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• W2108839621 abstract "Cystic fibrosis (CF), the most common autosomal recessive disease in whites, is caused by mutations in the CF transmembrane conductance regulator (CFTR). So far, >1900 mutations have been described, most of which are nonsense, missense, and frameshift, and can lead to severe phenotypes, reducing the level of function of the CFTR protein. Synonymous variations are usually considered silent without pathogenic effects. However, synonymous mutations exhibiting exon skipping as a consequence of aberrant splicing of pre-mRNA differ. Herein, we describe the effect of the aberrant splicing of the c.273G>C (G91G) synonymous variation found in a 9-year-old white (ΔF508) patient affected by CF and pancreatitis associated with a variant in chymotrypsin C (CTRC). Magnetic resonance imaging showed an atrophic pancreatic gland with substitution of the pancreatic parenchyma with three cysts. Genetic examination revealed compound heterozygosity for the c.1521_1523delCTT (ΔF508) pathogenic variant and the c.273G>C (G91G) variant in CFTR. Sweat test results confirmed the diagnosis of CF. We have thus identified a synonymous variation (G91G) causing the skipping of exon 3 in a CF patient carrying the ΔF508 mutation. However, the clinical phenotype with pancreatic symptoms encouraged us to investigate a panel of pancreas-related genes, which resulted in finding a known sequence variation inside CTRC. We further discuss the role of these variants and their possible interactions in determining the current phenotype. Cystic fibrosis (CF), the most common autosomal recessive disease in whites, is caused by mutations in the CF transmembrane conductance regulator (CFTR). So far, >1900 mutations have been described, most of which are nonsense, missense, and frameshift, and can lead to severe phenotypes, reducing the level of function of the CFTR protein. Synonymous variations are usually considered silent without pathogenic effects. However, synonymous mutations exhibiting exon skipping as a consequence of aberrant splicing of pre-mRNA differ. Herein, we describe the effect of the aberrant splicing of the c.273G>C (G91G) synonymous variation found in a 9-year-old white (ΔF508) patient affected by CF and pancreatitis associated with a variant in chymotrypsin C (CTRC). Magnetic resonance imaging showed an atrophic pancreatic gland with substitution of the pancreatic parenchyma with three cysts. Genetic examination revealed compound heterozygosity for the c.1521_1523delCTT (ΔF508) pathogenic variant and the c.273G>C (G91G) variant in CFTR. Sweat test results confirmed the diagnosis of CF. We have thus identified a synonymous variation (G91G) causing the skipping of exon 3 in a CF patient carrying the ΔF508 mutation. However, the clinical phenotype with pancreatic symptoms encouraged us to investigate a panel of pancreas-related genes, which resulted in finding a known sequence variation inside CTRC. We further discuss the role of these variants and their possible interactions in determining the current phenotype. Cystic fibrosis (CF; Online Mendelian Inheritance in Man no. 219700, http://www.ncbi.nlm.nih.gov/omim), the most common autosomal recessive disease in whites,1Welsh M.J. Tsui L.C. Boat T.F. Beaudet A.I. Cystic fibrosis.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill, New York1995: 3799-3876Google Scholar is classically described as chronic obstructive pulmonary disease with disseminated bronchiectasis,2Pignatti P.F. Bombieri C. Marigo C. Benetazzo M. Luisetti M. Increased incidence of cystic fibrosis gene mutations in adults with disseminated bronchiectasis.Hum Mol Genet. 1995; 4: 635-639Crossref PubMed Scopus (152) Google Scholar, 3Girodon E. Cazeneuve C. Lebargy F. Chinet T. Costes B. Ghanem N. Martin J. Lemay S. Scheid P. Housset B. Bignon J. Goossens M. CFTR gene mutations in adults with disseminated bronchiectasis.Eur J Hum Genet. 1997; 5: 149-155Crossref PubMed Scopus (150) Google Scholar exocrine pancreatic insufficiency,4Cohn J.A. Friedman K.J. Noone P.G. Knowels M.R. Silverman L.M. Jowel P.S. Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis.N Engl J Med. 1998; 339: 653-658Crossref PubMed Scopus (805) Google Scholar, 5Sharer N. Schawrz M. Malone G. Howarth A. Painter J. Super M. Brazanga J. Mutations of the cystic fibrosis gene in patients with chronic pancreatitis.N Engl J Med. 1998; 339: 645-652Crossref PubMed Scopus (797) Google Scholar and elevation of sodium and chloride concentrations in the sweat.6Rosentein B.J. Zeitlin P.L. Cystic fibrosis.Lancet. 1998; 351: 277-281Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar At the genetic level, CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, located on the long arm of chromosome 7, encoding an activated chloride channel in epithelial cell membrane and regulating other membrane transport proteins. This wide distribution accounts for the multiorgan involvement of CF. To date, >1900 mutations have been described, 98% of which are point mutations or microdeletions/insertions,7Bobadilla J.L. Macek Jr., M. Fine J.P. Farrell P.M. Cystic fibrosis: a worldwide analysis of CFTR mutations-correlations with incidence data and application to screening.Hum Mutat. 