Genotype–phenotype correlation in cystic fibrosis: The role of modifier genes

More than 1,000 mutations have been identified in the cystic fibrosis (CF) transmembrane regulator (CFTR) disease gene. The impact of these mutations on the protein and the wide spectrum of CF phenotypes prompted a series of Genotype–Phenotype correlation studies. The CFTR genotype is invariably correlated with pancreatic status—in about 85% of cases with pancreatic insufficiency and in about 15% of cases with pancreatic sufficiency. The correlations between the CFTR genotype and pulmonary, liver, and gastrointestinal expression are debatable. The heterogeneous phenotype in CF patients bearing the same genotype or homozygotes for nonsense mutations implicated environmental and/or genetic factors in the disease. However, the discordant phenotype observed in CF siblings argued against a major role of environmental factors and suggested that genes other than CFTR modulate the CF phenotype. A locus that modulates gastrointestinal expression was identified in mice and subsequently in humans. By analyzing nine CF patients discordant for meconium ileus we were able to show that this locus had a dominant effect. Moreover, in a collaborative study we found a higher rate of polymorphisms in β‐defensin genes 1 and 2 in CF patients and in controls. In another multicenter study mutations in α‐1 antitrypsin (A1AT) and mannose binding lectin genes were found to be independent risk factors for liver disease in CF patients. The body of evidence available suggests that the variegated CF phenotype results from complex interactions between numerous gene products. © 2002 Wiley‐Liss, Inc.     

UMD‐CFTR: A database dedicated to CF and CFTR‐related disorders

With the increasing knowledge of cystic fibrosis (CF) and CFTR‐related diseases (CFTR‐RD), the number of sequence variations in the CFTR gene is constantly raising. CF and particularly CFTR‐RD provide a particular challenge because of many unclassified variants and identical genotypes associated with different phenotypes. Using the Universal Mutation Database (UMD^®) software we have constructed UMD‐CFTR (freely available at the URL: http://www.umd.be/CFTR/ ), the first comprehensive relational CFTR database that allows an in‐depth analysis and annotation of all variations identified in individuals whose CFTR genes have been analyzed extensively. The system has been tested on the molecular data from 757 patients (540 CF and 217 CBAVD) including disease‐causing, unclassified, and nonpathogenic alterations (301 different sequence variations) representing 3,973 entries. Tools are provided to assess the pathogenicity of mutations. UMD‐CFTR also offers a number of query tools and graphical views providing instant access to the list of mutations, their frequencies, positions and predicted consequences, or correlations between genotypes, haplotypes, and phenotypes. UMD‐CFTR offers a way to compile not only disease‐causing genotypes but also haplotypes. It will help the CFTR scientific and medical communities to improve sequence variation interpretation, evaluate the putative influence of haplotypes on mutations, and correlate molecular data with phenotypes. Hum Mutat 31:1011–1019, 2010. © 2010 Wiley‐Liss, Inc.     

Complex cystic fibrosis allele R334W-R1158X results in reduced levels of correctly processed mRNA in a pancreatic sufficient patient

CFTR alleles containing two mutations have been very rarely found in cystic fibrosis (CF) patients. They provide an opportunity to study the effect of two in cis-interacting gene defects on gene expression. Here, we describe a three-generation CF family with a complex CFTR allele that has not been previously described, containing the missense mutation R334W in exon 7 and the nonsense mutation R1158X in exon 19. Lymphocyte RNA analysis showed that (1) the mRNA corresponding to the complex allele is present although at markedly reduced levels; and (2) the nonsense mutation does not lead to detectable skipping of exon 19. The clinical picture of the patients with the genotype R334W-R1158X/ΔF508 is characterized by pancreatic sufficiency and an atypical course of the disease. © 1996 Wiley-Liss, Inc.     

Carrier frequency of CFTR variants in the non-Caucasian populations by genome aggregation database (gnomAD)-based analysis

The complexity in the molecular diagnosis of Cystic Fibrosis (CF) also depends on the variable prevalence/incidence of the disease associated with the wide CFTR allelic heterogeneity among different populations. In fact, CF incidence in Asian and African countries is underestimated and the few patients reported so far have rare or unique CFTR pathogenic variants. To obtain insights into CF variants profile and frequency, we used the large population sequencing data in the Genome Aggregation Database (gnomAD). We selected 207 CF-causing/varying clinical consequence variants from CFTR2 database and additional 15 variants submitted to the ClinVar database. Only 14 of these variants were found in the East-Asian population, while for South-Asian and African populations we identified 43 and 52 variants, respectively, confirming the peculiarity of the CFTR allelic spectrum with only few population-specific variants. These data could be used to optimize CFTR carrier screening in non-Caucasian subjects, choosing between the full gene sequencing and cost and time-effective targeted panels.     

