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Absence of motilin gene mutations in infantile hypertrophic pyloric stenosis

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Erythromycin treatment before 2 weeks of age has been shown to increase the risk of infantile hypertrophic pyloric stenosis (IHPS) up to 10 times. Erythromycin is a motilin agonist, a hormone that induces gastrointestinal contractions. The purpose of
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  Absence of motilin gene mutations in infantilehypertrophic pyloric stenosis Anna Svenningsson a,b , Kristina Lagerstedt a ,Mir Davood Omrani a , Agneta Nordenskjöld a,b, ⁎ a   Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76 Stockholm, Sweden  b  Division of Pediatric Surgery, Astrid Lindgren's Hospital, Karolinska University Hospital, 171 76 Stockholm, Sweden Key words: Erythromycin;Infantile hypertrophic pyloric stenosis;Motilin;V15A polymorphism AbstractBackground:  Erythromycin treatment before 2 weeks of age has been shown to increase the risk of infantile hypertrophic pyloric stenosis (IHPS) up to 10 times. Erythromycin is a motilin agonist, ahormone that induces gastrointestinal contractions. The purpose of this study was to investigate if mutations in the motilin gene (  MLN  ) cause IHPS or if the V15A polymorphism in  MLN   is associatedwith the disease. Methods:  The  MLN   was screened for mutations, and the V15A polymorphism was determined in a totalof 57 patients with IHPS using polymerase chain reaction and DNA sequencing. The polymorphismgenotype and allele frequencies among the patients were compared with 184 controls. Results:  We detected 3 different, not previously reported,  MLN   sequence variants in 4 patients. One of these variants results in an amino acid exchange in the motilin signal peptide (A25G). All 3 detectedsequence variants were also found in controls or were not inherited with the disease. We found nosignificant association between the V15A polymorphism and the disease. Conclusions:  We have excluded the  MLN   coding region as a major cause of IHPS. Future studies willevaluate the importance of this metabolic pathway in the pathogenesis of IHPS.© 2008 Elsevier Inc. All rights reserved. 1. Background The etiology of infantile hypertrophic pyloric stenosis(IHPS) is still unknown, but a genetic contribution to thedisease is well established. A familial association is known inabout 13% to 15% of cases [1,2], and there are a few reportsof large families demonstrating an autosomal dominant modeof inheritance [3-5]. However, the inheritance pattern cannot  be explained by Mendelian genetics. Instead, IHPS isregarded as a complex disease, where a combination of  both genetic and environmental factors is of importance for development of the disease [6-8].During a pertussis epidemic at a US community hospital,a sudden increased incidence of IHPS was detected amonginfants born at the hospital at that time and who had beentreated prophylactically with erythromycin [9]. Additionalstudies have shown that erythromycin treatment before2 weeks of age increased the risk for IHPS up to 10 times[10,11]. Erythromycin acts as a motilin agonist competitively ⁎  Corresponding author. CMM L8:02, Karolinska UniversityHospital, 171 76 Stockholm, Sweden. Tel.: +46 8 517 764 08;fax: +46 8 517 736 20.  E-mail address:  agneta.nordenskjold@ki.se (A. Nordenskjöld).www.elsevier.com/locate/jpedsurg0022-3468/$  –  see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.jpedsurg.2007.10.006Journal of Pediatric Surgery (2008)  43 , 443 – 446   binding to the motilin receptor in the gastrointestinal tract where it induces motor activity in a dose-dependent manner [12,13]. Low doses of erythromycin induce intense contrac-tions migrating through the intestine, so-called migratingmotor complex (MMC). Higher doses, as used for antibiotic purposes, induce strong antral contractions in the stomachthat do not migrate to the duodenum [14-16]. Well-knownadverse effects of erythromycin treatment are abdominal painand nausea, probably because of the abnormal motility