2002; 19: 575-606Crossref PubMed Scopus (798) Google Scholar with ΔF508 being the most frequent worldwide.8Wilschanski M. Zielenski J. Markiewicz D. Tsui L.C. Corey M. Levison H. Durie P.R. Correlation of sweat chloride concentration with classes of the cystic fibrosis transmembrane conductance regulator gene mutations.J Pediatr. 1995; 127: 705-710Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar Nonsense, missense, and frameshift mutations, and genomic rearrangements, according to their consequences on CFTR function, are usually considered severe mutations because they reduce the level of normally functioning CFTR at the apical membrane. In this setting, synonymous variations play a significant role: most of them are known to be innocuous polymorphisms because the amino acid chain remains unchanged. Moreover, the nucleotide substitution sometimes may be located in critical regions affecting the splicing mechanisms, leading to the well-recognized effect of exon skipping, as reported for the synonymous variant (G893G) causing aberrant splicing of exon 15.9Faa' V. Coiana A. Incani F. Costantino L. Cao A. Rosatelli M.C. A synonymous mutation in the CFTR gene causes aberrant splicing in an Italian patient affected by a mild form of cystic fibrosis.J Mol Diagn. 2010; 12: 380-383Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar The relative impact of the CFTR genotype on clinical phenotype is organ specific: patients with mild mutations (classes IV and V) have a pancreas-sufficient phenotype with high probability of developing pancreatitis.10Chen J.M. Fèrec C. Chronic pancreatits: genetics and pathogenesis.Annu Rev Genomics Hum Genet. 2009; 10: 63-87Crossref PubMed Scopus (101) Google Scholar In human exocrine pancreas, CFTR is predominantly expressed at the apical plasma membrane of the ductal cells and controls cAMP-mediated bicarbonate secretion into the duct lumen. The major effect of CFTR in the pancreas is to dilute and alkalinize the protein-rich acinar secretion into the duct lumen, thereby preventing the formation of protein plugs that predispose to pancreatic injury. The phenotype complexity of the disease comes from the interactions among the involved genes.11Rosendahl J. Landt O. Bernadova J. Kovacs P. Teich N. Bodeker H. Keim V. Ruffert C. Mossner J. Kage A. Stumvoll M. Groneberg D. Kruger R. Luck W. Treiber M. Becker M. Witt H. CFTR, SPINK1, CTRC and PRSS1 variants in chronic pancreatitis: is the role of mutated CFTR overestimated?.Gut. 2013; 62: 582-592Crossref PubMed Scopus (134) Google Scholar Chronic pancreatitis (CP) is a persistent inflammation of the pancreas characterized by repeated attacks of abdominal pain, irreversible morphological changes, and impairment of both exocrine and endocrine functions.12Chen J.M. Fèrec C. Genetics and pathogenesis of chronic pancreatitis: the 2012 update.Clin Res Hepatol Gastroenterol. 2012; 36: 334-340Crossref PubMed Scopus (45) Google Scholar The identification of mutations in protease, serine, 1 (PRSS1), serine protease inhibitor, Kazal type 1 (SPINK1), and chymotrypsin C (CTRC) has established the role of prematurely activated trypsin in CP patients. Herein, we describe the effect of the aberrant splicing of the c.273G>C (G91G) synonymous variation found in an Italian patient affected by CF and pancreatitis associated with a variant in the CTRC (ΔF508). Accordingly, the segregation analysis of all variants within the family members was provided to better recognize the genotype-phenotype correlation. A comparative study, to investigate the pancreas-associated genes, was also performed in two previously reported CF patients having the same functional defect affecting CFTR.13Ramalho A.S. Beck S. Farinha C.M. Methods for RNA extraction, cDNA preparation and analysis of CFTR transcripts.J Cyst Fibros. 2004; 3: 11-15Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar The patient was the second son of a couple of healthy unrelated parents and was born at term from cesarean section (weight at birth, 2.4 kg). At the age of 9 years, he manifested fever (40°C) with vomiting and severe abdominal pain. Blood analysis showed neutrophilia (17.3 × 103 U/μL), hyperamylasemia (840 UI/L), and hyperlipasemia (913 UI/L). Abdominal ultrasound showed diffuse disomogeneity of the pancreas. Because of the persistence of abdominal pain, he started therapy with octreotide, with resolution of symptoms even after resumption of oral feeding. Afterward, he showed persistently high pancreatic enzymes (both amylase and lipase at approximately 550 UI/L) without symptoms since the age of 11 years when he showed recurrence of severe abdominal pain. Abdominal computed tomography scans showed multiple hypodense areas substituting