The L441P Mutation of Cystic Fibrosis Transmembrane conductance Regulator and its Molecular Pathogenic Mechanisms in a Korean Patient with Cystic Fibrosis

Cystic fibrosis (CF) is an autosomal recessive disorder usually found in populations of white Caucasian descent. CF is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. A 5-yr-old Korean girl was admitted complaining of coughing and greenish sputum. Chest radiographs and computed tomographic (CT) scan revealed diffuse bronchiectasis in both lungs. The patient had chronic diarrhea and poor weight gain, and the abdominal pancreaticobiliary CT scan revealed atrophy of the pancreas. Finally, CF was confirmed by the repeated analysis of the quantitative pilocarpine iontophoresis test. The chloride concentration of sweat samples taken from both forearms of the pateint was an average of 88.7 mM/L (normal value <40 mM/L). After a comprehensive search for mutations in the CFTR gene, the patient was found to carry the non-synonymous L441P mutation in one allele. Molecular physiologic analysis of the L441P mutation of CFTR revealed that the L441P mutation completely abolished the CFTR Cl^- channel activity by disrupting proper protein folding and membrane trafficking of CFTR protein. These results confirmed the pathogenicity of the L441P mutation of CFTR circulating in the Korean population. The possibility of CF should be suspected in patients with chronic bronchiectasis, although the frequency of CF is relatively rare in East Asia.     

Analysis of DEFB1 regulatory SNPs in cystic fibrosis patients from North-Eastern Italy

Cystic fibrosis (CF) transmembrane regulator protein (CFTR) gene is undoubtedly the main genetic factor involved in the modulation of CF phenotype. However, other factors such as human defensins and the genes encoding for these antimicrobial peptides have been hypothesized as possible modifiers influencing airways infection in CF patients, but their role in the pathogenesis of lung disease is still debated. Since DEFB1 gene encoding for human beta-defensin 1 displays features such as antimicrobial or chemotactic activity playing a role in inflammation, it has been considered as a possible candidate CF modifier gene. We analysed three single nucleotide polymorphisms (SNPs) in the 5′-untranslated region of the DEFB1 gene (namely g-52G>A, g-44C>G and g-20G>A) in a group of 62 CF patients from North Eastern Italy, and in 130 healthy controls, with the aim of verifying the possible association of these functional SNPs with the pulmonary phenotype of CF patients. DEFB1 SNPs have been genotyped by using Taqman allele-specific fluorescent probes and a real-time PCR platform. No significant differences were found for allele, genotype and haplotype frequencies of DEFB1 g-52G>A, g-44C>G and g-20G>A SNPs in CF patients stratified for Pseudomonas aeruginosa infection, as well as in patients with a severe and mild clinical phenotype or in patients stratified for CFTR genotypes. DEFB1 allele, genotype and haplotype frequencies of CF patients globally considered were similar to those of healthy controls. Our findings are discordant with respect to another recent study performed on CF patients coming from Southern Italy, probably due to different ethnicity of the patients.     

Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis

The large number of possible disease-causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for mutations in the CFTR gene can be both substantially facilitated and made virtually complete, by two-dimensional DNA electrophoretic separation of polymerase chain reaction (PCR) amplified exons on the basis of size and basepair sequence in denaturing gradient gels. Under a single optimized set of conditions we were able to obtain a pattern of spots representing all 27 exons of the CFTR gene and to readily detect 17 out of 17 identified sequence variations in 9 different exons in DNA from 11 CF patients and carriers. Our results demonstrate the potential of 2-dimensional DNA electrophoresis for comprehensive mutation analysis of the CFTR gene. The approach serves as a model for comprehensive diagnosis of the many other large disease genes for which a variety of mutations have also which been reported. © 1996 Wiley-Liss, Inc.     

Clinical and molecular characterization of the potential CF disease modifier syntaxin 1A

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator gene (CFTR). Disease severity in CF varies greatly, and sibling studies strongly indicate that genes other than CFTR modify disease outcome. Syntaxin 1A (STX1A) has been reported as a negative regulator of CFTR and other ion channels. We hypothesized that STX1A variants act as a CF modifier by influencing the remaining function of mutated CFTR. We identified STX1A variants by genomic resequencing patients from the Bernese CF Patient Data Registry and applied linear mixed model analysis to establish genotype–phenotype correlations, revealing STX1A rs4363087 (c.467−38A>G) to significantly influence lung function. The same STX1A risk allele was recognized in the European CF Twin and Sibling Study (P=0.0027), demonstrating that the genotype–phenotype association of STX1A to CF disease severity is robust enough to allow replication in two independent CF populations. rs4363087 is in linkage disequilibrium to the exonic variant rs2228607 (c.204C>T). Considering that neither rs4363087 nor rs2228607 changes the amino-acid sequence of STX1A, we investigated their effects on mRNA level. We show that rs2228607 reinforces aberrant splicing of STX1A mRNA, leading to nonsense-mediated mRNA decay. In conclusion, we demonstrate the clinical relevance of STX1A variants in CF, and evidence the functional relevance of STX1A variant rs2228607 at molecular level. Our findings show that genes interacting with CFTR can modify CF disease progression.     

