Meconium Ileus Caused by Mutations in GUCY2C, Encoding the CFTR-Activating Guanylate Cyclase 2C
2012; Elsevier BV; Volume: 90; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2012.03.022
ISSN1537-6605
AutoresHila Romi, Idan Cohen, Daniella Landau, Suliman Alkrinawi, Baruch Yerushalmi, Reli Hershkovitz, Nitza Newman-Heiman, Garry R. Cutting, Rivka Ofir, Sara Sivan, Ohad S. Birk,
Tópico(s)Neonatal Respiratory Health Research
ResumoMeconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage. Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage. Meconium ileus (MI), intestinal obstruction by inspissated meconium in the distal ileum and cecum, develops in utero and presents shortly after birth as failure to pass meconium.1Eggermont E. Gastrointestinal manifestations in cystic fibrosis.Eur. J. Gastroenterol. Hepatol. 1996; 8: 731-738PubMed Google Scholar Some 80% of MI cases are caused by cystic fibrosis transmembrane conductance regulator (CFTR [MIM 602421]) mutations.2Rosenstein B.J. Langbaum T.S. Incidence of meconium abnormalities in newborn infants with cystic fibrosis.Am. J. Dis. 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Chai-Am E. Zirkin H. Bar-Ziv J. Freud E. Familial meconium ileus with normal sweat electrolytes.Clin. Pediatr. (Phila.). 1985; 24: 460-462Crossref PubMed Scopus (8) Google Scholar Intestinal biopsy done in three of the affected individuals demonstrated normal ganglions and cholinergic neurons. On the basis of the extended pedigree (Figure 1A , family 1), we assumed a founder effect. Following Soroka Medical Center institutional review board approval and informed consent, DNA samples of 11 affected and 26 nonaffected individuals from the kindred were obtained. Homozygosity of affected individuals at the CFTR locus was ruled out via polymorphic markers D7S2460, D7S677, and D7S655 within CFTR as previously described25Birnbaum R.Y. Zvulunov A. Hallel-Halevy D. Cagnano E. Finer G. Ofir R. Geiger D. Silberstein E. Feferman Y. Birk O.S. Seborrhea-like dermatitis with psoriasiform elements caused by a mutation in ZNF750, encoding a putative C2H2 zinc finger protein.Nat. 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Lethal Congenital Contractural Syndrome Type 2 (LCCS2) is Caused by a Mutation in ERBB3 (Her3), a Modulator of the PI3K/Akt Pathway.Am. J. Hum. Genet. 2007; 81: 589-595Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar and identified a single locus of homozygosity on chromosome 12p13 (spanning 9.5 Mb between markers D12S366 and D12S310) that was common to all affected individuals. Fine mapping of the locus was done with polymorphic markers as previously described26Narkis G. Ofir R. Manor E. Landau D. Elbedour K. Birk O.S. Lethal Congenital Contractural Syndrome Type 2 (LCCS2) is Caused by a Mutation in ERBB3 (Her3), a Modulator of the PI3K/Akt Pathway.Am. J. Hum. Genet. 2007; 81: 589-595Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar and narrowed down the locus to 4 Mb between markers D12391 and Ch12_TG (Figure 1B). Maximal two-point LOD score (SUPERLINK)27Fishelson M. Geiger D. Optimizing exact genetic linkage computations.J. Comput. Biol. 2004; 11: 263-275Crossref PubMed Scopus (73) Google Scholar was 4.1 (theta = 0) at D12S1580. Of the 40 genes within that locus, our S2G software28Gefen A. Cohen R. Birk O.S. Syndrome to gene (S2G): In-silico identification of candidate genes for human diseases.Hum. Mutat. 2010; 31: 229-236Crossref PubMed Scopus (20) Google Scholar, 29Cohen R. Gefen A. Elhadad M. Birk O.S. CSI-OMIM—Clinical Synopsis Search in OMIM.BMC Bioinformatics. 2011; 12: 65-67Crossref PubMed Scopus (10) Google Scholar identified GUCY2C (MIM 601330) as the primary candidate gene. Sequencing of the entire coding sequence and exon-intron boundaries of GUCY2C (NM_004963.3) identified a single homozygous mutation, c.1160A>G, leading to p.(Asp387Gly) amino acid substitution in the encoded protein. The mutation was not found in any SNP or mutation database and was common to all affected individuals (Figure 2). At the position of the c.1160A>G mutation, BanI restriction analysis gave differential cleavage products for the wild-type and mutant sequences (207 bp in the wild-type versus 134 bp and 73 bp fragments in the mutant; PCR amplification primers: forward 5′-TCCAACTTATCTGTCAGGCAAA-3′; reverse 5′-GTTACCCCTCCTCACCCAGT-3′). This differential BanI restriction analysis was used to test the 37 DNA samples in family 1 as well as controls. Of the 24 nonaffected individuals in the kindred, three offspring of a consanguineous marriage and one obligatory carrier were homozygous for the mutation. However, partial penetrance of the phenotype was evident: one of the three that had been examined by ultrasonography late in pregnancy had unequivocal intrauterine sonographic evidence of meconium ileus, yet did pass stools unassisted after birth. Of 240 unrelated Bedouin controls, three3 were found to be heterozygous for the mutation and none were homozygous. The penetrance of the postnatal MI phenotype in c.1160A>G homozygous individuals was 73% (11 affected/15 mutants).Figure 2The c.1160A>G and c.2270dupA GUCY2C Mutations: Homozygosity for the Wild-Type AlleleShow full caption(A–F) Heterozygosity for the wild-type (A and D) and the mutated alleles (B and E) and homozygosity for the mutated allele (C and F) in normal obligatory carriers and affected individuals of families 1 (A, B, and C) and 2 (D, E, and F). (G) The p.Asp387Gly amino acid substitution: multiple alignment and conservation analysis (ClustalW) of human GUCY2C with its orthologs. (the asterisk [∗] represents the mutated amino acid).