almost completely glandular parenchyma, with dilatation of the Wirsung duct and segmental stenosis of the lateral ducts. Magnetic resonance imaging after secretin stimulus showed an atrophic pancreatic gland and confirmed substitution of pancreatic parenchyma with three cysts ranging from 2 to 6.5 cm and irregular Wirsung duct caliber with tortuous course and multiple stenotic tracts. Considering the persistence of pain, he underwent endoscopic retrograde cholangiopancreatography showing that the Wirsung duct was interrupted after 3 cm by a pseudocyst that was in contact with the posterior stomach wall. As abdominal pain dramatically increased, he began therapy with ketorolac, protease inhibitors, and even morphine, with partial resolution. Drainage of the cyst was then performed with open gastrocystostomy. After surgery, the boy exhibited pain resolution. He resumed oral feeding 1 month after surgery without recurrence of symptoms. Pancreatic ultrasound showed disappearance of the cysts. Sweat test results confirmed diagnosis of classic CF (Cl, 78 mmol/L; sweat, 110 mg). Fecal elastase was normal in several determinations (>500 ng/g stools). When this article was submitted, the boy was 14 years old, and in good clinical and nutritional conditions, his weight being at 75th percentile and height at 90th percentile. No respiratory tract symptoms were present when this article was submitted. Pulmonary computed tomography showed minimal bronchiectasis, airways were chronically colonized by Staphylococcus aureus, and the mean forced expiratory volume in the first second was 101% of predicted value. He was on a free diet, and he was following a therapy with proton pump inhibitors, ursodeoxycholic acid, and specific therapy to prevent and cure pulmonary CF disease. Genetic examination revealed compound heterozygosity for the c.1521_1523delCTT (ΔF508) mutation and the c.273G>C (G91G) variant in CFTR. Informed consent was obtained from the patient’s parents. The study was approved by the ethical committee scientific board of Bambino Gesù Children’s Hospital (Rome, Italy) and was conducted in accordance with the Declaration of Helsinki. Genomic DNA was extracted from peripheral blood using the commercial kit High Pure PCR Template Preparation (Roche Diagnostics, Mannheim, Germany). Mutations and Tn locus were screened by means of the InnoLipa 17 + Tn update, 19, and Regional assay (Fujirebio Europe, Gent, Belgium), covering 85% of all known pathogenic variants, following the manufacturer's protocol. Complete analysis of the CFTR, including promoter, all coding regions, intronic and exonic junctions, intervening sequence (IVS)6, IVS10, and IVS19 intronic regions containing known splicing variation, and the 5′ and 3′ untranslated regions, has been performed with the Sanger method using the Big-Dye Termination cycle sequencing kit (Applied Biosystems, Foster City, CA). CFTR deletion analysis was performed using the SALSA P091-B CFTR multiplex ligation-dependent probe amplification kit (MRC-Holland, Amsterdam, the Netherlands). Amplified products were capillary electrophoresed on the ABI-Prism 3130 XL Genetic Analyzer (Applied Biosystems) and analyzed with the GeneScan software version 4.0 (Applied Biosystems). Data were processed with the Coffalyser software version 8.0 (MRC-Holland). Nasal epithelial cells were collected from the nasal epithelium by gently brushing the inferior turbinates of both nostrils using a cytobrush. RNA was extracted by means of guanidinium isothiocyanate/phenol method RNAzol (Invitrogen Life Technologies, Groningen, the Netherlands). Total RNA (1 μg) was reverse transcribed using hexanucleotide primers and a high-capacity cDNA Archive kit (Applied Biosystems). cDNA was amplified by PCR in six overlapping fragments spanning the entire gene using FastStart Taq DNA Polymerase (Roche).13Ramalho A.S. Beck S. Farinha C.M. Methods for RNA extraction, cDNA preparation and analysis of CFTR transcripts.J Cyst Fibros. 2004; 3: 11-15Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar The PCR thermal cycling conditions (Applied Biosystems) were as follows: initial denaturation at 95°C for 6 minutes; 30 to 36 cycles at 95°C for 30 seconds, annealing at 60°C for 1 minute, and extension at 72°C for 2 minutes, followed by a final extension at 72°C for 8 minutes. The PCR was tested by running 5 μL of the product on a 1.5% agarose gel, as previously described.14Tomaiuolo A.C. Sirleto P. Centrone C. Surace C. Alghisi F. Petrocchi S. Lombardo A. Rossi M. Torricelli F. Lucidi V. Angioni A. Characterization of a novel isolated deletion of the exon 3 within the CFTR gene: relevance for phenotypic expression and genetic counseling.Clin Biochem. 