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Meconium Ileus Caused by Mutations in GUCY2C, Encoding the CFTR-Activating Guanylate 2C

Hila Romi,1,6 Idan Cohen,1,6 Daniella Landau,2 Suliman Alkrinawi,2 Baruch Yerushalmi,2 Reli Hershkovitz,3 Nitza Newman-Heiman,2 Garry R. Cutting,4 Rivka Ofir,1 Sara Sivan,1 and Ohad S. Birk1,5,*

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modi- fier . 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 whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide , or by an external ligand, (E. coli) heat-stable 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 , it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.

Meconium ileus (MI), intestinal obstruction by inspissated homozygosity on 12p13 (spanning 9.5 Mb meconium in the distal ileum and cecum, develops in between markers D12S366 and D12S310) that was utero and presents shortly after birth as failure to pass common to all affected individuals. Fine mapping of the meconium.1 Some 80% of MI cases are caused by cystic locus was done with polymorphic markers as previously fibrosis transmembrane conductance regulator (CFTR described26 and narrowed down the locus to 4 Mb between [MIM 602421]) mutations.2,3 In fact, 15%–20% of infants markers D12391 and Ch12_TG (Figure 1B). Maximal two- with cystic fibrosis (CF [MIM 219700]) develop MI as point LOD score (SUPERLINK)27 was 4.1 (theta ¼ 0) at a presenting symptom.1 The predisposition to MI in CF is D12S1580. Of the 40 genes within that locus, our S2G soft- genetically determined;4–8 some CFTR mutations lead to ware28,29 identified GUCY2C (MIM 601330) as the primary MI at a higher incidence than others.6,9–17 This predisposi- candidate . Sequencing of the entire coding sequence tion is modified by yet other, unidentified genes, residing and exon-intron boundaries of GUCY2C (NM_004963.3) in several genomic loci.16–22 Non-CF MI can be caused by identified a single homozygous mutation, c.1160A>G, an array of etiologies, from defects in intestinal innerva- leading to p.(Asp387Gly) amino acid substitution in the tion to pancreatic insufficiency and meconium plug encoded protein. The mutation was not found in any syndrome, to various anorectal malformations.23 SNP or mutation database and was common to all affected We have previously described inbred consanguineous individuals (Figure 2). At the position of the c.1160A>G Bedouin kindred with autosomal-recessive MI, a normal mutation, BanI restriction analysis gave differential sweat test, and no further clinical stigmata of CF.24 Intes- cleavage products for the wild-type and mutant sequences tinal biopsy done in three of the affected individuals (207 bp in the wild-type versus 134 bp and 73 bp fragments demonstrated normal ganglions and cholinergic neurons. in the mutant; PCR amplification primers: forward 50- On the basis of the extended pedigree (Figure 1A, family 1), TCCAACTTATCTGTCAGGCAAA-30; reverse 50-GTTACC we assumed a founder effect. Following Soroka Medical CCTCCTCACCCAGT-30). This differential BanI restriction Center institutional review board approval and informed analysis was used to test the 37 DNA samples in family 1 consent, DNA samples of 11 affected and 26 nonaffected as well as controls. Of the 24 nonaffected individuals in individuals from the kindred were obtained. Homozy- the kindred, three offspring of a consanguineous marriage gosity of affected individuals at the CFTR locus was ruled and one obligatory carrier were homozygous for the muta- out via polymorphic markers D7S2460, D7S677, and tion. However, partial penetrance of the phenotype was D7S655 within CFTR as previously described25,26 (data evident: one of the three that had been examined by ultra- not shown). Genome-wide linkage analysis (ABI PRISM sonography late in pregnancy had unequivocal intra- Linkage Mapping Set, Applied Biosystems) was done as uterine sonographic evidence of meconium ileus, yet did previously described,25,26 and identified a single locus of pass stools unassisted after birth. Of 240 unrelated Bedouin

1The Morris Kahn Laboratory of Human Genetics, National Institute for Biotechnology in the Negev, Ben Gurion University, Beer-Sheva 84105, Israel; 2Division of Pediatrics, Soroka Medical Center, Beer-Sheva 84101, Israel; 3Division of obstetrics and gynecology, Soroka Medical Center, Beer-Sheva 84101, Israel; 4Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore MD 21287, USA; 5Genetics Institute, Soroka Medical Center, Beer-Sheva 84101, Israel 6These authors contributed equally *Correspondence: [email protected] DOI 10.1016/j.ajhg.2012.03.022. Ó2012 by The American Society of Human Genetics. All rights reserved.

