Cystic fibrosis gene mutations and pancreatitis risk: relation to epithelial ion transport and trypsin inhibitor gene mutations.

BACKGROUND & AIMS: Nonalcoholic chronic pancreatitis is usually idiopathic and often associated with cystic fibrosis gene (CFTR) mutations. It is unknown whether pancreatitis risk correlates with having 1 or 2 CFTR mutations, abnormal epithelial ion transport, or mutations of other genes. METHODS: We tested 39 patients with idiopathic chronic pancreatitis (mean age at diagnosis, 33 years) for common mutations of CFTR and of genes encoding a trypsin inhibitor (PSTI) and trypsinogen (PRSS1). To exclude hereditary pancreatitis, we initially relied on family history and subsequently tested for PRSS1 mutations. Twenty subjects were tested for rare CFTR mutations (DNA sequencing) and 11 were tested for extrapancreatic CFTR function (clinical and physiologic evaluation). RESULTS: Mutations were identified in 24 of 39 subjects. Nine patients had cystic fibrosis-causing mutations, 8 of whom also had mild-variable mutations. Eight others had only mild-variable mutations. Nine subjects had the N34S PSTI mutation and 1 had hereditary pancreatitis (R122H, PRSS1). Pancreatitis risk was increased approximately 40-fold by having 2 CFTR mutations (P < 0.0001), 20-fold by having N34S (P < 0.0001), and 900-fold by having both (P < 0.0001). Subjects with 2 CFTR mutations had abnormal nasal epithelial ion transport and clinical findings suggesting residual CFTR function between that in cystic fibrosis and in carriers. By contrast, subjects with only PSTI mutations had normal CFTR function. CONCLUSIONS: CFTR-related pancreatitis risk correlates with having 2 CFTR mutations and reduced extrapancreatic CFTR function. The N34S PSTI mutation increased risk separately. Testing for pancreatitis-associated CFTR and PSTI genotypes may be useful in nonalcoholic pancreatitis.

In-frame deletions of BRCA1 may define critical functional domains.

The identification of genomic rearrangements involving more than 0.5 kb of the BRCA1 gene has confirmed a more complex mutation spectrum than was initially appreciated. Genomic rearrangements in BRCA1 represent 15% of all mutations in a group of French and American breast and ovarian cancer families and 36% of all mutations in a group of Dutch families. The rearrangements described to date range in size from 510 bp to 23.8 kb, are found throughout the gene, and are most frequently attributable to homologous recombination. We describe the identification of rearrangements in two breast and ovarian cancer families that involve 3.4 and 11.5 kb of the BRCA1 gene and span multiple exons but maintain the reading frame. Both gene rearrangements appear to result from Alu-mediated homologous recombination and have been detected by using a combination of protein truncation analysis and Southern blot analysis. These rearrangements result in the loss of amino acids that lie at the carboxy-terminus of the protein and that have previously been shown to have functional significance. Because these rearrangements result in the deletion of exons but maintain the reading frame, they may provide insights into specific regions and amino acids that have functional significance for the BRCA1 protein.

Lung disease associated with the IVS8 5T allele of the CFTR gene.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. The 5T allele in intron 8 (IVS8) causes abnormal splicing in the CFTR gene, and is associated with lung disease when it occurs in cis with a missense mutation in the CFTR gene, R117H. However, the 5T variant alone has not been reported to cause lung disease. We describe two adult female patients with CF-like lung disease associated with the 5T allele. One patient's genotype is 5T-TG12-M470V/5T-TG12-M470V, and the other is DeltaF508/5T-TG12-M470V; full sequencing of the CFTR gene revealed no other mutation on the same allele as the 5T variant. The levels of full-length CFTR mRNA in respiratory epithelia were very low in these patients (11 and 6%, respectively, of total CFTR mRNA expression). Both patients had defective CFTR-mediated chloride conductance in the sweat ductal and/or acinar epithelia (sweat chloride, mmol/L, mean +/- SEM: 40.0 +/- 5.0 [n = 8 samples] and 80. 0 +/- 3.5 [n = 6 samples]) and airway epithelia (mV, mean +/- SEM CFTR-mediated Cl(-) conductance of 1.2 +/- 2.2 [n = 5 studies] and -6.75 +/- 8.1 [n = 4 studies]). These data suggest that the 5T polythymidine tract sequence on specific haplotype backgrounds (TG12 and M470V) may cause a low level of full-length functional CFTR protein and CF-like lung disease.

An Alu-mediated 7.1 kb deletion of BRCA1 exons 8 and 9 in breast and ovarian cancer families that results in alternative splicing of exon 10.

