Relative Contribution of Genetic and Nongenetic Modifiers to Intestinal Obstruction in Cystic Fibrosis
2006; Elsevier BV; Volume: 131; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2006.07.016
ISSN1528-0012
AutoresScott M. Blackman, Rebecca Deering–Brose, Rita McWilliams, Kathleen Naughton, Barbara Coleman, Teresa Y.Y. Lai, Marilyn Algire, Suzanne E. Beck, Julie Hoover‐Fong, Ada Hamosh, M. Daniele Fallin, Kristen M. West, Dan E. Arking, Aravinda Chakravarti, David J. Cutler, Garry R. Cutting,
Tópico(s)Infant Nutrition and Health
ResumoBackground & Aims: Neonatal intestinal obstruction (meconium ileus [MI]) occurs in 15% of patients with cystic fibrosis (CF). Our aim was to determine the relative contribution of genetic and nongenetic modifiers to the development of this major complication of CF. Methods: A total of 65 monozygous twin pairs, 23 dizygous twin/triplet sets, and 349 sets of siblings with CF were analyzed for MI status, significant covariates, and genome-wide linkage. Results: Specific mutations in the CF transmembrane conductance regulator (CFTR), the gene responsible for CF, correlated with MI, indicating a role for CFTR genotype. Monozygous twins showed substantially greater concordance for MI than dizygous twins and siblings (P = 1 × 10−5), showing that modifier genes independent of CFTR contribute substantially to this trait. Regression analysis revealed that MI was correlated with distal intestinal obstruction syndrome (P = 8 × 10−4). Unlike MI, concordance analysis indicated that the risk for development of distal intestinal obstruction syndrome in CF patients is caused primarily by nongenetic factors. Regions of suggestive linkage (logarithm of the odds of linkage >2.0) for modifier genes that cause MI (chromosomes 4q35.1, 8p23.1, and 11q25) or protect from MI (chromosomes 20p11.22 and 21q22.3) were identified by genome-wide analyses. These analyses did not support the existence of a major modifier gene on chromosome 19 in a region previously linked to MI. Conclusions: The CFTR gene along with 2 or more modifier genes are the major determinants of intestinal obstruction in newborn CF patients, whereas intestinal obstruction in older CF patients is caused primarily by nongenetic factors. Background & Aims: Neonatal intestinal obstruction (meconium ileus [MI]) occurs in 15% of patients with cystic fibrosis (CF). Our aim was to determine the relative contribution of genetic and nongenetic modifiers to the development of this major complication of CF. Methods: A total of 65 monozygous twin pairs, 23 dizygous twin/triplet sets, and 349 sets of siblings with CF were analyzed for MI status, significant covariates, and genome-wide linkage. Results: Specific mutations in the CF transmembrane conductance regulator (CFTR), the gene responsible for CF, correlated with MI, indicating a role for CFTR genotype. Monozygous twins showed substantially greater concordance for MI than dizygous twins and siblings (P = 1 × 10−5), showing that modifier genes independent of CFTR contribute substantially to this trait. Regression analysis revealed that MI was correlated with distal intestinal obstruction syndrome (P = 8 × 10−4). Unlike MI, concordance analysis indicated that the risk for development of distal intestinal obstruction syndrome in CF patients is caused primarily by nongenetic factors. Regions of suggestive linkage (logarithm of the odds of linkage >2.0) for modifier genes that cause MI (chromosomes 4q35.1, 8p23.1, and 11q25) or protect from MI (chromosomes 20p11.22 and 21q22.3) were identified by genome-wide analyses. These analyses did not support the existence of a major modifier gene on chromosome 19 in a region previously linked to MI. Conclusions: The CFTR gene along with 2 or more modifier genes are the major determinants of intestinal obstruction in newborn CF patients, whereas intestinal obstruction in older CF patients is caused primarily by nongenetic factors. Cystic fibrosis (CF [Mendelian Inheritance in Man 219700]) is an autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR [Mendelian Inheritance in Man 602421]). Variation in the CFTR genotype has been associated with some aspects of the CF phenotype such as pancreatic status.1Kristidis P. Bozon D. Corey M. Markiewicz D. Rommens J. Tsui L.C. Durie P. Genetic determination of exocrine pancreatic function in cystic fibrosis.Am J Hum Genet. 