The American Journal of Human Genetics 90, 893–899, May 4, 2012 893 Figure 1. Family Trees and Linkage Analysis Results (A) The Israeli-Bedouin Kindred affected with non-CF MI; family 1 and family 2 are not related. (B) Linkage analysis results (chromosome 12p13, partial pedigree of family 1): the haplotype showing the homozygosity region is boxed. Physical locations of the markers are shown. Individuals VII:8, VII:9 defined the upper boundary of the locus and individual VII:23 defined the lower boundary. For tested individuals, the c.1160A>G mutation status of each allele is marked as wild-type (WT) or mutant (M). controls, three3 were found to be heterozygous for the matic guanylyl cyclase domain.30,31 The extracellular mutation and none were homozygous. The penetrance of domain of GUCY2C is targeted by either the endogenous the postnatal MI phenotype in c.1160A>G homozygous ligands, guanylin and related peptide uroguanylin, or individuals was 73% (11 affected/15 mutants). by an external ligand, E. coli heat-stable enterotoxin GUCY2C encodes guanylyl cyclase 2C, a regulator of STa.30,32–35 Binding of the physiological ligands or of STa ion and fluid balance in the intestine and harbors an to the extracellular domain of GUCY2C activates the intra- N-terminal extracellular ligand-binding domain, a single cellular cyclase domain, which catalyzes the synthesis of transmembrane domain, and a C-terminal intracytoplas- the second messenger cyclic GMP (cGMP) from GTP.31–35

894 The American Journal of Human Genetics 90, 893–899, May 4, 2012 Figure 2. The c.1160A>G and c.2270dupA GUCY2C Mutations: Homozy- gosity for the Wild-Type Allele (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 align- ment 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 (SMART).

GUCY2C is predominantly local- ized at the apical brush border mem- brane of the intestinal : specific STa-binding sites are present in apical membranes of intestinal epi- thelium from the duodenum to the distal colon.36,37 The highest density of GUCY2C receptor molecules is found in the proximal small intestine and decreases progressively distally.36 The endogenous peptides guanylin and uroguanylin have a lower affinity for GUCY2C than do the STa peptides and are produced within the in- testinal mucosa to serve as paracrine and autocrine regulators of intestinal fluid and electrolyte secretion.38 The toxic E. coli STa peptide is a major pathogen in humans that causes acute and secretory diarrhea in in- fants, travelers, and domestic animals and has a geographic distribution pri- marily in developing countries:39,40 when targeted by exogenous STa, GUCY2C is overactivated and intra- cellular cGMP is induced, resulting in imbalance in the secretion of fluids and chloride from intestinal cells and culminating in severe secretory diar- rhea in both humans and mice.31,41–43 Protein levels of GUCY2C receptors are highest in newborns and neonates (in which STa-mediated diarrhea is Elevated levels of cGMP in response to ligand binding more frequent and more severe)44, and their affinity and cross-activate cGMP-dependent G II density rapidly decrease with increasing age in humans,45 (PKGII); this activation leads to phosphorylation and as well as in mice,46 rats,47 and pigs.48 subsequent opening of the CFTR.31 This signaling cascade The p.(Asp387Gly) substitution (Figure 3G) found in the leads to chloride and bicarbonate secretion, ultimately affected individuals is in a conserved aspartate residue of driving the paracellular movement of sodium into the GUCY2C that is within one of the two essential regions of intestinal lumen and regulating intestinal ion and water its extracellular ligand-binding domain (Figure 3H) and is transport.31 immediately adjacent to seven other amino acids that are

The American Journal of Human Genetics 90, 893–899, May 4, 2012 895 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 instruc- tions, verified by immunoblot analysis as previously described,25 and quantified with a densitometer (Multigage software, Fujifilm). FACS analysis of the HEK293:hGCC and