pattern induced by high doses of erythromycin [14].Motilin is a 22-amino acid polypeptide expressed inenteroendocrine cells in the upper part of the small intestine[17]. It is considered as a key factor in the regulation andinitiation of MMCs, the purpose of which is to propelundigested food in the forward direction down the gastro-intestinal tract. Plasma levels of motilin undergo cyclicfluctuations during fasting with peak levels correlating withthe onset of MMCs in the gastric antrum [18]. Exogenousmotilin infusion is shown to induce premature MMC activity[19]. Release of motilin is stimulated by gastrin-releasing peptide and inhibited by pancreatic polypeptide, somatosta-tin, and secretin [20].Motilin is synthesized as part of a 115-amino acid prohormone consisting of a 25-amino acid signal peptide, the22-amino acid motilin sequence, and a 66-amino acidcarboxy-terminal motilin-associated peptide separated fromthe motilin sequence by a lysine-lysine dipeptide (Fig. 1).The motilin hormone is released after proteolytic cleavage of the prohormone [20,21]. The motilin gene (  MLN  ) is locatedon chromosome 6p21.2-p21.3 and consists of 5 exons, of which the first is noncoding. The motilin sequence isencoded by exons 2 and 3; the signal peptide by exon 2; andthe motilin-associated peptide encoded by exons 3, 4, and 5[20,22]. The motilin gene exons contain 2 reported singlenucleotide polymorphisms located in the noncoding exon 1(rs2281818) and the coding exon 2 (rs2281820). The polymorphism in exon 2 gives the variation of C to T at  position 44, [c.44C N T], exchanging a valine to alanine in thesignal peptide (V15A). No mutations have so far beenreported in the human  MLN  .The purpose of this study was to investigate if mutationsin the coding region of   MLN   cause IHPS or whether there isan association between the V15A polymorphism in  MLN  and the disease. 2. Material Fifty-seven IHPS patients treated with pyloromyotomy at the Pediatric Surgery Departments in Stockholm (Sweden)and Gothenburg (Sweden) were included in the study.Thirty-five (33 males, 2 females) of these were sporadiccases, and 22 (16 males, 6 females) had a family history of at least one more affected relative. Pedigrees are available onrequest. One hundred eighty-four Swedish blood donors(95 males, 89 females) served as a control group. GenomicDNA from both patients and controls was extracted from blood using standard protocols. The study was approved bythe regional ethics committee of the Karolinska Institutet (Stockholm, Sweden). 3. Methods The 5 exons were amplified with polymerase chainreaction (PCR); primer sequences are shown in Table 1.The PCRs were performed in a total volume of 50  μ Lcontaining 1× PCR Buffer II (Applied Biosystems, Foster City, Calif), 1.5 mmol/L MgCl 2  (Applied Biosystems), 0.1mmol/L dNTP, 2.5 U AmpliTaq Gold (Applied Biosys-tems), 0.1  μ mol/L of each primer (ordered from ThermoHybaid Ashford, Middlesex, UK), and approximately Fig. 1  I, Structure of the motilin gene, location of the V15A polymorphism, and its relation to the prohormone on messenger RNA level. II,Localization of detected heterozygote sequence variants. The [c.74C N G] variant in exon 2 substitutes alanine to glycine (A25G) at position 25in the signal peptide. 444 A. Svenningsson et al.  100 ng of genomic DNA. The PCRs were cycled in a 15-minute 96°C initiation step followed by 35 cycles of 30-second 96°C denaturation, 30-second 56°C annealing, and55-second 72°C elongation before a final 2-minute 72°Cextension step. The PCR products were purified beforesequencing using QIAquick PCR purification kit (QIA-gene, VWR International AB, Stockholm, Sweden). DNAsequence reactions were performed using the ABI PRISMBigDye Terminator v1.1 Cycle Sequencing kit (AppliedBiosystems) according to the manufacturer's recommenda-tions and analyzed on ABI 310 and ABI 3110 automatedsequencers (Applied Biosystems). 