1992; 50: 1178-1184PubMed Google Scholar On the other hand, many other variable features of CF are not correlated, indicating that factors independent of CFTR genotype play a major role in the complications and course of this otherwise single-gene disorder.2Kerem E. Corey M. Kerem B.-S. Rommens J. Markiewicz D. Levison H. Tsui L.C. Durie P. The relation between genotype and phenotype in cystic fibrosis—analysis of the most common mutation (deltaF508).N Engl J Med. 1990; 323: 1517-1522Crossref PubMed Scopus (626) Google Scholar, 3Hamosh A. Corey M. Correlation between genotype and phenotype in patients with cystic fibrosis. The Cystic Fibrosis Genotype-Phenotype Consortium.N Engl J Med. 1993; 329: 1308-1313Crossref PubMed Scopus (553) Google Scholar Two such features are meconium ileus (MI), a form of intestinal obstruction observed in the neonatal period that occurs in 13%–20% of CF patients,3Hamosh A. Corey M. Correlation between genotype and phenotype in patients with cystic fibrosis. The Cystic Fibrosis Genotype-Phenotype Consortium.N Engl J Med. 1993; 329: 1308-1313Crossref PubMed Scopus (553) Google Scholar, 4Kerem E. Corey M. Kerem B.-S. Durie P. Tsui L.C. Levison H. Clinical and genetic comparisons of patients with cystic fibrosis, with or without meconium ileus.J Pediatr. 1989; 114: 767-773Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 5Cystic Fibrosis FoundationCystic Fibrosis Foundation Patient Registry Annual Data Report 2000. 2001Google Scholar, 6Lai H.J. Cheng Y. Cho H. Kosorok M.R. Farrell P.M. Association between initial disease presentation, lung disease outcomes, and survival in patients with cystic fibrosis.Am J Epidemiol. 2004; 159: 537-546Crossref PubMed Scopus (117) Google Scholar and distal intestinal obstruction syndrome (DIOS), a trait with clinical and pathologic similarities to MI that affects older CF patients.7Rosenstein B.J. Langbaum T.S. Incidence of distal intestinal obstruction syndrome in cystic fibrosis.J Pediatr Gastroenterol Nutr. 1983; 2: 299-301Crossref PubMed Google Scholar, 8Dray X. Bienvenu T. Desmazes-Dufeu N. Dusser D. Marteau P. Hubert D. Distal intestinal obstruction syndrome in adults with cystic fibrosis.Clin Gastroenterol Hepatol. 2004; 2: 498-503Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar Estimates of the prevalence of DIOS in CF patients vary considerably (2.4%–41.3%), probably because of differences in the diagnostic criteria, length of follow-up evaluation, and age range of the patients studied.7Rosenstein B.J. Langbaum T.S. Incidence of distal intestinal obstruction syndrome in cystic fibrosis.J Pediatr Gastroenterol Nutr. 1983; 2: 299-301Crossref PubMed Google Scholar, 8Dray X. Bienvenu T. Desmazes-Dufeu N. Dusser D. Marteau P. Hubert D. Distal intestinal obstruction syndrome in adults with cystic fibrosis.Clin Gastroenterol Hepatol. 2004; 2: 498-503Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 9Jaffe B.F. Graham III, W.P. Goldman L. Postinfancy intestinal obstruction in children with cystic fibrosis.Arch Surg. 1966; 92: 337-343Crossref PubMed Scopus (31) Google Scholar, 10O’Halloran S.M. Gilbert J. McKendrick O.M. Carty H.M. Heaf D.P. Gastrografin in acute meconium ileus equivalent.Arch Dis Child. 1986; 61: 1128-1130Crossref PubMed Scopus (51) Google Scholar Several reports indicate that the recurrence rate of MI in siblings is significantly higher than the prevalence of MI in unrelated patients, suggesting that factors shared among siblings contribute to the development of MI.4Kerem E. Corey M. Kerem B.-S. Durie P. Tsui L.C. Levison H. Clinical and genetic comparisons of patients with cystic fibrosis, with or without meconium ileus.J Pediatr. 1989; 114: 767-773Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 11Donnison A.B. Shwachman H. Gross R.E. A review of 164 children with meconium ileus seen at the Children’s Hospital Medical Center, Boston.Pediatrics. 1966; 37: 833-850PubMed Google Scholar, 12Allan J.R. Robbie M. Phelan P.D. Danks D.M. Familial occurrence of meconium ileus.Eur J Pediatr. 1981; 135: 291-292Crossref PubMed Scopus (45) Google Scholar, 13Picard E. Aviram M. Yahav Y. Rivlin J. Blau H. Bentur L. Avital A. Villa Y. Schwartz S. Kerem B. Kerem E. Familial concordance of phenotype and microbial variation among siblings with CF.Pediatr Pulmonol. 