Kenneth J. Friedman

Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis

BACKGROUND It is unknown whether genetic factors predispose patients to idiopathic pancreatitis. In patients with cystic fibrosis, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene typically cause pulmonary and pancreatic insufficiency while rarely causing pancreatitis. We examined whether idiopathic pancreatitis is associated with CFTR mutations in persons who do not have lung disease of cystic fibrosis. METHODS We studied 27 patients (mean age at diagnosis, 36 years), 22 of whom were female, who had been referred for an evaluation of idiopathic pancreatitis. DNA was tested for 17 CFTR mutations and for the 5T allele in intron 8 of the CFTR gene. The 5T allele reduces the level of functional CFTR and is associated with an inherited form of infertility in males. Patients with two abnormal CFTR alleles were further evaluated for unrecognized cystic fibrosis-related lung disease, and both base-line and CFTR-mediated ion transport were measured in the nasal mucosa. RESULTS Ten patients with idiopathic chronic pancreatitis (37 percent) had at least one abnormal CFTR allele. Eight CFTR mutations were detected (prevalence ratio, 11:1; 95 percent confidence interval, 5 to 23; P<0.001). In three patients both alleles were affected (prevalence ratio, 80:1; 95 percent confidence interval, 17 to 379; P<0.001). These three patients did not have lung disease typical of cystic fibrosis on the basis of sweat testing, spirometry, or base-line nasal potential-difference measurements. Nonetheless, each had abnormal nasal cyclic AMP-mediated chloride transport. CONCLUSION In a group of patients referred for evaluation of idiopathic pancreatitis, there was a strong association between mutations in the CFTR gene and pancreatitis. The abnormal CFTR genotypes in these patients with pancreatitis resemble those associated with male infertility.

Genetic Modifiers of Liver Disease in Cystic Fibrosis

CONTEXT A subset (approximately 3%-5%) of patients with cystic fibrosis (CF) develops severe liver disease with portal hypertension. OBJECTIVE To assess whether any of 9 polymorphisms in 5 candidate genes (alpha(1)-antitrypsin or alpha(1)-antiprotease [SERPINA1], angiotensin-converting enzyme [ACE], glutathione S-transferase [GSTP1], mannose-binding lectin 2 [MBL2], and transforming growth factor beta1 [TGFB1]) are associated with severe liver disease in patients with CF. DESIGN, SETTING, AND PARTICIPANTS Two-stage case-control study enrolling patients with CF and severe liver disease with portal hypertension (CFLD) from 63 CF centers in the United States as well as 32 in Canada and 18 outside of North America, with the University of North Carolina at Chapel Hill as the coordinating site. In the initial study, 124 patients with CFLD (enrolled January 1999-December 2004) and 843 control patients without CFLD were studied by genotyping 9 polymorphisms in 5 genes previously studied as modifiers of liver disease in CF. In the second stage, the SERPINA1 Z allele and TGFB1 codon 10 genotype were tested in an additional 136 patients with CFLD (enrolled January 2005-February 2007) and 1088 with no CFLD. MAIN OUTCOME MEASURES Differences in distribution of genotypes in patients with CFLD vs patients without CFLD. RESULTS The initial study showed CFLD to be associated with the SERPINA1 Z allele (odds ratio [OR], 4.72; 95% confidence interval [CI], 2.31-9.61; P = 3.3 x 10(-6)) and with TGFB1 codon 10 CC genotype (OR, 1.53; 95% CI, 1.16-2.03; P = 2.8 x 10(-3)). In the replication study, CFLD was associated with the SERPINA1 Z allele (OR, 3.42; 95% CI, 1.54-7.59; P = 1.4 x 10(-3)) but not with TGFB1 codon 10. A combined analysis of the initial and replication studies by logistic regression showed CFLD to be associated with SERPINA1 Z allele (OR, 5.04; 95% CI, 2.88-8.83; P = 1.5 x 10(-8)). CONCLUSIONS The SERPINA1 Z allele is a risk factor for liver disease in CF. Patients who carry the Z allele are at greater risk (OR, approximately 5) of developing severe liver disease with portal hypertension.

Correction of Aberrant Splicing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene by Antisense Oligonucleotides

The CFTR splicing mutation 3849 + 10 kb C → T creates a novel donor site 10 kilobases (kb) into intron 19 of the gene and is one of the more common splicing mutations that causes cystic fibrosis (CF). It has an elevated prevalence among patients with atypically mild disease and normal sweat electrolytes and is especially prominent in Ashkenazi Jews. This class of splicing mutations, reported in several genes, involves novel splice sites activated deep within introns while leaving wild-type splice elements intact. CFTR cDNA constructs that modeled the 3849 + 10 kb C → T mutation were expressed in 3T3 mouse fibroblasts and in CFT1 human tracheal and C127 mouse mammary epithelial cells. In all three cell types, aberrant splicing of CFTR pre-mRNA was comparable to that reported in vivo in CF patients. Treatment of the cells with 2′-O-methyl phosphorothioate oligoribonucleotides antisense toward the aberrant donor and acceptor splice sites or to the retained exon-like sequence, disfavored aberrant splicing and enhanced normal processing of CFTR pre-mRNA. This antisense-mediated correction of splicing was dose- and sequence-dependent and was accompanied by increased production of CFTR protein that was appropriately glycosylated. Antisense-mediated correction of splicing in a mutation-specific context represents a potential gene therapy modality with applicability to many inherited disorders.