(H) The p.Asn757Lysfs∗2 mutation: deleting the functional domain of the enzyme (SMART).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A–F) Heterozygosity for the wild-type (A and D) and the mutated alleles (B and E) and homozygosity for the mutated allele (C and F) in normal obligatory carriers and affected individuals of families 1 (A, B, and C) and 2 (D, E, and F). (G) The p.Asp387Gly amino acid substitution: multiple alignment and conservation analysis (ClustalW) of human GUCY2C with its orthologs. (the asterisk [∗] represents the mutated amino acid). (H) The p.Asn757Lysfs∗2 mutation: deleting the functional domain of the enzyme (SMART). GUCY2C encodes guanylyl cyclase 2C, a regulator of ion and fluid balance in the intestine and harbors an N-terminal extracellular ligand-binding domain, a single transmembrane domain, and a C-terminal intracytoplasmatic guanylyl cyclase domain.30Vaandrager A.B. 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Res. 2005; 65: 261-271Crossref PubMed Scopus (17) Google Scholar In fact, in the porcine GUCY2C ortholog, substitution by alanine residues of Asp387 together with two amino acid residues surrounding it, leads to a significant reduction in ligand-binding and to a reduction in guanylate cyclase activity.30Vaandrager A.B. Structure and function of the heat-stable enterotoxin receptor/guanylyl cyclase C.Mol. Cell. Biochem. 2002; 230: 73-83Crossref PubMed Scopus (112) Google Scholar, 49Hasegawa M. Shimonishi Y. Recognition and signal transduction mechanism of Escherichia coli heat-stable enterotoxin and its receptor, guanylate cyclase C.J. Pept. Res. 2005; 65: 261-271Crossref PubMed Scopus (17) Google Scholar To prove experimentally the effect of the p.(Asp387Gly) substitution on the guanylate cyclase activity of the GUCY2C protein, we transfected HEK293 cells (which normally do not express GUCY2C) with either the wild-type or the mutant GUCY2C. To that end, we first cloned the full-length wild-type 3.4 Kb human GUCY2C cDNA into the mammalian expression vector pcDNA3 (Invitrogen, Life Technologies), generating a construct we termed pcDNAhGCC. Site-directed mutagenesis was performed on the cloned wild-type GUCY2C and generated the mutant gene by overlapping PCR amplification: first, two PCR products, including the mutation site, were amplified with primers designated with the mutation site (one of the primers of each pair). Then, we further used these two PCR products as a template to amplify the whole fragment flanking the mutation region by using the other primers of each pair (primer sequences available upon request). This fragment was subcloned into the pGEM-T easy vector and confirmed by sequencing. The digestion of the resulting construct with EcoRV and BamHI yielded a fragment that contained the mutation region and was inserted in-frame into the corresponding sites in the pcDNAhGCC plasmid. This insertion generated the pcDNAhGCCA1207G clone containing the full length of the mutated GUCY2C (GUCY2CA1207G) cDNA. The final plasmid full sequences were verified by sequencing (data not shown). HEK293 cells were then stably transfected with constructs encoding either the wild-type or the mutant GUCY2C. Transfection was done with TransIT-LT1 reagent (Mirus) per kit instructions, verified by immunoblot analysis as previously described,25Birnbaum R.Y. Zvulunov A. Hallel-Halevy D. Cagnano E. Finer G. Ofir R. Geiger D. Silberstein E. Feferman Y. Birk O.S. Seborrhea-like dermatitis with psoriasiform elements caused by a mutation in ZNF750, encoding a putative C2H2 zinc finger protein.Nat. Genet. 2006; 38: 749-751Crossref PubMed Scopus (55) Google Scholar and quantified with a densitometer (Multigage software, Fujifilm). FACS analysis of the HEK293:hGCC and HEK293:hGCCD387G cell lines with the GUCY2C monoclonal antibody and second FITC antibody demonstrated that both the wild-type and the mutated GUCY2C were expressed on the plasma membrane (data not shown). Thus, we concluded that the mutation does not prevent expression of the ligand-stimulated form of the receptor on the cell surface. We proceeded to test the effect of the p.(Asp387Gly) substitution on guanylate cyclase catalytic activity. We selected three pairs of stable transfected cell lines that express equivalent levels of wild-type versus mutant proteins as demonstrated by immunoblot analysis (Figure 3) and verified quantitatively. Each of the three pairs of cell lines was analyzed (in triplicates) for guanylate cyclase catalytic activity, measuring the formation of intracellular cGMP levels in response to extracellular STa as previously described.34Schulz S. Green C.K. Yuen P.S. Garbers D.L. Guanylyl cyclase is a heat-stable enterotoxin receptor.Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (520) Google Scholar In essence, HEK293 cells were cultured in 24-well plates. After 24 hr, the culture medium was removed and the cells were incubated in 500 μl serum-free medium containing 100 μM 3-isobutylmethyl-1-xanthine (IBMX) (Sigma) for 10 min at 37°C in a 5% CO2 humidified incubator. Subsequently, 0.1 μM E. coli STa (Sigma) or double-distilled acidified water (pH 2.3) (STa dilution buffer) was added to each well, and incubations were continued for 15 min at 37°C. The reaction was stopped by aspiration of the incubation media and lysis of the cells by the addition of 500 μl 5% ice-cold trichloracetic acid (TCA) for 30 min at 4°C. The resulting lysates were collected to glass tubes and extracted twice with five volumes of ice-cold water-saturated ether to remove TCA. Residual ether was removed by evaporation in a chemical hood for additional 2 hr. Samp
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