2011; 44: 799-803Crossref PubMed Scopus (1) Google Scholar Targeted resequencing was performed using a uniquely customized design: TruSeq Custom Amplicon (Illumina, San Diego, CA) with the MiSeq sequencing platform (Illumina). TruSeq Custom Amplicon is a fully integrated DNA-to-data solution, including online probe design and ordering through the Illumina website, sequencing assay, automated data analysis, and offline software for reviewing results. Online probe design was performed by entering target genomic regions into Design Studio software version 1.6 (Illumina), as described elsewhere.15Lepri F.R. Scavelli R. Digilio M.C. Gnazzo M. Grotta S. Dentici M.L. Pisaneschi E. Sirleto P. Capolino R. Baban A. Russo S. Franchin T. Angioni A. Dallapiccola B. Diagnosis of Noonan syndrome and related disorders using target next generation sequencing.BMC Med Genet. 2014; 15: 14Crossref PubMed Scopus (50) Google Scholar The design was performed to generate a panel of six genes: CFTR (NM_000492.3), SPINK1 (NM_003122.3), PRSS1 (NM_002769.4), protease, serine, 2 (PRSS2) (NM_002770.2), CTRC (NM_007272.2), and calcium-sensing receptor (CASR) (NM_001178065.1). These genes were generated by University of California, Santa Cruz, Genome Browser Home (http://genome.ucsc.edu/cgi-bin/hgGateway, last accessed October 23, 2013), involved in CF and chronic pancreatitis, including 91 amplicons with a coverage of 100%. The choice of genes investigated in this panel was based on scientific evidence for their causative or predisposing role in the disease.10Chen J.M. Fèrec C. Chronic pancreatits: genetics and pathogenesis.Annu Rev Genomics Hum Genet. 2009; 10: 63-87Crossref PubMed Scopus (101) Google Scholar The MiSeq system provides fully integrated on-instrument data analysis software. Each single variant reported in the vCard output file has been evaluated for the coverage and the Q score and visualized via Integrative Genomics Viewer.16Helga T. James T. Robinson J.T. Mesirov J.P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration.Brief Bioinform. 2013; 14: 178-192Crossref PubMed Scopus (4884) Google Scholar, 17Robinson J.T. Thorvaldsdóttir H. Winckler W. Guttman M. Lander E.S. Getz G. Mesirov J.P. Integrative Genomics Viewer.Nat Biotechnol. 2011; 29: 24-26Crossref PubMed Scopus (7591) Google Scholar On the basis of the guidelines of the American College of Medical Genetics and Genomics,18Rehm H.L. Bale S.J. Bayrak-Toydemir P. Berg J.S. Brown K.K. Deignan J.L. Friez M.J. Funke B.H. Hegde M.R. Lyon E. ACMG clinical laboratory standards for next-generation sequencing.Genet Med. 2013; 15: 733-747Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar all regions that have been sequenced with a sequencing depth of <30 have been considered not suitable for analysis. Furthermore, we established a minimum threshold in Q score of 30 (base call accuracy, 99.9%). All mutations identified by MiSeq Reporter have been validated by Sanger sequencing using standard protocols. The patient studied using the commercial kit (Fujirebio Diagnostics, Malvern, PA) showed one CF-causing pathogenic variant revealing a genotype containing c.1521_1523delCTT (ΔF508)/unknown. Because he was a carrier for a single CF mutation, we sequenced CFTR and detected a second change, c.273G>C (G91G), which was a synonymous variation. Because this variation was located close to a splice site, we performed an in silico analysis using Human Splicing Finder software version 2.4.1 (http://www.umd.be/HSF, last accessed December 11, 2013), which suggested a low probability for the new GGC codon to play a role as donor splice site. To clarify the effect of c.273G>C at the transcription level, we investigated its effect at the level of mRNA. RNA was retrotranscribed into cDNA using primers covering the entire CFTR sequence and examined on agarose gel, which revealed two bands in the patient as against one band in the healthy control (Figure 1). The additional band of the patient was extracted, purified, and sequenced, revealing the loss of exon 3 (Figure 2).Figure 2The patient's CFTR gene relevant fragment. A: CFTR genomic DNA region spanning exons 2 to 4. B: cDNA of the same region after the exon 3 skipping. The nucleotide sequences of exons 2 and 4 are reported in blue and gray, respectively. C: Electropherogram of patient's DNA showing the exon 3 skipping with the juxtaposition of the exons 2 and 4 and the patient's amino acid residues of exons 2 and 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because of the pancreatic phenotype, the patient was further studied by next-generation sequencing (NGS) using a TruSeq Custom Amplicon panel containing the genes (CFTR, SPINK1, PRSS1, PRSS2, CTRC, and CASR) described in the literature as associated with CP.10Chen J.M. Fèrec C. Chronic pancreatits: genetics and pathogenesis.Annu Rev Genomics Hum Genet. 2009; 10: 63-87Crossref PubMed Scopus (101) Google Scholar The analysis of these genes revealed inside the CTRC the known sequence variation c.703G>A (V235I).19Rosendahl J. Witt H. Szmola R. Bhatia E. Ózsvári B. Landt O. Schulz H.U. Gress T.M. Pfützer R. Löhr M. Kovacs P. Blüher M. Stumvoll M. Choudhuri G. Hegyi P. te Morsche R.H.M. Drenth J.P.H. Truninger K. Macek J.M. Puhl G. Witt U. Schmidt H. Büning C. Ockenga J. Kage A. Groneberg D.A. Nicke R. Berg T. Wiedenmann B. Bödeker H. Keim V. Mössner J. Teich N. Sahin-Tóth M. Chymotrypsin C (CTRC) alterations that diminish activity or secretion are associated with chronic pancreatitis.Nat Genet. 