Rapid characterization of the variable length polythymidine tract in the cystic fibrosis (CFTR) gene: Association of the 5T allele with selected CFTR mutations and its incidence in atypical sinopulmonary disease

The CFTR intron 8 variable length polythymidine tract modulates the cystic fibrosis (CF) phenotype associated with the mutation R117H. To explore whether other mutations reside on multiple intron 8 backgrounds with discernible impacts on phenotype, we developed an allele‐specific PCR assay to characterize this locus. Our approach types samples rapidly without the use of radioisotopes. Polythymidine alleles were identified for mutations either associated with a wide range of clinical phenotypes (R117H, R347P, G85E, D1152H, R334W, 2789+5 G>A, 3849+10kb C>T), and/or located at hypermutable CpG loci (R117H, 3849+10kb C>T, R553X, R334W, S945L and R75Q). R117H was detected in cis with each of three alleles (5T, 7T, 9T) at the intron 8 locus. The novel R117H‐9T association was detected in a 10‐month old African‐American male with borderline‐to‐mildly elevated sweat chloride values (˜50–66 mEq/L). All other mutations studied were associated with 7T except 3849+10kb C>T, which was detected on both 7T and 9T backgrounds, but not 5T. Three individuals with a ΔF508/3849+10kb C>T genotype were 9T,9T and had pancreatic sufficiency and normal sweat chloride values, whereas 15 others who carried 3849+10kb C>T on a 7T background had variable pancreatic function (sufficient, n = 12, insufficient, n = 3), and variable sweat chloride values (normal, n = 12, elevated, n = 3). Surprisingly, when not associated with known CFTR mutations, 5T was detected with elevated frequency among individuals with sinopulmonary disease of ill‐defined etiology, but with some characteristics of variant CF. In summary, the 5T allele was not found in cis with CF‐causing mutations besides R117H, but an elevated 5T allele frequency in variant CF patients suggests 5T may be associated with disease in some situations. Hum Mutat 10:108–115, 1997.

Identification of a splice site mutation (2789+5 G>A) associated with small amounts of normal CFTRmRNA and mild cystic fibrosis

A splicing mutation was identified at the +5 position of the splice donor site of exon 14b of CFTR in CF patients in a consanguineous family that is remarkable for unusually mild disease. Quantitative studies of nasal epithelial mRNA revealed that homozygotes for the spice site mutation produced approximately 4% of the normal amount of normally‐spliced CFTR. We propose that this small amount of normally spliced mRNA is associated with synthesis of some normal CFTR protein, and accounts for the mild phenotype. Further characterization of epithelial function and clinical phenotype in patients bearing this form of mutation, termed a type V mutation, will be useful in determining the level of CFTR associated with amelioration of lung disease. Hum Mutat 9:332–338, 1997.

Developing a sustainable process to provide quality control materials for genetic testing

Purpose: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community.Methods: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps.Results: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step.Conclusions: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

A CFTR mutation (D1152H) in a family with mild lung disease and normal sweat chlorides.