4. Results We detected one heterozygote missense sequence variant in exon 2, [c.74C N G], in a familial case of IHPS. This variant results in an exchange of the signal peptide's last amino acidalanine to glycine (A25G) and is to our knowledge not  previously described. One of the probands nonaffected sonshad inherited the [c.74C N G] variant, but the affected son hadnot (Fig. 2). All 3 sons in this family were heterozygote for another sequence variant in the noncoding exon 1, [c.-66C N T], inherited from the nonaffected mother. A hetero-zygote sequence variant in the noncoding part of exon 5,[c.489C N T], was found in 2 sporadic cases. Screening thecontrols for these variants revealed that 1 of 184 washeterozygote for the exon 2 [c.74C N G] variant, 3 of 184 wereheterozygote for the exon 1 [c.-66C N T] sequence variant,and 15 of 184 heterozygote for the exon 5 [c.489C N T]sequence variant.To evaluate a possible association between the V15A polymorphism and the disease, we compared the 44C allelefrequency and genotype distribution at position 44 between patients and controls. Allele frequency for 44C was 64.9% in patients with IHPS compared with 60.9% in controls, whichis not a significant difference ( χ 2 test,  P   N  .05). Thedistribution of genotypes among patients with IHPS was not significantly different from that among controls. Dividing theIHPS group into familial and sporadic cases revealed ahigher frequency of the C allele among the familial casescompared to sporadic (70.5% and 61.4%, respectively), but this difference was not significant (Table 2). 5. Discussion The  MLN   is a candidate gene for IHPS because treatment with the motilin agonist erythromycin gives an increased risk for IHPS. In this study, mutation screening of the motilingene was performed in 57 patients with IHPS. Threedifferent, not previously reported, heterozygote sequencevariants were found in 4 patients. These variants were either also found in controls or not inherited with disease in afamilial case. In addition, we found no significant association between allele frequency of the V15A polymorphism and thedisease. These results do not support the hypothesis that the  MLN   is involved in the etiology of IHPS, but its relevance onthe other hand cannot be excluded because of the complexityof inheritance of complex disorders. Also, we have not excluded a heterozygote deletion of the  MLN   or looked for  putative mutations that could alter transcription or stability of the  MLN   transcript. Other factors that may be involved areother genes in the motilin metabolic pathway as well as themotilin receptor. In addition, one can speculate about anepigenetic mechanism that might explain the sex differencesin inheritance pattern because the risk to develop IHPS for asonof a womanwith IHPS is 19%, and therisk for a daughter of a man with IHPS is only 2% [6]. Interestingly, a recent report stated that the motilin concentration in blood isincreased in lactating women, and also that breast milk  Fig. 2  Inheritance of detected  MLN   sequence variants ina family. Table2  Genotype distribution for the V15A polymorphism in patients with IHPS and controlsCC (n [%]) CT (n [%]) TT (n [%])IHPS group (N = 57) 26 (45.6) 22 (38.6) 9 (15.8)IHPS familialcases (N = 22)11 (50.0) 9 (40.9) 2 (9.1)IHPS sporadiccases (N = 35)15 (42.9) 13 (37.1) 7 (20.0)Control group(N = 184)65 (35.3) 94 (51.1) 25 (13.6) Table 1  Primer sequencesExon 1 fwd 5 ′ -TCAAAGGTTAATGGGCTCCAA-3 ′ Exon 1 rev 5 ′ -TTCTGCTTTAGGGCTTATGCT-3 ′ Exon 2 fwd 5 ′ -GTACCCCAAACTTAGTGACAA-3 ′ Exon 2 rev 5 ′ -GTCCTGGTCCTTTACACTTTC-3 ′ Exon 3 fwd 5 ′ -GGGTTGAGGGAGGGCGGTTTT-3 ′ Exon 3 rev 5 ′ -ATCCTAGGACACTCTGAGGCT-3 ′ Exon 4 fwd 5 ′ -CTCTCCACCTCTCCCTCGTTG-3 ′ Exon 4 rev 5 ′ -AGCAGGTATCACAGCATAGCC-3 ′ Exon 5 fwd 5 ′ -GATGGTACTGAGCGCAAACCT-3 ′ Exon 5 rev 5 ′ -TGGCCAACTTTCTCTGAGCTT-3 ′ fwd indicates forward; rev, reverse. 