2008; 40: 78-82Crossref PubMed Scopus (299) Google Scholar A comprehensive list of the variants found in all of the investigated genes is reported in Table 1. SPINK1 and PRSS2 exhibited wild-type features only. Additional experiments were planned to analyze, with the same TruSeq Custom Amplicon panel, two previously reported CF patients, a male and a female, bearing the following genotype, c.1521_1523delCTT (ΔF508)/c.197_274+461del538 (CFTRdele3), resulting in the skipping of exon 3.14Tomaiuolo A.C. Sirleto P. Centrone C. Surace C. Alghisi F. Petrocchi S. Lombardo A. Rossi M. Torricelli F. Lucidi V. Angioni A. Characterization of a novel isolated deletion of the exon 3 within the CFTR gene: relevance for phenotypic expression and genetic counseling.Clin Biochem. 2011; 44: 799-803Crossref PubMed Scopus (1) Google Scholar These patients had an important involvement of the pulmonary district, moderate reduction of pancreas function, and no evidence of pancreatitis at follow-up. Sequencing analysis did not detect any pathogenic variants among the panel of the pancreatic genes (Tables 2 and 3).Table 1Variants Detected in the Patient by TSCA Panel as Reported by Amplicon Viewer SoftwareGenesPatient IDExon/intronCoordinateTypeAlleleQ scoreCoverageVariant frequencyVariationProteinCFTR22203117149196SNPhet995140.519c.273G>Cp.Gly91GlyIVS7117176568Deletionhom09250.944c.744-33_744-30delGATTIVS7117176738SNPhet9918220.496c.869+11C>TIVS9117188660Insertionhet01600.26c.1210-34 TG[10]; [11]∗c.1210-12T[9]; [7] poly T tract.IVS10117188682SNPhet991600.441c.1210-13G>T11117199533SNPhom991311c.1408G>Ap.Val470Met11117199644Deletionhet02570.572c.1521_1523delCTTp.Phe508del15117235055SNPhet99950.505c.2562T>Gp.Thr854ThrPRSS12220IVS4142460261SNPhom992001c.455-21T>A4142460313SNPhet992000.513c.486T>Cp.Asp162Asp5142460865SNPhet99650.585c.738T>Cp.Asn246AsnCTRC2220715772155SNPhet992160.501c.703G>Ap.Val235IleIVS715772057SNPhet995930.486c.640-35G>TCASR2220IVS3121976253SNPhom991500.993c.492+19G>AIVS3121976255SNPhet991500.573c.492+21G>A7122003045SNPhom991411c.2274C>Gp.Pro758Pro7122003832SNPhom99321c.3061C>Gp.Gln1011Glu†p.Gln1011Glu is a known polymorphism in CASR.20Het, heterozygous; hom, homozygous; IVS, intervening sequence; SNP, single-nucleotide polymorphism; TSCA, TruSeq Custom Amplicon.∗ c.1210-12T[9]; [7] poly T tract.† p.Gln1011Glu is a known polymorphism in CASR.20Muddana V. Lamb J. Greer J.B. Elinoff B. Hawes R.H. Cotton P.B. Anderson M.A. Brand R.E. Slivka A. Whitcomb D.C. Association between calcium sensing receptor gene polymorphisms and chronic pancreatitis in a US population: role of serine protease inhibitor Kazal 1 type and alcohol.World J Gastroenterol. 2008; 14: 4486-4491Crossref PubMed Scopus (61) Google Scholar Open table in a new tab Table 2Variants Detected in the c.1521_1523delCTT (ΔF508)/c.197_274+461del538 (CFTRdele3) Male Patient by TSCA Panel as Reported by Amplicon Viewer SoftwareGenesPatient IDExon/intronCoordinateTypeAlleleQ scoreCoverageVariant frequencyVariationProteinCFTR1301IVS7117176568Deletionhom011331c.744-33_744-30delGATTIVS7117176738SNPhet9922280.5c.869+11C>TIVS9117188660Insertionhet014800.15c.1210-34TG[10]∗c.1210-12[9]; [9] poly T tract.IVS10117188682SNPhet9912830.5c.1210-13G>T11117199533SNPhom991491c.1408G>Ap.Val470Met11117199644Deletionhet02870.572c.1521_1523delCTTp.Phe508del15117235055SNPhet9914200.505c.2562T>Gp.Thr854ThrIVS17117246636SNPhet9926550.5c.2909-92G>A27117307108SNPhet9949980.5c.4389G>Ap.Gln1463Gln3′UTR117308413SNPhet9942570.5c.*1251C>TPRSS11301IVS4142460261SNPhom9932411c.455-21T>A4142460313SNPhet9932411c.486T>Cp.Asp162Asp5142460865SNPhet9942881c.738T>Cp.Asn246AsnCTRC1301IVS415769139SNPhet993030.5c.356+71G>ACASR13017122003832SNPhom9942011c.3061C>Gp.Gln1011Glu†p.Gln1011Glu is a known polymorphism in CASR.20Het, heterozygous; hom, homozygous; IVS, intervening sequence; SNP, single-nucleotide polymorphism; TSCA, TruSeq Custom Amplicon; UTR, untranslated region.∗ c.1210-12[9]; [9] poly T tract.† p.Gln1011Glu is a known polymorphism in CASR.20Muddana V. Lamb J. Greer J.B. Elinoff B. Hawes R.H. Cotton P.B. Anderson M.A. Brand R.E. Slivka A. Whitcomb D.C. Association between calcium sensing receptor gene polymorphisms and chronic pancreatitis in a US population: role of serine protease inhibitor Kazal 1 type and alcohol.World J Gastroenterol. 2008; 14: 4486-4491Crossref PubMed Scopus (61) Google Scholar Open table in a new tab Table 3Variants Detected in the c.1521_1523delCTT (ΔF508)/c.197_274+461del538 (CFTRdele3) Female Patient by TSCA Panel as Reported by Amplicon Viewer SoftwareGenesPatientExon/intronCoordinateTypeAlleleQ scoreCoverageVariant frequencyVariationProteinCFTR0852IVS7117176568Deletionhom033990.495c.744-33_744-30delGATTIVS7117176738SNPhet9950000.519c.869+11C>TIVS9117188660Insertionhet999940.429c.1210-34TG[10]∗c.1210-12[9]; [9]poly T tract.IVS10117199457SNPhet997290.538c.1393-61A>G11117199533SNPhom997290.549c.1408G>Ap.Val470Met11117199644Deletionhet014290.526c.1521_1523delCTTp.Phe508delIVS11117218159SNPhet9932590.429c.1909-9634T>AIVS11117218575SNPhet99340.441c.1909-9218G>ASPINK108523′UTR147204093SNPhet994720.424c.