To the Editor: Over 1000 mutations in the cystic fibrosis transmembrane regulator (CFTR) gene have been reported to cause cystic fibrosis (CF); however, only a few are associated with mild disease (1, 2). We report a novel mutation (D1152H) in three siblings with unusually mild CF, surviving past age 64 with pancreatic exocrine sufficiency, normal sweat chloride (Cl) concentrations, and reduced CFTR-mediated Cl conductance across the nasal epithelium. Direct sequencing of a positive heteroduplex band (3) in exon 18 of the propositus revealed a heterozygous G-to-C base change at base number 3586 of the CFTR cDNA sequence. This nucleotide change results in an aspartic acid to histidine substitution at position 1152 of the mature protein (D1152H). The clinical characteristics of the three siblings with the clinical syndrome of CF are summarized in Table 1. The study protocol was approved by the Institutional Review Board and informed consent was obtained. Although these patients had a relatively benign long-term clinical course and repeatedly normal sweat Cl values, each had a history of pulmonary symptoms, predominantly cough and intermittent episodes of ‘bronchitis’ dating from early childhood or adolescence; and later in life, there was evidence of airflow obstruction present on spirometric testing. Each had mucoid Pseudomonas aeruginosa cultured from sputum samples, and chest radiographs were compatible with CF with predominant upper lung zone bronchiectasis. Pancreatic exocrine function was preserved in the two subjects who were tested. Each male sibling with CF was infertile. One of the siblings had an episode of acute pancreatitis at age 64 that was of undefined etiology, despite extensive evaluation. All three had recurrent rhinosinus disease, either nasal polyps and/or recurrent sinusitis. Finally, the propositus had an open lung biopsy at age 54 (in 1978) that was compatible with pathologic features of CF lung disease. A fourth sibling died at age 25 (in 1948) with complications of a pulmonary/intestinal syndrome compatible with CF, but no DNA was available for mutation analysis. Medical records of her clinical disease have been destroyed, and the only information available was verbal recollection from family members and her family doctor and the death certificate. The three siblings with the clinical syndrome of CF were compound heterozygotes, D1152H/G542X. None of the six siblings, who were carriers or normal at each allele, had any symptomatology that was suggestive of CF, and each had a normal chest radiograph. Results of sweat gland and nasal potential difference (PD) measurements are summarized in Table 2. The sweat ductal PD measurement for the propositus was normal, which is consistent with normal concentrations of Cl in sweat. In contrast, neither of the two siblings tested secreted sweat in response to intradermal injection of isoproterenol (4). The nasal bioelectric properties of the two patients studied were also abnormal, consistent with those seen in mild CF patients (5, 6). Mutations with residual function are associated with milder disease presentations and have been classified as type IV mutations (7, 8). Recent work by Vankeerberghen et al. has demonstrated reduced whole cell Cl currents when a D1152H-bearing CFTR cDNA is expressed in Xenopus oocytes, confirming that D1152H is a type IV mutation (9). Since our initial report to the Cystic Fibrosis Gene Analysis Consortium (10, 11), D1152H has been detected frequently in men with congenital bilateral absence of vas deferens (CBAVD) from three continents (12–16). One large study found D1152H to be the fourth most common CFTR mutation in men with CBAVD (17). These men are commonly ascertained through infertility clinics and initially reported to have no Clin Genet 2005: 68: 88–90 Copyright # Blackwell Munksgaard 2005 Printed in Singapore. All rights reserved CLINICAL GENETICS doi: 10.1111/j.1399-0004.2005.00459.x

Cystic fibrosis transmembrane-conductance regulator mutations among African Americans.