HEK293:hGCCD387G lines with the GUCY2C mono- clonal 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) Figure 3. Functional Analysis of the c.1160A>G Mutation that substitution on catalytic activity. We Produces p.Asp387Gly selected three pairs of stable transfected cell lines that (A and B) Immunoblot analysis showing equal amounts of wild- express equivalent levels of wild-type versus mutant pro- type (GCC) and mutant (GCCD387G) protein in stable transfected teins as demonstrated by immunoblot analysis (Figure 3) HEK293 cell lines used for testing GUCY2C activity. (C) GUCY2C activity in the presence (þSta) or absence (no Sta) of and verified quantitatively. Each of the three pairs of cell E. coli heat-stable enterotoxin STa, tested in HEK293 cells stably lines was analyzed (in triplicates) for guanylate cyclase cata- transfected with either empty pcDNA vector (negative control) lytic activity, measuring the formation of intracellular GUCY2C or wild-type (GCC) or mutant (GCCD387G) . Each experi- 5 cGMP levels in response to extracellular STa as previously ment represents mean standard deviation of three repeats. 34 Similar results obtained for three different pairs of cell lines stably described. In essence, HEK293 cells were cultured in overexpressing equal amounts of mutant and wild-type GUCY2C. 24-well plates. After 24 hr, the culture medium was removed and the cells were incubated in 500 ml serum-free medium containing 100 mM 3-isobutylmethyl-1-xanthine (IBMX) 49 cardinal to ligand binding. In fact, in the porcine (Sigma) for 10 min at 37 C in a 5% CO2 humidified incu- GUCY2C ortholog, substitution by alanine residues of bator. Subsequently, 0.1 mM E. coli STa (Sigma) or double- Asp387 together with two amino acid residues surrounding distilled acidified water (pH 2.3) (STa dilution buffer) was it, leads to a significant reduction in ligand-binding and to added to each well, and incubations were continued for a reduction in guanylate cyclase activity.30,49 15 min at 37C. The reaction was stopped by aspiration of To prove experimentally the effect of the p.(Asp387Gly) the incubation media and lysis of the cells by the addition substitution on the guanylate cyclase activity of the of 500 ml 5% ice-cold trichloracetic acid (TCA) for 30 min GUCY2C protein, we transfected HEK293 cells (which at 4C. The resulting lysates were collected to glass tubes normally do not express GUCY2C) with either the wild- and extracted twice with five volumes of ice-cold water- type or the mutant GUCY2C. To that end, we first cloned saturated ether to remove TCA. Residual ether was removed the full-length wild-type 3.4 Kb human GUCY2C cDNA by evaporation in a chemical hood for additional 2 hr. into the mammalian expression vector pcDNA3 (Invitro- Samples were then frozen at 20C overnight and lyophi- gen, Life Technologies), generating a construct we termed lized. After lyopilization, samples were resuspended in ace- pcDNAhGCC. Site-directed mutagenesis was performed tic acid and acetylated with a mixture of acetic anhydride on the cloned wild-type GUCY2C and generated the and triethylamine to increase the sensitivity of the assay. mutant gene by overlapping PCR amplification: first, two Cyclic GMP production was measured with a commercially PCR products, including the mutation site, were amplified available cGMP Enzyme Immunoassay kit (Biomedical with primers designated with the mutation site (one of the Technologies) according to the manufacturer’s specifica- primers of each pair). Then, we further used these two PCR tions. All samples were analyzed in triplicate. Significant products as a template to amplify the whole fragment activation of guanylate cyclase activity was seen in flanking the mutation region by using the other primers HEK293 cells harboring the wild-type construct (HEK293: of each pair (primer sequences available upon request). GCC) in the presence of STa (Figure 3), comparable to that This fragment was subcloned into the pGEM-T easy vector seen in previously published studies.34 STa-mediated acti- and confirmed by sequencing. The digestion of the result- vation of guanylate cyclase in cells transfected with the ing construct with EcoRV and BamHI yielded a fragment mutant construct (HEK293:GCCD387G) was ~60% lower that contained the mutation region and was inserted in- than that in cells harboring the wild-type construct frame into the corresponding sites in the pcDNAhGCC (Figure 3). plasmid. This insertion generated the pcDNAhGCCA1207G The association of GUCY2C with the MI phenotype clone containing the full length of the mutated GUCY2C was further substantiated by our finding of a different GUCY2C GUCY2C ( A1207G) cDNA. The final plasmid full sequences deleterious homozygous mutation in a severely were verified by sequencing (data not shown). HEK293 affected individual in another, unrelated kindred (Figure 1,