*131G>A5′UTR147211393SNPhet9913530.974c.-253T>CPRSS10852IVS4142460261SNPhom9913780.999c.455-21T>A4142460313SNPhet9913780.984c.486T>Cp.Asp162Asp5142460865SNPhet9911780.986c.738T>Cp.Asn246AsnCTRC0852315767036SNPhet99610.984c.180C>Tp.Gly60GlyCASR0852IVS3121976253SNPhom9913250.998c.492+19G>AIVS3121980284SNPhet9910710.488c.493-91C>T7122003045SNPhom9927401c.2274C>Gp.Pro758Pro7122003832SNPhom9918351c.3061C>Gp.Gln1011Glu†p.Gln1011Glu is a known polymorphism in CASR.20Het, heterozygous; hom, homozygous; IVS, intervening sequence; SNP, single-nucleotide polymorphism; TSCA, TruSeq Custom Amplicon; UTR, untranslated region.∗ c.1210-12[9]; [9]poly T tract.† p.Gln1011Glu is a known polymorphism in CASR.20Muddana V. Lamb J. Greer J.B. Elinoff B. Hawes R.H. Cotton P.B. Anderson M.A. Brand R.E. Slivka A. Whitcomb D.C. Association between calcium sensing receptor gene polymorphisms and chronic pancreatitis in a US population: role of serine protease inhibitor Kazal 1 type and alcohol.World J Gastroenterol. 2008; 14: 4486-4491Crossref PubMed Scopus (61) Google Scholar Open table in a new tab Het, heterozygous; hom, homozygous; IVS, intervening sequence; SNP, single-nucleotide polymorphism; TSCA, TruSeq Custom Amplicon. Het, heterozygous; hom, homozygous; IVS, intervening sequence; SNP, single-nucleotide polymorphism; TSCA, TruSeq Custom Amplicon; UTR, untranslated region. Het, heterozygous; hom, homozygous; IVS, intervening sequence; SNP, single-nucleotide polymorphism; TSCA, TruSeq Custom Amplicon; UTR, untranslated region. The genetic analysis was also extended to the patient's parents and brother of the current case, and we found G91G and V235I in his father, and ΔF508 and V235I in his brother displaying a normal phenotype without clinical consequences; instead, his mother resulted only in ΔF508 carrier (Figure 3). These results indicated that the G91G and ΔF508 pathogenic variants are in trans and confirmed the diagnosis of CF, suggesting a role for the CTRC variant in influencing the onset of CP. Synonymous variations consisting of a single-base substitution generate a codon that does not change the encoded amino acid and are usually considered as silent variations without pathogenic consequences. Herein, we describe the effect on the splicing process of the synonymous change c.273G>C (G91G) located near the splicing donor site of exon 3 detected in a CF patient with CP, and the role of the c.703G>A (V235I) variant on CTRC in contributing to the development of the pancreatic phenotype. By using an in silico approach with Human Splicing Finder, we have found that the new codon has a low probability to act as donor splice site. The study performed on mRNA demonstrated that this mutation produces the skipping of exon 3, leading to loss of function in the protein. Exon 3 encodes for a polypeptide that concurs to the assembly of the first membrane spanning domain and is involved in the entire formation of the proper chloride channel structure. In a previous report,14Tomaiuolo A.C. Sirleto P. Centrone C. Surace C. Alghisi F. Petrocchi S. Lombardo A. Rossi M. Torricelli F. Lucidi V. Angioni A. Characterization of a novel isolated deletion of the exon 3 within the CFTR gene: relevance for phenotypic expression and genetic counseling.Clin Biochem. 2011; 44: 799-803Crossref PubMed Scopus (1) Google Scholar we described two CF patients, a male and a female, both CFTR compound heterozygous c.1521_1523delCTT (ΔF508)/c.197_274+461del538 (CFTRdele3). We demonstrated, by using the same RNA approach, that the partial deletion of exon 3 produced a complete skipping of the coding region, leading to a common phenotype showing a marked decline of the respiratory functions due to massive bronchiectasis and recurrent infections. Late onset and moderate reduction of pancreatic function, without signs of pancreatitis, were also additional findings observed at the clinical presentation. The current case revealed the abdominal symptoms when he was 9 years old, and the CFTR genotype showed a c.1521_1523delCTT (ΔF508)/c.273G>C (G91G) association. RNA studies clearly evidenced that the G91G mutation is pathogenic and affects the splicing function, resulting in the complete skipping of exon 3. On the basis of the molecular results, we expected to find similar phenotypic features compared to the previously reported cases. On the contrary, we can assume that the sole characteristic they share is the late onset of the clinical presentation. It is well known that CF patients, having the same genotype, may express relevant different phenotypes; this is particularly true for family members, sisters and/or brothers, homozygous for ΔF508 mutation showing marked