To the Editor:Cystic fibrosis (CF) is less common in African Americans than in Caucasians of northern European descent, with an estimated incidence of 1/15,300 (Hamosh et al., in pressxSee all ReferencesHamosh et al., in press), although the severity of the disease is comparable across racial lines. Macek et al. (1997)xIdentification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%. Macek, M Jr, Mackova, A, Hamosh, A, Hilman, BC, Selden, RF, Lucotte, G, Friedman, KJ et al. Am J Hum Genet. 1997; 60: 1122–1127PubMedSee all ReferencesMacek et al. (1997) recently reported in the Journal the identification of several CF transmembrane conductance regulator (CFTR) mutations of noteworthy prevalence in blacks. This information will help clinical laboratories to improve the sensitivity of CF mutation testing for African American patients.We have identified a CFTR mutation in exon 7 in two unrelated individuals of African American descent who were not included in the study by Macek et al. The mutation, ΔF311, results in the loss of a phenylalanine residue in the fifth transmembrane domain of the CFTR protein. One of our patients is a 4-year-old African American female who presented, at age 7 mo, with hypochloremic metabolic alkalosis and dehydration. She was subsequently found to have sweat chloride values on two occasions of 75 and 83 mEq/liter. Her lung disease is mild, with only slight peribronchial thickening on chest x-ray, and she had Staphylococcus aureus in her sputum at age 3 years. She is considered pancreatic sufficient, on the basis of qualitative fecal fat analysis. Without pancreatic-enzyme supplementation, she has maintained a normal growth pattern, with height and weight at the 50th percentile. Mutation testing determined her genotype to be ΔF508/ΔF311. The ΔF311 allele was first detected by the appearance of a distinct heteroduplex pattern when PCR product encompassing exon 7 was electrophoresed on 10% polyacrylamide. The mutation was identified as ΔF311 by dideoxy sequencing. No additional ΔF311 alleles have been found after a screening of a further 271 patient samples (~8.5% African American) at the University of North Carolina in Chapel Hill.The second patient was referred for genetic testing because of abnormal fetal ultrasound findings. The patient was a 25-year-old (G2 P0 SAB1) African American. An ultrasound performed at 17 wk gestation identified a fetus with a Dandy-Walker malformation and an echogenic bowel. Follow-up ultrasound at 18.2 wk gestation confirmed the CNS abnormalities and a grade II echogenic bowel. The patient was counseled with regard to the numerous causes of Dandy-Walker malformations, as well as with regard to the causes of echogenic bowel, including CF. The fetal karyotype was normal, but maternal and fetal CF testing identified a heteroduplex pattern identical to the ΔF311 heterozygote positive control (Mutation Detection Enhancement gel system; FMC BioProducts). Patient DNA mixed with equal amounts of ΔF311 control DNA showed the same heteroduplex pattern as did either the patient DNA sample or the ΔF311 heterozygote DNA sample alone, suggesting that these abnormal alleles were identical. DNA sequencing using the ABI 377 nucleic acid sequencer subsequently confirmed this sequence change to be the ΔF311 mutation in heterozygous form.Maternal cell contamination was ruled out by MCT-118 genotyping. The father of the fetus was not available for testing, and no other CF mutation or abnormal heteroduplex pattern was detected in the fetal sample. Because of the presence of the Dandy-Walker malformation, and prior to the CF results being provided to the patient, the patient elected to terminate the pregnancy. An autopsy was not performed, and fetal tissue was not available for confirmation of the amniocentesis results.ΔF311 was first reported in a 2-year-old boy with a positive albumin-meconium test at birth and with repeatedly elevated sweat tests by age 4 mo (Meitinger et al. 1993xIn frame deletion (ΔF311) within a short trinucleotide repeat of the first transmembrane region of the cystic fibrosis gene. Meitinger, T, Golla, A, Dorner, C, Deufel, A, Aulehla-Scholz, C, Bohm, I, Reinhardt, D et al. Hum Mol Genet. 1993; 2: 2173–2174Crossref | PubMed | Scopus (1)See all ReferencesMeitinger et al. 1993). His other mutation is ΔF508. Prophylactic treatment with both pancreatic enzymes and mucolytic agents to deter lung disease has prevented the onset of either pulmonary or pancreatic symptoms in his first 6 years. The authors of that study did not identify any other individuals with this mutation, after screening an additional 205 CF chromosomes by SSCP (T. Meitinger, personal communication). This patient is of Bavarian Caucasian descent, and his pancreatic disease is distinct from that of the patient seen at the University of North Carolina in Chapel Hill (UNC), obscuring any correlation between ΔF311 and a particular phenotype. Apparent clinical dissimilarities among these three patients might be attributable to undefined aspects of either the genetic background or the environment, but low numbers prevent the drawing of conclusions along racial or other lines. Interestingly, the two individuals whom we describe, as well as the index case, each harbor a distinct ΔF311-associated haplotype (1 1 2, 1 2 2, and 2 1 2) defined by the flanking markers, XV2c-KM19-J3.11, suggesting that this mutation has occurred more than once. The multiple origins of ΔF311 suggest that it might be found on additional chromosomes, but this would not be limited to African American patients.ΔF311 has thus been identified in two individuals of African American ancestry. In this racial group, this mutation appears to be more common than any CFTR mutation except DF508, compared with other alleles also identified in Caucasians. Among the 23 African American CF patients genotyped at UNC, the inclusion of ΔF311 increased total mutation-detection rates by ~2%. On the basis of the criteria established by Macek et al., we feel that molecular diagnostic laboratories should consider the inclusion of ΔF311 in the development of CF mutation-testing panels tailored to African Americans.

Cystic fibrosis carrier screening in a North American population

Purpose:The aim of this study was to compare the mutation frequency distribution for a 32-mutation panel and a 69-mutation panel used for cystic fibrosis carrier screening. Further aims of the study were to examine the race-specific detection rates provided by both panels and to assess the performance of extended panels in large-scale, population-based cystic fibrosis carrier screening. Although genetic screening for the most common CFTR mutations allows detection of nearly 90% of cystic fibrosis carriers, the large number of other mutations, and their distribution within different ethnic groups, limits the utility of general population screening.Methods:Patients referred for cystic fibrosis screening from January 2005 through December 2010 were tested using either a 32-mutation panel (n = 1,601,308 individuals) or a 69-mutation panel (n = 109,830).Results:The carrier frequencies observed for the 69-mutation panel study population (1/36) and Caucasian (1/27) and African-American individuals (1/79) agree well with published cystic fibrosis carrier frequencies; however, a higher carrier frequency was observed for Hispanic-American individuals (1/48) using the 69-mutation panel as compared with the 32-mutation panel (1/69). The 69-mutation panel detected ~20% more mutations than the 32-mutation panel for both African-American and Hispanic-American individuals.Conclusion:Expanded panels using race-specific variants can improve cystic fibrosis carrier detection rates within specific populations. However, it is important that the pathogenicity and the relative frequency of these variants are confirmed.Genet Med 16 7, 539–546.