896 The American Journal of Human Genetics 90, 893–899, May 4, 2012 family 2): a single case who had severe non-CF MI and was any features of the CF phenotype other than MI. Interest- born to Bedouin parents that were first cousins. The MI ingly, SNP rs9300298 at the 12p13.3 locus has been phenotype in that individual was severe and required associated with the MI phenotype in CF.22 However, this surgery. The sweat test was normal, and homozygosity of SNP is located ~13 Mb from GUCY2C, and further detailed the affected individual at the CFTR locus was ruled out, association studies of this locus should be done to test whereas homozygosity at the GUCY2C locus was demon- whether GUCY2C is a likely modifier of the MI phenotype strated (different haplotype than in family 1, data not in CF. shown). Sequence analysis of GUCY2C identified a single Finally, we would like to note that mouse experiments homozygous mutation, c.2270dupA (Figure 2D–F), not suggest a likely evolutionary advantage of GUCY2C hetero- found in 240 Bedouin nonrelated controls or in the zygous mutant individuals of the desert-dwelling Bedouin two healthy siblings of the affected individual. This community: in line with the partial penetrance of the c.2270dupA insertion mutation results in a premature human phenotype we describe, null mutant mice lacking stop codon two amino acids following the insertion functional GUCY2C protein have been generated and (p.Asn757Lysfs*2) and fully abrogates the guanylate cyclase are with no evident clinical phenotype when unchal- catalytic domain (Figure 2H). It should be noted that both lenged.31,42 However, although wild-type mice challenged the c.1160A>G mutation and the c.2270dupA insertion with the E. coli interotoxin STa die of severe secretory / mutation were not reported to date in either the HapMap diarrhea, Gucy2c null mutant mice are protected from or the 1000 genomes databases. this detrimental effect. Moreover, even the heterozygous The fact that downregulation of GUCY2C leads to a mutant mice demonstrate lower diarrhea-related mortality CF-like intestinal phenotype is in line with previous data rates than the wild-type mice.31,42 As the human GUCY2C demonstrating that the secretory effect of guanylin is abol- mutations found are in Bedouin kindred whose habitat ished by CFTR blockers.50,51 Moreover, anti-CFTR anti- is the desert, one can speculate that heterozygosity of bodies have been shown to prevent a cGMP-induced GUCY2C mutations might have led to selective advantage increase in chloride secretion,52 and reduction of CFTR at times of infectious diarrhea. expression in colonic carcinoma cells with antisense oligo- nucleotides to CFTR mRNA diminished STa-induced chlo- Acknowledgments ride secretion. In fact, cells that do not express CFTR do not respond to cGMP until transfected with CFTR cDNA.41 This work was funded through a grant from the U.S. Cystic Fibrosis Further support for the interaction between GUCY2C Foundation. We thank the Morris Kahn Family Foundation for the and CFTR comes from studies of mutant mice: mice lack- kind support. Facilities used were donated in part by the Wolfson ing functional CFTR have virtually no response to an Foundation and the Wolfson Family Charitable Trust. G.R.C. was supported through National Institutes of Health grants NHLBI agonist elevating cGMP or cAMP concentrations,38 and þ HL068927 and NIDDK DK 044003. heterozygous CFTR / mice demonstrate reduction of 53 STa secretory effect by about 50%. Because both cGMP Received: January 30, 2012 and cAMP secretory responses are impaired, CFTR null Revised: February 29, 2012 mice, unlike GUCY2C null mice, suffer from severe Accepted: March 28, 2012 54 obstructions that result in high mortality. It should be Published online: April 19, 2012 noted, though, that no complementation studies were done with CFTR and GUCY2C mutant mice to prove their Web Resources interaction in vivo. Finally, in patients with CF treated with STa and cGMP analogs, the failure to induce chloride The URLs for data presented herein are as follows: and water secretion results in resistance to STa-induced 1000 Genomes, http://www.1000genomes.org/ 55 diarrhea. Further in-vivo evidence for the connection BLAST, http://www.ncbi.nlm.nih.gov/blast/ between GUCY2C and CFTR-associated effects comes ClustalW2–Multiple Sequence Alignment, http://www.ebi.ac.uk/ from studies of mice lacking functional PKGII, known to Tools/msa/clustalw2/ mediate the GUCY2C effect on CFTR:56 in PKGII / HapMap, http://hapmap.ncbi.nlm.nih.gov/ mice, the secretory response of the intestine to STa is mark- Online Mendelian Inheritance in Man (OMIM), http://omim.org/ edly reduced.57 As in GUCY2C-deficient mice, mice lack- Primer3, http://frodo.wi.mit.edu/primer3 ing functional PKGII are otherwise healthy, indicating SMART - Simple modular architecture research tool, http://smart. that other transduction pathways, such as cAMP-protein embl-heidelberg.de/ SUPERLINK, http://bioinfo.cs.technion.ac.il/superlink/ kinase A, might compensate for a cGMP-signaling deficit Tandem Repeats Finder, http://tandem.bu.edu/trf/trf.html in mice. UCSC Genome Browser, http://www.genome.ucsc.edu/ Although GUCY2C is highly expressed in the intestine, its expression levels in the exocrine pancreas and in the lungs are far lower,36,37 and CFTR activity in those other References 1 CF-related tissues is dependent on other activators. 1. Eggermont, E. (1996). Gastrointestinal manifestations in Thus, affected individuals in this study do not demonstrate cystic fibrosis. Eur. J. Gastroenterol. Hepatol. 8, 731–738.

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