2004; 38: 292-297Crossref PubMed Scopus (20) Google Scholar However, it is not known whether familial concordance of MI is the result of shared genetic or environmental factors or a combination of both. Evidence for genetic factors underlying intestinal obstruction has been derived from studies of the CF mouse model. The majority of homozygous CFTR null mice have a severe and usually fatal intestinal obstruction early in life.14Snouwaert J.N. Brigman K.K. Latour A.M. Malouf N.N. Boucher R.C. Smithies O. Koller B.H. An animal model for cystic fibrosis made by gene targeting.Science. 1992; 257: 1083-1088Crossref PubMed Scopus (769) Google Scholar By capitalizing on strain-specific differences in survival among CFTR null mice, Rozmahel et al15Rozmahel R. Wilschanski M. Matin A. Plyte S. Oliver M. Auerbach W. Moore A. Forstner J. Durie P. Nadeau J. Bear C. Tsui L.C. Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor.Nat Genet. 1996; 12: 280-287Crossref PubMed Scopus (361) Google Scholar were able to locate a modifier locus to the distal portion of mouse chromosome 7. Several other loci for survival from intestinal obstruction in CF mice have since been identified.16Haston C.K. Tsui L.C. Loci of intestinal distress in cystic fibrosis knockout mice.Physiol Genomics. 2003; 12: 79-84PubMed Google Scholar Zielenski et al17Zielenski J. Corey M. Rozmahel R. Markiewicz D. Aznarez I. Casals T. Larriba S. Mercier B. Cutting G.R. Krebsova A. Macek Jr, M. Langfelder-Schwind E. Marshall B.C. DeCelie-Germana J. Claustres M. Palacio A. Bal J. Nowakowska A. Ferec C. Estivill X. Durie P. Tsui L.C. Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13.Nat Genet. 1999; 22: 128-129Crossref PubMed Scopus (195) Google Scholar postulated that the locus on chromosome 7 responsible for murine intestinal obstruction may contain a gene that is responsible for MI in CF patients. This theory was tested by genotyping 188 CF sibling pairs and parents using 9 microsatellite markers that spanned 7.65 Mb on chromosome 19q13, a region of conserved synteny with the segment of mouse chromosome 7 that was linked to intestinal obstruction. Several markers from this region showed significant linkage with the MI phenotype consistent with the presence of an MI-modifier gene, which was named CF Modifier 1 (CFM1 [Mendelian Inheritance in Man 603855]) within this region. However, the gene (or genes) within the CFM1 region that modifies MI has not been identified. The CF Twin and Sibling Study in the United States is currently recruiting patients to assess genetic and nongenetic contribution to disease variation in CF. Monozygous (MZ) and dizygous (DZ) twins can differentiate the relative contribution of genetic factors because twins have a high degree of shared environment, but MZ and DZ twins differ in their degree of gene sharing (100% vs 50%).18MacGregor A.J. Snieder H. Schork N.J. Spector T.D. Twins Novel uses to study complex traits and genetic diseases.Trends Genet. 2000; 16: 131-134Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 19Hammond C.J. Snieder H. Spector T.D. Gilbert C.E. Genetic and environmental factors in age-related nuclear cataracts in monozygotic and dizygotic twins.N Engl J Med. 2000; 342: 1786-1790Crossref PubMed Scopus (188) Google Scholar, 20Bronsveld I. Mekus F. Bijman J. Ballmann M. Greipel J. Hundrieser J. Halley D.J. Laabs U. Busche R. De Jonge H.R. Tummler B. Veeze H.J. Residual chloride secretion in intestinal tissue of deltaF508 homozygous twins and siblings with cystic fibrosis The European CF Twin and Sibling Study Consortium.Gastroenterology. 2000; 119: 32-40Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 21Selmi C. Mayo M.J. Bach N. Ishibashi H. Invernizzi P. Gish R.G. Gordon S.C. Wright H.I. Zweiban B. Podda M. Gershwin M.E. Primary biliary cirrhosis in monozygotic and dizygotic twins: genetics, epigenetics, and environment.Gastroenterology. 