445Absence of motilin gene mutations in IHPS  contains motilin [23]. This might be important from a physiologic view because the maturation of the neonatalgastrointestinal tract may be affected by motilin. Further studies with comparison of breast milk motilin levels between mothers to children with IHPS and controls would be interesting. Acknowledgments We thank the patients for their cooperation. This studywas supported by grants from the Swedish Research Council,HRH Crown Princess Lovisa Foundation, Magnus BergvallFoundation, Marcus Borgström Foundation, KarolinskaInstitutet, and Foundation Frimurare Barnhuset. A. Sven-ningsson is a recipient of scholarships from FoundationSamariten, Foundation Frimurare Barnhuset, and SwedishSociety of Medicine.We would like to express our gratitude to Sofia Perssonfor DNA sampling and to Cat Halthur, Cecilia Fagerblad,and Fredrik Lundberg for excellent technical assistance inDNA sequencing. References [1] Jedd MB, Melton III JI, Griffin MR, et al. Factors associated withinfantile hypertrophic pyloric stenosis. Am J Dis Child 1988;142:334-7.[2] Stang H. Pyloric stenosis associated with erythromycin ingestedthrough breastmilk. Minn Med 1986;69:669-70, 682.[3] Fried K, Aviv S, Nisenbaum C. Probable autosomal dominant infantile pyloric stenosis in a large kindred. Clin Genet 1981;20(5):328-30.[4] Soderhall C, Nordenskjold A. Neuronal nitric oxide synthase, nNOS,is not linked to infantile hypertrophic pyloric stenosis in three families.Clin Genet 1998;53(5):421-2.[5] Capon F, Reece A, Ravindrarajah R, et al. Linkage of monogenicinfantile hypertrophic pyloric stenosis to chromosome 16p12-p13 andevidence for genetic heterogeneity. Am J Hum Genet 2006;79(2):378-82.[6] Carter CO, Evans KA. Inheritance of congenital pyloric stenosis.J Med Genet 1969;6:233-54.[7] Mitchell LE, Risch N. The genetics of infantile hypertrophic pyloricstenosis: a reanalysis. Am J Dis Child 1993;147:1203-11.[8] Chakraborty R. The inheritance of pyloric stenosis explained by amultifactorial threshold model with sex dimorphism for liability. Genet Epidemiol 1986;3:1-15.[9] Honein MA, Paulozzi LJ, Himelright IM, et al. Infantile hypertrophic pyloric stenosis after pertussis prophylaxis with erythromycin: a casereview and cohort study. Lancet 1999;354:2101-5.[10] Cooper WO, Griffin MR, Arbogast P, et al. Very early exposure toerythromycin and infantile hypertrophic pyloric stenosis. Arch Pediatr Adolesc Med 2002;156:647-50.[11] Mahon BE, Rosenman MB, Kleiman MB. Maternal and infant use of erythromycin and other macrolide antibiotics as risk factors for infantile hypertrophic pyloric stenosis. J Pediatr 2001;139(3):380-4.[12] Peeters T, Matthijs G, Depoortere I, et al. Erythromycin is a motilinreceptor agonist. Am J Physiol 1989;257:G470-4.[13] Jadcherla S, Klee G, Berseth C. Regulation of migrating motor complexes by motilin and pancreatic polypeptide in human infants.Pediatr Res 1997;42:365-9.[14] Sarna SK, Soergel KH, Koch TR, et al. Gastrointestinal motor effectsof erythromycin in human. Gastroenterology 1991;101:1488-96.[15] Tack J, Janssens J, Vantrappen G, et al. Effect of erythromycin ongastric motility in controls and diabetic gastroparesis. Gastroenterol-ogy 1992;103:72-9.[16] Kawamura O, Sekiguchi T, Kusano M, et al. Effect of erythromycin oninterdigestive gastrointestinal contractile activity and plasma concen-tration in humans. Dig Dis Sci 1993;38:870-6.[17] Pearse A, Polak J, Bloom S, et al. Enterochromaffin cells of themammalian small intestine as the source of motilin. Virchows Arch BCell Pathol 1974;16:111-20.[18] Bormans V, Peeters TL, Janssens J, et al. In man, only activity frontsthat srcinate in the stomach correlate with motilin peaks. Scand JGastroenterol 1987;22:781-4.[19] Vantrappen G, Janssens J, Peeters TL, et al. Motilin and theinterdigestive migrating motor complex in man. Dig Dis Sci 1979;24:497-500.[20] Daikh DI, Douglass JO, Adelman JP. Structure and expression of thehuman motilin gene. DNA 1989;8(8):615-21.[21] Seino Y, Tanaka K, Takeda J, et al. Sequence of an intestinal cDNAencoding human motilin precursor. FEBS Lett 1987;223:74-6.[22] Yano H, Seino Y, Fujita J, et al. Exon-intron organization, expression,and chromosomal localization of the human motilin gene. FEBS Lett 1989;249:248-52.[23] Liu J, Qiao X, Qian W, et al. Motilin in human milk and its elevated plasma concentration in lactating women. J Gastroenterol Hepatol2004;19(10):1187-91. 446 A. Svenningsson et al.
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