Constitutive large deletions and duplications of BRCA1 resulting from Alu-mediated recombination account for a significant proportion of disease-causing mutations in breast and/or ovarian cancer families. Using Southern blot analysis and a protein truncation test (PTT), we have identified a 7.1 kb germline deletion in two families with breast and ovarian cancer. This deletion, which includes exons 8 and 9 and leads to a frameshift at the mRNA level, appears to result from homologous recombination between closely related Alu repeats, one in intron 7 and one in intron 9. In addition to the transcript without exons 8 and 9, analysis of RNA by protein truncation test from individuals with the deletion also identified the presence of alternative splicing of exon 10 from the mutant allele, which results in a transcript that lacks exons 8, 9, and 10. Of interest is that the two American families who carry this deletion are of northern European ancestry and share a common haplotype, suggesting that this deletion may represent a founder mutation. Genes Chromosomes Cancer 28:300-307, 2000.

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.

HFE genotyping using multiplex allele-specific polymerase chain reaction and capillary electrophoresis.

CONTEXT: Hereditary hemochromatosis is recognized as one of the most common autosomal recessive disorders, with a prevalence of 1 in 200 to 400 in the white population. Early detection and treatment are completely effective in preventing pathology. It is anticipated that testing for hereditary hemochromatosis will increase, as will the need for a technology that can handle the demand. OBJECTIVE: To describe a high-throughput, single-tube, allele-specific multiplex polymerase chain reaction assay for identifying the 2 mutations in the HFE gene associated with hereditary hemochromatosis. DESIGN: Fluorescence-labeled polymerase chain reaction products from a multiplex polymerase chain reaction are analyzed by automated capillary electrophoresis. DATA ANALYSIS: The assay was validated by analysis of 25 blinded samples, and results were concordant with an established laboratory assay. CONCLUSION: The assay described offers a significant improvement over manual laboratory assays in throughput, reduced technologist time, and cost.

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.

Is the hemochromatosis gene a modifier locus for cystic fibrosis?

The variable clinical manifestations of cystic fibrosis (CF) suggest the influence of modifier genes. For example, meconium ileus is present in approximately 10-15% of neonates with cystic fibrosis; however, the genetic and, or environmental factors that determine whether an individual will develop this complication have not been determined. We propose the HFE gene as a candidate modifier locus for CF based on (1) the suggestion of an association between the HLA loci and CF phenotypes; (2) the location of the HFE gene near the HLA loci and; (3) the similarity between the gastrointestinal manifestations of hereditary hemochromatosis and CF. We have determined the frequency of the C282Y and H63D mutations in a group of 89 CF patients who were homozygous for delta F508 and for whom meconium ileus status was known. The carrier frequency of C282Y among the CF patients with meconium ileus was significantly different from that of our unaffected control group (19.4% versus 7.7%). However, the difference between the meconium ileus and the nonmeconium ileus groups was not significant (19.4% versus 10.3%). There was no difference in the frequency of the H63D among the three groups that were studied. These data are suggestive of a relationship between the development of meconium ileus or other gastrointestinal diseases in CF and the HFE gene. Further study of a larger group of patients is warranted.

Identification of a splice site mutation (2789 +5 G > A) associated with small amounts of normal CFTR mRNA 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.

Direct detection of mutations in the breast and ovarian cancer susceptibility gene BRCA1 by PCR-mediated site-directed mutagenesis.

The tumor suppressor genes BRCA1 and BRCA2, which confer increased susceptibility to breast and (or) ovarian cancer, were recently identified. Mutation analysis of BRCA1 has demonstrated significant allelic heterogeneity; however, some distinct mutations have been detected in unrelated individuals. The most notable is the 185delAG mutation, which occurs at an estimated frequency of approximately 1% in individuals of Ashkenazi Jewish descent [1]. Although consensus has not been reached regarding clinical testing for mutations in BRCA1, a tiered strategy may be appropriate, in which direct testing for the more common mutations is one component. Specific alleles can be detected by using PCR-mediated site-directed mutagenesis (PSM), which alters the PCR products derived from either the wild-type or mutant allele to create or destroy a restriction endonuclease recognition site. Recognition sites are introduced by a base substitution in one of the primers. The alleles are then resolved by electrophoresis of the digested PCR products. We have applied this technique to the detection of four BRCA1 mutations: 185delAG, 5382insC, E1250X, and R1443X. Another mutation, 1294de140, can be resolved from the wild-type allele by high-resolution gel electrophoresis alone. The PSM technique is sensitive, does not require radioactivity, and is specific for individual mutations.

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 or 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, 3845 + 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 African-American male with borderline-to-mildly elevated sweat chloride values (approximately 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 delta F508/3849 + 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.

Screening Young syndrome patients for CFTR mutations.