2004; 127: 485-492Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar On the other hand, siblings have the same degree of gene sharing as DZ twins (50%), but a lower degree of shared environment, so that a comparison of DZ twins and siblings can estimate environmental contribution. Although recruitment for the CF Twin and Sibling study is not complete, MI and DIOS are sufficiently frequent to perform an analysis of concordance rates among the patients recruited to date. We also performed an initial genome-wide linkage analysis for MI including a re-evaluation of the CFM1 locus. The CF Twin and Sibling Study is a collaborative effort to collect detailed clinical information and DNA samples from affected siblings and their parents. Informed consent was obtained from all subjects before enrollment in the study. Enrollment was based on conclusive diagnosis of CF.22Rosenstein B.J. Cutting G.R. The diagnosis of cystic fibrosis: a consensus statement.J Pediatr. 1998; 132: 589-595Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar The diagnosis of MI was based on the presence of the following features in the newborn period: lack of passage of stool within 24 hours after birth, evidence of obstruction on abdominal radiograph (ground-glass appearance of intestine, air-fluid levels, and/or intra-abdominal calcifications), evidence of colonic abnormality (microcolon on radiograph), and treatment for obstruction (enema or surgery). Some cases were complicated by bilious vomiting, perforation, and/or atresia of the jejunum or ileum. The diagnosis of DIOS was based on the clinical impression of the reporting CF center. A required feature was intestinal obstruction requiring treatment beyond laxatives (eg, oral polyethylene glycol solution, enema, or surgery); radiologic documentation of DIOS was obtained for some, but not all, patients. Pancreatic status was obtained from patient medical records. Pancreatic-insufficient status was determined by a fecal fat test, a fecal elastase test, the appearance of the stool pattern, the presence of oil in the stool, the patient’s growth pattern, and/or the complaint of abdominal pains. Genomic DNA was isolated from whole blood using the phenol/chloroform procedure.23Cutting G.R. Antonarakis S.E. Buetow K.H. Kasch L.M. Rosenstein B.J. Kazazian Jr, H.H. Analysis of DNA polymorphism haplotypes linked to the cystic fibrosis locus in North American Black and Caucasian families supports the existence of multiple mutations of the cystic fibrosis gene.Am J Med Genet. 1989; 44: 307-318Google Scholar The zygosity status of twins was determined by AmpFISTR Profiler (Applied Biosystems, Foster City, CA). CFTR genotype was obtained from the patient’s medical record. In cases in which CFTR genotyping had not been performed or was incomplete, DNA samples were typed for 58 CFTR alleles by using the Roche polymerase chain reaction–based Line Probe Assay (Roche Molecular Systems, Alameda, CA).24Wang X. Myers A. Saiki R.K. Cutting G.R. Development and evaluation of a PCR-based, line probe assay for the detection of 58 alleles in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.Clin Chem. 2002; 48: 1121-1123PubMed Google Scholar DNA samples with 1 or more unidentified CFTR mutations after typing with the Line Probe Assay were subjected to DNA sequencing of all coding regions of CFTR and flanking introns.25Groman J.D. Meyer M.E. Wilmott R.W. Zeitlin P.L. Cutting G.R. Variant cystic fibrosis phenotypes in the absence of CFTR mutations.N Engl J Med. 2002; 347: 401-407Crossref PubMed Scopus (158) Google Scholar For genome-wide linkage studies, patients were genotyped by the Marshfield Genotyping Center for 402 polymorphic short tandem repeat (STR) markers with an average spacing of 10 centimorgans (cM), or 7.5 megabases (Mb). The average rate of genotypes determined for the 1161 individuals was 95% (range, 70%–99.8%); identified Mendelian errors numbered 642 (1.1%). The average heterozygosity in the study population was 75% (range, 57%–89%). Single nucleotide polymorphism (SNP) typing was performed using the Centaurion 100K SNP set from Affymetrix (Santa Clara, CA)26Matsuzaki H. Dong S. Loi H. Di X. Liu G. Hubbell E. Law J. Berntsen T. Chadha M. Hui H. Yang G. Kennedy G.C. Webster T.A. Cawley S. Walsh P.S. Jones K.W. Fodor S.P. Mei R. Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays.Nat Methods. 