RESEARCH LETTER

A Post GWAS Association Study of SNPs Associated With Cleft Lip With or Without Cleft Palate in Submucous Cleft Palate Rudolf Reiter,1 Sibylle Brosch,1 Ingrid Goebel,2,3 Kerstin U. Ludwig,4,5 Anja Pickhard,6 Josef Ho¨gel,2 Guido Schlo¨mer,7 Elisabeth Mangold,4 Christian Kubisch,2,3 and Guntram Borck2* 1

Section of Phoniatrics and Pedaudiology, Department of Otolaryngology - Head and Neck Surgery, University of Ulm, Ulm, Germany

2

Institute of Human Genetics, University of Ulm, Ulm, Germany

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Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Institute of Human Genetics, University of Bonn, Bonn, Germany

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Department of Genomics, Life and Brain Center, University of Bonn, Bonn, Germany

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Department of Otolaryngology - Head and Neck Surgery, Technical University Munich, Munich, Germany Department of Cranio-Maxillo-Facial Surgery, University of Ulm, Ulm, Germany

7

Manuscript Received: 14 July 2014; Manuscript Accepted: 30 October 2014

TO THE EDITOR:

How to Cite this Article:

Submucous cleft palate (SMCP) is a subgroup of orofacial clefts with insufficient median fusion of the muscles of the soft palate hidden under the mucosa. It has an estimated prevalence of 1:1,250–1:5,000 [Gosain et al., 1996]. The diagnosis of SMCP is often delayed although velopharyngeal insufficiency typically leads to open nasal speech and Eustachian tube dysfunction with conductive hearing loss [Reiter et al., 2011]. Orofacial clefts can be classified as cleft lip, cleft lip with or without cleft palate (CL/P) of the primary and secondary palate, and cleft palate only (CPO), which affects the secondary palate. Anatomically, SMCP is a subgroup of CPO, of which it may represent a minor form. However, whether CL/P on the one hand and CPO and SMCP on the other hand represent genetically related entities is largely unknown. Orofacial clefts including SMCP can occur as an isolated anomaly or as part of malformation syndromes [Leslie and Marazita, 2013]. The 22q11.2 deletion syndrome (22q11.2DS; OMIM#188400 and #192430) represents one of the most common examples of a syndrome in which SMCP is frequently seen. In a study of over 300 patients with 22q11.2DS, 77% had cleft palate and of these, 68% were a SMCP [Friedman et al., 2011]. In addition, in TP63-associated ankyloblepharonectodermal defects-cleft lip/palate syndrome (AEC syndrome; OMIM#106260), almost all patients have an orofacial cleft of which 17% were noted to have SMCP [Cole et al., 2009]. Notably, either SMCP or CL/P can be seen as part of several syndromes, such as 22q11.2DS, AEC syndrome, and IRF6 or GRHL3-associated van der Woude syndrome (OMIM#119300 and #606713, respectively) [Kondo et al., 2002; Peyrard-Janvid et al., 2014], suggesting that the same genetic cause may contribute to both types of clefting. The OMIM database of Mendelian disorders lists SMCP as a clinical

Reiter R, Brosch S, Goebel I, Ludwig KU, Pickhard A, Ho¨gel J, Schlo¨mer G, Mangold E, Kubisch C, Borck G. 2015. A Post GWAS association study of SNPs associated with cleft lip with or without cleft palate in submucous cleft palate. Am J Med Genet Part A 167A:670–673.

Ó 2015 Wiley Periodicals, Inc.

finding of approximately 40 distinct syndromes. Yet, in approximately 70% of cases, SMCP is an isolated finding. Nonsyndromic orofacial clefts are regarded as multifactorial disorders with diverse genetic and environmental factors contributing to their occurrence [Jugessur et al., 2009; Dixon et al., 2011; Reiter et al., 2014]. In contrast to CL/P, the genetic architecture of CPO and SMCP is poorly understood. Although previous candidate gene-based association studies have identified SMCP candidate loci [Reiter et al., 2012], no genome wide association study (GWAS) in SMCP has been performed to date. Conversely, several GWAS and meta-analyses have identified common variants associated with CL/P [Birnbaum et al., 2009; Beaty et al., 2010; Conflict of interest: none.  Correspondence to: Guntram Borck, Institute of Human Genetics, University of Ulm, AlbertEinstein-Allee 11, 89081 Ulm, Germany. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): DOI 10.1002/ajmg.a.36891

