ADIPOR2 gene, resulting in unequal phenotypic expression among CF patients. Regarding CF and meconium ileus, two mutations of copy number variation (the 315 base deletions and the insertion of 134 bases) in the ADIPOR2 gene were identified [15].…”