2004; 1: 109-111Crossref PubMed Scopus (354) Google Scholar; 90,689 markers met quality criteria for inclusion (minor allele frequency at least 1%; genotype obtained in at least 85% of samples) and yielded an average marker spacing of 23.6 kb. The average rate of genotypes determined for the 86 individuals was 96% (range, 80%–100%); identified Mendelian errors numbered 2115 (0.03%). The average heterozygosity was 30%. For the chromosome 19–specific map, patients were genotyped at 7 STR markers spanning 6.5 Mb of chr19q13: D19S211, D19S217, D19S219, D19S112, D19S412, D19S902, and D19S604, yielding an average marker spacing of 1.1 Mb. STR markers were sized using an ABI Prism 310 DNA Sequencer or an ABI 3100 DNA Sequencer with GENESCAN analysis software version 3.1.2 (Applied Biosystems, Foster City, CA). Genotyping was controlled internally by genotyping individuals of the same family on the same run. Any ambiguous genotypes and genotypes that were not consistent with Mendelian inheritance were retyped. Concordance for disease (MI or DIOS) was calculated by the number of pairs concordant for disease divided by the total number of pairs in which at least one member had disease. Concordance for lack of disease was calculated by the number of pairs in which neither member had disease divided by the total number of pairs in which at least one member did not have disease. Heritability estimates were estimated as described by Falconer27Falconer D.S. Inheritance of liability to certain diseases estimated from incidence among relatives.Ann Hum Genet. 1965; 29: 51-71Crossref Scopus (1105) Google Scholar and Falconer and Mackay.28Falconer D.S. Mackay T.F.C. Heritability. Introduction to quantitative genetics. 4th ed. Pearson Education Limited, Essex1996: 160-183Google Scholar For DIOS concordance calculations, siblings were used as a proxy for DZ twins by correcting the DIOS status for age difference: the DIOS status of the older sibling when he/she was the age of the younger sibling was used. Power calculation was performed using DSTPLAN (M. D. Anderson Cancer Center, Houston, TX). One set of nonidentical triplets in which 1 member had MI and none had DIOS was counted as a single MI discordant pair and as a single pair without DIOS. Three families had a set of MZ twins and a sibling all with CF and contributed to both the MZ and sibling pair concordance calculations. With one exception, the MZ twin set was concordant for disease (MI or DIOS) or lack of disease, and thus was counted as a single individual within a sibling pair. The exception was an MZ twin pair discordant for DIOS with a sibling without DIOS; this set was counted as a single discordant sibling pair. Statistical calculations were performed using STATA (StataCorp, College Station, TX). For all tests, a P value of less than .05 was considered statistically significant. In case of small cell sizes, the Fisher exact test was used (http://www.unc.edu/∼preacher/fisher/fisher.htm). The Student t test was used to evaluate means derived from normally distributed continuous data. Multiple logistic regression was performed with STATA using clinical data from the CF Twin and Sibling Study. Each clinical variable was tested for correlation with MI along with age and sex in a 3-variable logistic model. Variance was estimated using the robust Huber/White/sandwich estimator, and observations within each family were not considered to be independent. Factors tested individually for correlation with MI were age, sex, DIOS, surgery for DIOS, pancreatic insufficiency, number of ΔF508 alleles, sweat chloride, positive cultures for Pseudomonas aeruginosa, mucoid pseudomonas, antibiotic-resistant pseudomonas, Burkholderia cepacia, atypical mycobacteria, aspergillus, Staphylococcus aureus, methicillin-resistant S aureus, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae, streptococcus, sinus disease, surgery for sinus disease, nasal polyps, surgery for nasal polyps, increased transaminase levels, pancreatitis, surgery for pancreatitis, diabetes, cholelithiasis, cholecystectomy, gastroesophageal reflux, Nissen fundoplication, gastrostomy tube placement, rectal prolapse, appendiceal disease, failure to thrive, steatorrhea, neonatal jaundice, average forced expiratory volume in 1 second (CF-specific percentile score29Kulich M. Rosenfeld M. Campbell J. Kronmal R. Gibson R.L. Goss C.H. Ramsey B. Disease-specific reference equations for lung function in patients with cystic fibrosis.Am J Respir Crit Care Med. 2005; 172: 