Targeted mutation screening panels expose systematic population bias in detection of cystic fibrosis risk

PurposeCarrier screening for mutations contributing to cystic fibrosis (CF) is typically accomplished with panels composed of variants that are clinically validated primarily in patients of European descent. This approach has created a static genetic and phenotypic profile for CF. An opportunity now exists to reevaluate the disease profile of CFTR at a global population level.MethodsCFTR allele and genotype frequencies were obtained from a nonpatient cohort with more than 60,000 unrelated personal genomes collected by the Exome Aggregation Consortium. Likely disease-contributing mutations were identified with the use of public database annotations and computational tools.ResultsWe identified 131 previously described and likely pathogenic variants and another 210 untested variants with a high probability of causing protein damage. None of the current genetic screening panels or existing CFTR mutation databases covered a majority of deleterious variants in any geographical population outside of Europe.ConclusionsBoth clinical annotation and mutation coverage by commercially available targeted screening panels for CF are strongly biased toward detection of reproductive risk in persons of European descent. South and East Asian populations are severely underrepresented, in part because of a definition of disease that preferences the phenotype associated with European-typical CFTR alleles.     

mRNA‐based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity

Even with advent of next generation sequencing complete sequencing of large disease‐associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease‐causing mutations. Moreover, with the advent of mutation‐based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost‐effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA‐based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis.     

Gastrointestinal Pathology in Juvenile and Adult CFTR-Knockout Ferrets

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.Cystic fibrosis (CF) is the most common life-threatening, autosomal recessive, genetic disorder among Caucasians, occurring in approximately 1 in 3500 births. Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that disrupt function of this chloride channel cause abnormalities in electrolyte and fluid movement across many epithelia of the body, leading to viscous, poorly hydrated, secretions.¹ Although chronic bacterial infections in the lung are the most significant cause of mortality in CF, pathology in multiple other organs contributes to the progression of disease and overall health of CF patients. These organs include the intestine, pancreas, liver, and gallbladder for which clinical and/or histological disease is seen in CF patients at frequencies of 10% to 90%.^1–4 In the current study, we evaluated gastrointestinal disease in juvenile and adult CFTR-knockout ferrets.Mouse models of CF have been critical to our understanding of CFTR function in several organs, however, CF mice fail to develop spontaneous disease in the lung and pancreas, and have relatively minor disease of the liver and gallbladder.^5,6 The recent creation of new CF pig and ferret models has provided the field with new tools to dissect CF disease pathophysiology and the factors that influence disease severity in CF patients.^7,8 Comparisons between CF mouse, pig, and ferret models have clearly demonstrated that species-specific differences in organ physiology and CFTR biology influence the extent of pathology in major organs affected in CF.^9,10 For example, all newborn CF models have intestinal pathology that manifests as meconium ileus at birth in the case of CF pigs (100% of animals)¹¹ and CF ferrets (75% of animals),¹² or as intestinal obstruction at weaning in the case of CF mice.⁵ By contrast, pancreatic phenotypes at birth are highly variable between species with disease being most severe in CF pigs,¹¹ less severe in CF ferrets,^12,13 and absent in CF mice.⁵ Similarly, CF pigs demonstrate histopathology in the gallbladder and liver at birth,¹¹ whereas disease in these organs is relative minor in newborn CF ferrets^12,13 and CF adult mice.^5,6 Elucidating the differences in the severity of CF gastrointestinal disease at birth and in disease progression between the various species may aid in dissecting genetic and environmental factors that influence gastrointestinal disease severity in CF patients.Here, we report the phenotype of gastrointestinal organs (pancreas, liver, gallbladder, stomach, and intestine) in older CF animals reared on antibiotics until 6 months of age, or the time at which they were euthanized due to severity of disease. We have also evaluated the extent of bacterial overgrowth in the CF intestine and the types of bacterial flora found in the intestine of non-CF and CF animals. Our findings demonstrate that juvenile and adult CF ferrets naturally acquire gastrointestinal disease at frequencies similar to that observed in CF patients. Of great interest, a small subset of CF animals was pancreatic sufficient from birth, implicating modifier genes that can compensate for the loss of CFTR in the exocrine pancreas. These studies suggest that the CF ferret model could be useful for testing therapies aimed at gastrointestinal organs and dissecting how genetic variation influences disease in CF.     