lung damage and a strong pancreatic compromission.21Kerem 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 (delta F508).N Engl J Med. 1990; 323: 1517-1522Crossref PubMed Scopus (624) Google Scholar Usually, these differences have been explained by the involvement of environmental factors or, more likely, by the influence of modifier genes.22Cutting G.R. Modifier genes in Mendelian disorders: the example of cystic fibrosis.Ann N Y Acad Sci. 2010; 1214: 57-69Crossref PubMed Scopus (195) Google Scholar Sometimes, the proper characteristics of the mutation may address a particular phenotype, as described for the c.3454G>C (D1152H) mutation; in a family study, the mother and her son were both D1152H homozygous, but only the son had complete CF, whereas the mother was affected by recurrent episodes of chronic pancreatitis.23Alghisi F. Bella S. Lucidi V. Angioni A. Tomaiuolo A.C. D'Apice M.R. Gambardella S. Novelli G. Phenotypic variability in a family with pancreatitis and cystic fibrosis sharing common mild CFTR mutation: report on CFTR mutations and their phenotypic variability.Pancreas. 2009; 38: 109-110Crossref PubMed Scopus (4) Google Scholar The relevant role of CFTR for the pancreatic phenotype has been clearly elucidated by demonstration of its high activity at the apical plasma membrane of the ductal pancreatic cells. Moreover, an increasing amount of evidence has shifted the attention to a distinct group of genes, highly expressed in the pancreatic tissue, strictly interacting with CFTR. By using an NGS approach, we analyzed the entire panel of the pancreas-related genes and we found a missense variation, c.703G>A (V235I), inside the CTRC. The CTRC is located on chromosome 1p36.21, includes eight exons,24Zhou J. Sahin-Tóth M. Chymotrypsin C. (CTRC) mutations in chronic pancreatitis.J Gastroenterol Hepatol. 2011; 26: 1238-1246Crossref PubMed Scopus (56) Google Scholar and encodes for chymotrypsin C, a pancreatic digestive enzyme that promotes degradation and inactivation of all human trypsin and trypsinogen isoforms.19Rosendahl J. Witt H. Szmola R. Bhatia E. Ózsvári B. Landt O. Schulz H.U. Gress T.M. Pfützer R. Löhr M. Kovacs P. Blüher M. Stumvoll M. Choudhuri G. Hegyi P. te Morsche R.H.M. Drenth J.P.H. Truninger K. Macek J.M. Puhl G. Witt U. Schmidt H. Büning C. Ockenga J. Kage A. Groneberg D.A. Nicke R. Berg T. Wiedenmann B. Bödeker H. Keim V. Mössner J. Teich N. Sahin-Tóth M. Chymotrypsin C (CTRC) alterations that diminish activity or secretion are associated with chronic pancreatitis.Nat Genet. 2008; 40: 78-82Crossref PubMed Scopus (299) Google Scholar, 25Szmola R. Sahin-Tóth M. Chymotrypsin C (caldecrin) promotes degradation of human cationic trypsin: identity with Rinderknecht's enzyme Y.Proc Natl Acad Sci U S A. 2007; 104: 11227-11232Crossref PubMed Scopus (117) Google Scholar Because human trypsinogens are prone to autoactivation, chymotrypsin C exerts a protective role against pancreatitis. Variations in CTRC are responsible for a growing amount of trypsin concentration, increasing the risk of CP.24Zhou J. Sahin-Tóth M. Chymotrypsin C. (CTRC) mutations in chronic pancreatitis.J Gastroenterol Hepatol. 2011; 26: 1238-1246Crossref PubMed Scopus (56) Google Scholar The V235I variation has been described in a patient with CP carrying the c.760C>T (R254W) on the other allele. The familial transmission from two healthy parents was also documented. Functional studies demonstrated that the V235I variation leads to the down-regulation of chymotrypsin C activity, resulting in an abnormal amount of trypsinogen.19Rosendahl J. Witt H. Szmola R. Bhatia E. Ózsvári B. Landt O. Schulz H.U. Gress T.M. Pfützer R. Löhr M. Kovacs P. Blüher M. Stumvoll M. Choudhuri G. Hegyi P. te Morsche R.H.M. Drenth J.P.H. Truninger K. Macek J.M. Puhl G. Witt U. Schmidt H. Büning C. Ockenga J. Kage A. Groneberg D.A. Nicke R. Berg T. Wiedenmann B. Bödeker H. Keim V. Mössner J. Teich N. Sahin-Tóth M. Chymotrypsin C (CTRC) alterations that diminish activity or secretion are associated with chronic pancreatitis.Nat Genet. 2008; 40: 78-82Crossref PubMed Scopus (299) Google Scholar Gene-gene interaction is a relevant factor to take into account in relationship to the onset of the pathogenic pancreatic process. A higher risk of disease has been documented for trans-heterozygote carriers of variants in different pancreatitis-associated genes.12Chen J.M. Fèrec C. Genetics and pathogenesis of chronic pancreatitis: the 2012 update.Clin Res Hepatol Gastroenterol. 2012; 36: 334-340Crossref PubMed Scopus (45) Google Scholar Trans-heterozygosity has been extensively studied in CP, where a variable frequency, ranging from 6.5% to approximately 9% of patients, was reported in two large series.12Chen J.M. Fèrec C. Genetics and pathogenesis of chronic pancreatitis: the 2012 update.Clin Res Hepatol Gastroenterol. 2012; 36: 334-340Crossref PubMed Scopus (45) Google Scholar, 26Masson E. Chen J.M. Scotet V. Le Maréchal C. Férec C. Association of rare chymotrypsinogen C (CTRC) gene variations in patients with idiopathic chronic pancreatitis.Hum Genet. 