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671 We selected one SNP from each of six genomic loci with a strong and replicated association with CL/P in GWAS: rs560426 at 1p22.1, rs2013162 at 1q32.2, rs987525 at 8q24.21, rs7078160 at 10q25.3, rs227731 at 17q22, and rs13041247 at 20q12 [Birnbaum et al., 2009; Beaty et al., 2010; Mangold et al., 2010]. These six loci represent all loci with a published genome wide significant association in a CL/P GWAS as of the start of our study in 2012. Genotyping was performed by direct sequencing of PCR products containing the respective SNP. We have genotyped six SNPs in 119 German patients with SMCP and compared the genotypes with 383 population-based controls. None of the SNPs showed association with SMCP, neither after correction for multiple testing nor nominally. Results of single marker associations are shown in Table I. One limitation of our study is the relatively small sample size although we believe our sample is among the largest ones reported to date for this rare disorder. Association studies in rare diseases [Bezzina et al., 2013] can be confronted with insufficient statistical power to detect associations at least for SNPs with modest to small effect size. However, simulation shows that our study was adequately powered on the nominal significance level of 0.05: assuming odds ratios of 2.0 for heterozygotes and 4.0 for homozygotes at rs987525, our study had a power of detection of association of >0.85 in each case. Thus, the failure to detect an association with top CL/P-associated SNPs in our study might represent a true finding and may thus indicate that there are genuine differences in the genetic architecture of SMCP as compared to CL/P. Indeed, from an anatomical and pathophysiological point of view, SMCP is believed to be more closely related to CPO than to CL/P [Reiter et al., 2014]. Consistently, several SNPs associated with CL/P in GWAS did not show a positive association signal when evaluated in CPO [Birnbaum et al., 2009]. While a single GWAS in CPO has tentatively implicated gene x environment interactions [Beaty et al., 2011], no locus with genome wide significant association has been reported in CPO to date. Similarly, there may be genetic

Mangold et al., 2010; Ludwig et al., 2012], including a key susceptibility SNP on chromosome 8 (rs987525) that is consistently associated with CL/P across different populations but shows no association with CPO [Birnbaum et al., 2009; Grant et al., 2009; Ludwig et al., 2012; Uslu et al., 2014]. To start to dissect the genetic contribution to SMCP and to investigate possible overlaps with CL/ P risk alleles we have performed an association study of SNPs identified in CL/P GWAS in an ethnically homogeneous group of 119 individuals with SMCP and 383 controls. Clinical data collection of patients with SMCP was performed according to the International Consortium for Oral Clefts Genetics [Mitchell et al., 2002]. We enrolled 119 unrelated patients with SMCP at two tertiary referral centers in Germany (102 from Ulm and 17 from Bonn). Patients recruited in Ulm originated mainly from South-Western Germany and were seen at the Section of Phoniatrics and Pedaudiology and the Department of CranioMaxillo-Facial Surgery of the University Hospital Ulm for surgical repair of SMCP. In Bonn, affected individuals were recruited for the ongoing project of GWAS in orofacial clefts, but SMCP had not been included in previous GWAS that were performed in CL/ P only [Birnbaum et al., 2009; Mangold et al., 2010; Ludwig et al., 2012]. All patients and their parents were of German origin. Sixtysix affected individuals were male and 53 female, and the mean age was 18.7 years (range 1–74 years). Clinical assessment was carried out by an ENT surgeon, specialist for phoniatrics, surgeon for oral and maxillofacial surgery and, if necessary, by a medical geneticist to rule out a possible syndrome. Medical records were reviewed and a detailed questionnaire was filled out to identify possible additional malformations. Three hundred eighty three individuals (182 male and 201 female) of German descent were selected from the population-based Heinz Nixdorf Recall cohort study (Risk Factors, Evaluation of Coronary Calcium and Lifestyle) as controls [Schmermund et al., 2002; Birnbaum et al., 2009; Mangold et al., 2010]. The study was approved by the local Ethics Committee. Written informed consent was obtained from all patients or their parents.