885-891Crossref PubMed Scopus (54) Google Scholar), average body mass index Z-score, age at diagnosis of CF, lung transplantation, number of pulmonary exacerbations in the past 1 or last 5 years, and medication compliance. Factors significantly correlated with MI in the 3-variable analysis were tested for inclusion in a multivariate logistic regression model. Factors were removed if they did not contribute significantly to the risk of MI and their inclusion did not improve the model (likelihood ratio test), or if they were correlated strongly with another covariate. In the final multivariate model, the 3 parameters regarding number of ΔF508 alleles were correlated (as expected), but no other pair of factors had a correlation coefficient of greater than 0.25. The genome-wide and chromosome 19–specific STR marker data were screened for Mendelian inconsistencies by PedCheck30O’Connell J.R. Weeks D.E. PedCheck: a program for identification of genotype incompatibilities in linkage analysis.Am J Hum Genet. 1998; 63: 259-266Abstract Full Text Full Text PDF PubMed Scopus (1822) Google Scholar and SIB-PAIR 0.99.9 (Queensland University of Medical Research, Queensland, Australia)31Duffy D.L. SIB-PAIR 0.99.9: a program for elementary genetic analysis. Queensland University of Medical Research, Queensland, Australia2004Google Scholar; inconsistent markers were retyped or eliminated. Single- and multipoint, parametric and nonparametric linkage analyses were performed using GENEHUNTER32Kruglyak L. Daly M.J. Reeve-Daly M.P. Lander E.S. Parametric and nonparametric linkage analysis: a unified multipoint approach.Am J Hum Genet. 1996; 58: 1347-1363PubMed Google Scholar and MERLIN.33Abecasis G.R. Cherny S.S. Cookson W.O. Cardon L.R. Merlin—rapid analysis of dense genetic maps using sparse gene flow trees.Nat Genet. 2002; 30: 97-101Crossref PubMed Scopus (2783) Google Scholar Parametric analyses were performed with dominant, additive, and recessive models of inheritance with penetrance set at 0.80 and phenocopy rate set at 0.10 (both derived from observed rates of MI; see later), and with alternative penetrance values of 0.60 and 1.00 and phenocopy values of 0.00 and 0.20. Analysis using MERLIN included error detection through identification of improbable recombination events. For analysis of high-density SNP data, MERLIN again was used; for this analysis, linkage disequilibrium between markers was modeled by clustering correlated markers.34Abecasis G.R. Wigginton J.E. Handling marker-marker linkage disequilibrium: pedigree analysis with clustered markers.Am J Hum Genet. 2005; 77: 754-767Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar For the 7-STR map of chromosome 19, SIB-PAIR 0.99.931Duffy D.L. SIB-PAIR 0.99.9: a program for elementary genetic analysis. Queensland University of Medical Research, Queensland, Australia2004Google Scholar was used to perform identity-by-descent analyses. If parental genotype data were not available, then identical-by-descent was estimated from identical-by-state. ASP 26.07.200135Krawczak M. ASP—a simulation-based power calculator for genetic linkage studies of qualitative traits, using sib-pairs.Hum Genet. 2001; 109: 675-677Crossref PubMed Scopus (6) Google Scholar was used to simulate (n = 1000) the statistical power of our dataset under a dominant (disease allele frequency = 0.10), recessive (disease allele frequency = 0.43), or additive model (disease allele frequency = 0.10), with the disease gene located at a recombination fraction θ = 0.01 from the simulated marker and a phenocopy rate of 0.05. The earlier-described disease allele frequencies were calculated assuming Hardy–Weinberg equilibrium, a trait prevalence of 15% (derived from the observed rate of MI), and a trait penetrance of 80% (derived from the concordance rate in MZ twins). With disease allele frequency defined as q, calculations were as follows: q2 × 0.8 = 0.15 giving q = 0.43 for recessive; (1 − p2) × 0.8 = 0.15 giving p = 0.90 and q = 0.10 for dominant; (2pq + 2q2) × 0.8 = 0.15 giving q = 0.10 for additive. For all 3 models, the number of alleles and the allele frequencies calculated by GENEHUNTER for marker D19S112 were used (13 alleles: 0.0029, 0.1556, 0.0039, 0.0616, 0.0352, 0.2076, 0.2074, 0.0020, 0.1301, 0.1223, 0.0577, 0.0108, and 