2008; 123: 83-91Crossref PubMed Scopus (128) Google Scholar Among the 22 cases with digenic genotypes (two pathological genotypes in two different genes) described by Masson et al,26Masson E. Chen J.M. Scotet V. Le Maréchal C. Férec C. Association of rare chymotrypsinogen C (CTRC) gene variations in patients with idiopathic chronic pancreatitis.Hum Genet. 2008; 123: 83-91Crossref PubMed Scopus (128) Google Scholar 18 involved a pathological CFTR genotype, whereas the association with a pathological CTRC genotype was found in three patients. CTRC and SPINK1 trans-heterozygosity was detected, in the same study, in three other cases, including a patient with V235I variation. Depending on the gene and the type of sequence variation, digenic genotypes have been considered able, in some instances, to explain as the disease developed.12Chen J.M. Fèrec C. Genetics and pathogenesis of chronic pancreatitis: the 2012 update.Clin Res Hepatol Gastroenterol. 2012; 36: 334-340Crossref PubMed Scopus (45) Google Scholar CF patients with symptomatic chronic pancreatitis are infrequent and reveal an estimated overall occurrence of approximately 1.2% of cases.27De Boeck K. Weren M. Proesmans M. Kerem E. Pancreatitis among patients with cystic fibrosis: correlation with pancreatic status and genotype.Pediatrics. 2005; 115: 463-469Crossref Scopus (120) Google Scholar Large studies performed to investigate the involvement of pancreas-related genes are not available in the literature and usually the findings are restricted to a rare case report.28Kopp B.T. Pastore M.T. Sturm A.C. Holtzlander M.J. Westman J.A. A novel exon duplication of the cystic fibrosis transmembrane conductance regulator in a patient presenting with adult-onset recurrent pancreatitis.Pancreas. 2011; 40: 773-777Crossref PubMed Scopus (2) Google Scholar To evaluate their genetic setting, we also examined the previously reported patients with the same CFTR functional defect but different phenotype using the NGS panel with the most frequently CP-associated genes. The sequencing analysis results did not give indication of any significant sequence variation, clearly excluding a trans-heterozygous condition. In addition, no differences have been found regarding the most common CFTR variants usually associated with pancreatitis. The evidence we provided remarks on the diversity among the phenotypes observed and appears to likely correlate to the genetic findings. In this background, the overlapping effect of the V235I on CTRC may contribute to unbalance the residual CFTR function, ultimately leading to the current unexpected phenotype. The case we described, apparently easy to make a diagnosis, demonstrated the importance to use different tools to correctly recognize the significance of unusual genetic variations. Patients with diverse phenotypes showing common genotypes should be addressed to investigate organ-specific genes and, in this context, the use of NGS to perform multiple gene analysis is the right way to obtain faster and effective results. After this manuscript was accepted, we observed an additional case, a female with CF showing the same functional defect as the patient described in the present report. She presented the classic CF phenotype without signs of chronic pancreatitis. In accordance with the previously reported cases exhibiting the discordant phenotype, study by NGS of the panel of pancreas-related genes did not reveal any significant genetic variant. We thank Dr. Lucy Costantino for help with expression studies." @default.
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• W2108839621 title "Relationship between CFTR and CTRC Variants and the Clinical Phenotype in Late-Onset Cystic Fibrosis Disease with Chronic Pancreatitis" @default.
• W2108839621 cites W1608808389 @default.
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• W2108839621 cites W1998319454 @default.
• W2108839621 cites W2001869932 @default.
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• W2108839621 cites W2021341670 @default.
• W2108839621 cites W2057152418 @default.
• W2108839621 cites W2071386895 @default.
• W2108839621 cites W2080130466 @default.
• W2108839621 cites W2083214486 @default.
• W2108839621 cites W2085404502 @default.
• W2108839621 cites W2103879666 @default.
• W2108839621 cites W2104953797 @default.
• W2108839621 cites W2111543096 @default.
• W2108839621 cites W2117131162 @default.
• W2108839621 cites W2138362876 @default.
• W2108839621 cites W2139574634 @default.
• W2108839621 cites W2142682052 @default.
• W2108839621 cites W2155759872 @default.
• W2108839621 cites W2165956362 @default.
• W2108839621 cites W2166863205 @default.
• W2108839621 cites W2313829157 @default.
• W2108839621 cites W2330410882 @default.
• W2108839621 cites W2337925307 @default.
• W2108839621 cites W4240997916 @default.
• W2108839621 cites W4252831606 @default.
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