Young syndrome is characterized by obstructive azoospermia associated with chronic sinobronchial disease of an infectious nature, but normal sweat-gland and pancreatic function as well as normal nasal potential differences. Congenital bilateral absence of the vas deferens (CBAVD) in some patients arises from mutations within the cystic fibrosis (CF) transmembrane regulator (CFTR) gene. Because of some similarities between Young syndrome, CF, and CBAVD, we evaluated 13 patients with Young syndrome, including screening for more than 30 different mutations within the CFTR gene. The mean age of the patients was 43 yr (range, 32 to 50 yr), and all were of northern European extraction. The sweat chloride concentration was normal in all patients (mean = 29 mEq/L; range, 8 to 43 mEq/L). Most had intermittent bronchial and sinus infections, but none was chronically colonized with Staphylococcus aureus or Pseudomonas aeruginosa. The FEV1 was normal or only mildly reduced in most patients (mean = 74%; range, 48 to 100% predicted). Of 26 Young syndrome chromosomes, we identified one with the recognized CF mutation delta F508. The incidence of CFTR mutations (1 in 26) did not differ significantly from the expected carrier frequency in this population. In summary, it is unlikely that the typical Young syndrome patient has a clinical disease associated with CFTR mutation on both alleles.

Distribution of 13 truncating mutations in the neurofibromatosis 1 gene.

Neurofibromatosis 1 (NF1) is a common genetic disorder characterized by abnormalities of tissues derived from the neural crest. To define germ-line mutations in the NF1 gene, we studied 20 patients with familial or sporadic cases of NF1 diagnosed clinically and one patient with only café-au-lait spots and no other diagnostic criteria. A protein truncation assay identified abnormal polypeptides synthesized in vitro from five RT-PCR products that represented the entire NF1 coding region. Truncated polypeptides were observed in 14 individuals. The mutations responsible for the generation of abnormal polypeptides were characterized by DNA sequencing. Thirteen previously unpublished mutations were characterized in the 14 individuals. The mutation 2027insC was observed in two unrelated individuals; the other 12 mutations were unique. The sequence changes included seven nonsense and four frameshift mutations that created premature translation termination signals, and two large in-frame deletions that led to the synthesis of truncated polypeptides. One of the mutations was found in the child with a single clinical diagnostic criterion, providing her with a presumptive diagnosis of NF1. Our results confirm that truncating mutations are frequent in both familial and sporadic NF1 cases. The identification of mutations in 14 of 21 individuals studied (67%) suggests that the use of protein truncation assays will rapidly accelerate the rate of identification of NF1 mutations. Because we scanned the entire NF1 coding region in each individual, the distribution of NF1 truncating mutations was discerned for the first time. The mutations were relatively evenly distributed throughout the coding region with no evidence for clustering.

A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations.

BACKGROUND: Many patients with chronic pulmonary disease similar to that seen in cystic fibrosis have normal (or nondiagnostic) sweat chloride values. It has been difficult to make the diagnosis of cystic fibrosis in these patients because no associated mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been identified. METHODS: We evaluated 23 patients with pulmonary disease characteristic of cystic fibrosis but with sweat chloride concentrations in the normal range. Mutations in the CFTR gene were sought by direct sequencing of polymerase chain reaction-amplified nasal epithelial messenger RNA and by testing the functioning of affected epithelium. RESULTS: A cytidine phosphate guanosine dinucleotide C-to-T point mutation in intron 19 of the CFTR gene, termed 3849 + 10 kb C to T, was identified in 13 patients from eight unrelated families. This mutation was found in patients from three different ethnic groups with three different extended haplotypes. The mutation leads to the creation of a partially active splice site in intron 19 and to the insertion into most CFTR transcripts of a new 84-base-pair "exon," containing an in-frame stop codon, between exons 19 and 20. Normally spliced transcripts were also detected at a level approximately 8 percent of that found in normal subjects. This mutation is associated with abnormal nasal epithelial and sweat acinar epithelial function. CONCLUSIONS: We have identified a point mutation in intron 19 of CFTR and abnormal epithelial function in patients who have cystic fibrosis-like lung disease but normal sweat chloride values. The identification of this mutation indicates that this syndrome is a form of cystic fibrosis. Screening for the mutation should prove diagnostically useful in this population of patients.

Association of pancreatic adenocarcinoma, mild lung disease, and delta F508 mutation in a cystic fibrosis patient.

A case of adenocarcinoma of the pancreas and mild lung disease in a 39-year-old man homozygous for the delta F508 cystic fibrosis mutation is presented. Cystic fibrosis is the most common lethal genetic disease in Caucasians, and is most commonly associated with severe obstructive lung disease. To our knowledge, this is only the fifth case of adenocarcinoma of the pancreas in a CF patient to be reported and the first case for which molecular data are available. The rare incidence of this type of malignancy in the general population suggests a possible association of CF with this malignant disease.

In situ amplification: detection of target sequences in whole tissues.

Using the polymerase chain reaction (PCR) specific gene target sequences can be routinely amplified from isolated nucleic acids. However, this does not allow the investigator the option of correlating the presence of amplified product with cellular localization. When PCR is performed in situ and coupled with either a direct or indirect method of detecting amplified product, a powerful investigative tool exists. Owing to variability in results, the features of in situ PCR which are most likely to contribute to these effects are described. Also described is an amplification chamber for localized in situ amplification (LISA) which utilizes tissue culture cloning rings and allows for the amplification of gene target sequences in specific regions of paraffin-embedded tissue sections.