0.0029). The total power of the study was calculated using restricted model estimates and the equation: [1 − (1 − concordant estimated power) (1 − discordant estimated power)] × 100% = % power. Clinical information collected from 65 pairs of MZ twins, 22 pairs of DZ twins, 1 set of nonidentical triplets, 14 single siblings, 365 sibling pairs (includes 4 sibling-MZ twin pairs), 29 sets of 3 siblings, and 1 set of 5 siblings (1009 patients total) was obtained from the CF Twin and Sibling Study. All participants met the diagnostic criteria for CF. These patients account for approximately 85% of all twins affected with CF and 35% of all families with 2 or more children affected with CF in the United States according to the CF Patient Registry maintained by the US CF Foundation. Of the 940 patients with information about MI, 160 had MI (17%); treatment was known in 159 of these: 108 patients had surgical treatment, 49 patients were treated with an enema, 1 patient had an enema and surgery, and 1 patient resolved spontaneously. Approximately 95% of CF patients have pancreatic insufficiency (PI), and prior studies of MI have shown a close correlation between pancreatic status and MI.1Kristidis P. Bozon D. Corey M. Markiewicz D. Rommens J. Tsui L.C. Durie P. Genetic determination of exocrine pancreatic function in cystic fibrosis.Am J Hum Genet. 1992; 50: 1178-1184PubMed Google Scholar Indeed, 159 of the 160 MI patients in this study were diagnosed with PI. As expected, pancreatic sufficiency (PS) was present at a higher rate in patients without MI (91 of 762; 12%; P = 3 × 10−7). Because MI correlates with pancreatic status, and pancreatic status correlates with CFTR genotype, we expected that MI also should correlate with CFTR genotype. As predicted, the common CF mutation ΔF508, which is highly associated with PI, was found at increased frequency in MI vs non-MI patients (79% vs 68%; P = 9 × 10−5), and homozygosity for ΔF508 also was increased in MI vs non-MI patients (64% vs 47%; P = 2 × 10−4). Furthermore, of the 39 CFTR genotypes found in patients with PS in this study, only 1 genotype (ΔF508/2184insA) also occurred in a patient with MI: the single MI patient who remains PS (complete list of CFTR mutations available from corresponding author). Intriguingly, 31 patients with PI carried one mutation observed in PS patients, and none of these patients had MI, whereas among 784 PI patients with no PS mutations, 153 had MI (P = .002). The latter observation suggests that CFTR genotype is more predictive of MI than pancreatic status. To test this concept, we selected only PI patients and recalculated the frequency of the ΔF508 mutation and the frequency of homozygosity for ΔF508 in MI vs non-MI patients. Both ΔF508 and homozygosity for ΔF508 were found at greater frequency in MI patients than in non-MI patients (ΔF508 allele frequency: 79% vs 73%, P = .02; ΔF508 homozygote frequency: 64% vs 54%, P = .01). Finally, we performed a multivariate regression analysis in which PI was subdivided based on the number of ΔF508 alleles. As shown in Table 1, PI with ΔF508 homozygosity was an independent risk factor for MI (odds ratio, 13.0). PI in the context of other CFTR genotypes had a large effect size (odds ratio, 7.8–8.4) but was of borderline significance. Thus, although PI is highly correlated with MI, CFTR genotype can decrease the association with MI (eg, PS mutations) or increase the association with MI (eg, ΔF508 homozygosity) in PI patients.Table 1Multivariate Regression Analysis of MI CovariatesOdds ratio95% Confidence intervalP valuePI with no ΔF508 alleles8.4 (0.9–77.2).061PI with one ΔF508 allele7.8 (1.0–61.2).050PI with two ΔF508 alleles13.0 (1.7–100).014DIOS treated nonsurgically2.52 (1.47–4.32).001DIOS treated with surgery24.6 (8.34–72.6)7 × 10−9Increased ALT/AST levelsaAlanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels greater than twice the normal upper limit at least twice.4.15 (2.25–7.67)5 × 10−6Current agebOdds ratio reflects decreased odds of MI for each year of age.0.93 (0.89–0.97).001Body mass index z-scorecOdds ratio reflects increased odds of MI for each unit decrease in body mass index z-score.1.34 (1.03–1.75).032B cepacia–positive culture3.33 (1.48–7.47).004a Alanine
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