Cystic fibrosis–related diabetes onset can be predicted using biomarkers measured at birth

PurposeCystic fibrosis (CF), caused by pathogenic variants in the CF transmembrane conductance regulator (CFTR), affects multiple organs including the exocrine pancreas, which is a causal contributor to cystic fibrosis–related diabetes (CFRD). Untreated CFRD causes increased CF-related mortality whereas early detection can improve outcomes.MethodsUsing genetic and easily accessible clinical measures available at birth, we constructed a CFRD prediction model using the Canadian CF Gene Modifier Study (CGS; n = 1,958) and validated it in the French CF Gene Modifier Study (FGMS; n = 1,003). We investigated genetic variants shown to associate with CF disease severity across multiple organs in genome-wide association studies.ResultsThe strongest predictors included sex, CFTR severity score, and several genetic variants including one annotated to PRSS1, which encodes cationic trypsinogen. The final model defined in the CGS shows excellent agreement when validated on the FGMS, and the risk classifier shows slightly better performance at predicting CFRD risk later in life in both studies.ConclusionWe demonstrated clinical utility by comparing CFRD prevalence rates between the top 10% of individuals with the highest risk and the bottom 10% with the lowest risk. A web-based application was developed to provide practitioners with patient-specific CFRD risk to guide CFRD monitoring and treatment.     

Uniparental inheritance of microsatellite alleles of the cystic fibrosis gene (CFTR): identification of a 50 kilobase deletion

More than 250 mutations have been detected in the cystic fibrosis(CF) transmembrane regulator (CFTR) gene, most of which aresingle point mutations or small deletions or insertions of afew nucleotides. Here we report the first large deletion identifiedin the CFTR gene, which involves 50 kb in two stretches of DNA:one of 10 kb from exon 4 to exon 7, and another of 40 kb, spanningexons 11 to 18. The deletion has been detected via uniparentalinheritance of CFTR microsatellite alleles (IVS17BTA and IVS17BCA)in 3 independent CF families. Clinical status of the 3 CF patients,of which two have the     

Complete ascertainment of intragenic copy number mutations (CNMs) in the CFTR gene and its implications for CNM formation at other autosomal loci

Over the last 20 years since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, more than 1,600 different putatively pathological CFTR mutations have been identified. Until now, however, copy number mutations (CNMs) involving the CFTR gene have not been methodically analyzed, resulting almost certainly in the underascertainment of CFTR gene duplications compared with deletions. Here, high‐resolution array comparative genomic hybridization (averaging one interrogating probe every 95 bp) was used to analyze the entire length of the CFTR gene (189 kb) in 233 cystic fibrosis chromosomes lacking conventional mutations. We succeeded in identifying five duplication CNMs that would otherwise have been refractory to analysis. Based upon findings from this and other studies, we propose that deletion and duplication CNMs in the human autosomal genome are likely to be generated in the proportion of approximately 2–3:1. We further postulate that intragenic gene duplication CNMs in other disease loci may have been routinely underascertained. Finally, our analysis of ±20 bp flanking each of the 40 CFTR breakpoints characterized at the DNA sequence level provide support for the emerging concept that non‐B DNA conformations in combination with specific sequence motifs predispose to both recurring and nonrecurring genomic rearrangements. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Regulation of epithelial ion channels by the cystic fibrosis transmembrane conductance regulator

In most epithelia ion transport is tightly regulated. One major primary target of such regulation is the modulation of ion channels. The present brief review focuses on one specific example of ion channel regulation by the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a cAMP-regulated Cl^- channel. Its defect leads to the variable clinical pictures of cystic fibrosis (CF), which today is understood as a primary defect of epithelial Cl^- channels in a variety of tissues such as the respiratory tract, intestine, pancreas, skin, epididymis, fallopian tube, and others. Most recent findings suggest that CFTR also acts as a channel regulator. Three examples are discussed by which CFTR regulates other Cl^- channels, K⁺ channels, and epithelial Na⁺ channels. From this perspective it is evident that CFTR may play a major role in the integration of cellular function.Abbreviations CF Cystic fibrosis - CFTR Cystic fibrosis transmembrane conductance regulator - IBMX Isobutylmethylxanthine - ICOR Intermediate conductance outwardly rectifying - MDR Multidrug resistance protein Supported by DFG: Gr 480/11     

Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study

PurposeMany regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology.MethodsAn NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen–positive samples with one CFTR mutation.ResultsThe NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified.ConclusionThe NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.