TABLE I. Association Results for Six CL/P Loci in SMCP

SNP rs560426 rs2013162 rs987525 rs7078160 rs227731 rs13041247 a

Chromosomal band (position hg19) 1p22.1 (chr1:94553438) 1q32.2 (chr1:209968684) 8q24.21 (chr8:129946154) 10q25.3 (chr10:118827560) 17q22 (chr17:54773238) 20q12 (chr20:39269074)

Gene ABCA4 intragenic

Allelesa A/G

IRF6 intragenic

A/C

21,53,45

55,184,132

gene desert

A/C

7,48,64

19,132,228

KIAA1598 intragenic

A/G

2,32,85

15,104,264

between NOG andC17orf67 nearMAFB

A/C

43,53,23

141,153,84

C/T

20,46,53

46,176,161

CL/P risk alleles are in bold. Allele counts are given in the format homozygous allele 1 / heterozygous / homozygous allele 2. OR, odds ratio; CI, confidence interval. b

Allele count controlsb (n ¼ 383) 124,170,87

Allele count casesb (n ¼ 119) 40,59,20

OR per allele (95% CI) 0.87 (0.66–1.16) 0.99 (0.73–1.34) 0.82 (0.59–1.15) 1.18 (0.81–1.78) 0.97 (0.74–1.28) 0.95 (0.71–1.29)

P trend 0.35 0.94 0.25 0.40 0.83 0.75

672 and environmental factors jointly influencing the risk of SMCP, such as gene x smoking interactions [Reiter et al., 2012]. The lower incidence of CPO and SMCP and their presumed higher genetic heterogeneity when compared to CL/P might be reasons for the paucity of CPO and SMCP risk alleles reported to date. To further explore the genetics of SMCP future work should be directed at the replication of more recent CL/P GWAS hits [Ludwig et al., 2012] in larger SMCP samples and a GWAS in nonsyndromic SMCP.

ACKNOWLEDGMENTS We thank all patients and their families for participation in the study. We thank Susanne Moebus and Bernhard Hosthemke who contributed data on the population-based controls. The Heinz Nixdorf Recall cohort was established with the generous support of the Heinz Nixdorf Foundation, Germany (Chairman: G. Schmidt).

REFERENCES Beaty TH, Murray JC, Marazita ML, Munger RG, Ruczinski I, Hetmanski JB, Liang KY, Wu T, Murray T, Fallin MD, Redett RA, Raymond G, Schwender H, Jin SC, Cooper ME, Dunnwald M, Mansilla MA, Leslie E, Bullard S, Lidral AC, Moreno LM, Menezes R, Vieira AR, Petrin A, Wilcox AJ, Lie RT, Jabs EW, Wu-Chou YH, Chen PK, Wang H, Ye X, Huang S, Yeow V, Chong SS, Jee SH, Shi B, Christensen K, Melbye M, Doheny KF, Pugh EW, Ling H, Castilla EE, Czeizel AE, Ma L, Field LL, Brody L, Pangilinan F, Mills JL, Molloy AM, Kirke PN, Scott JM, ArcosBurgos M, Scott AF. 2010. A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4. Nat Genet 42:525–529. Beaty TH, Ruczinski I, Murray JC, Marazita ML, Munger RG, Hetmanski JB, Murray T, Redett RJ, Fallin MD, Liang KY, Wu T, Patel PJ, Jin SC, Zhang TX, Schwender H, Wu-Chou YH, Chen PK, Chong SS, Cheah F, Yeow V, Ye X, Wang H, Huang S, Jabs EW, Shi B, Wilcox AJ, Lie RT, Jee SH, Christensen K, Doheny KF, Pugh EW, Ling H, Scott AF. 2011. Evidence for gene-environment interaction in a genome wide study of nonsyndromic cleft palate. Genet Epidemiol 35:469–478. Bezzina CR, Barc J, Mizusawa Y, Remme CA, Gourraud JB, Simonet F, Verkerk AO, Schwartz PJ, Crotti L, Dagradi F, Guicheney P, Fressart V, Leenhardt A, Antzelevitch C, Bartkowiak S, Borggrefe M, Schimpf R, Schulze-Bahr E, Zumhagen S, Behr ER, Bastiaenen R, Tfelt-Hansen J, Olesen MS, Kaab S, Beckmann BM, Weeke P, Watanabe H, Endo N, Minamino T, Horie M, Ohno S, Hasegawa K, Makita N, Nogami A, Shimizu W, Aiba T, Froguel P, Balkau B, Lantieri O, Torchio M, Wiese C, Weber D, Wolswinkel R, Coronel R, Boukens BJ, Bezieau S, Charpentier E, Chatel S, Despres A, Gros F, Kyndt F, Lecointe S, Lindenbaum P, Portero V, Violleau J, Gessler M, Tan HL, Roden DM, Christoffels VM, Le Marec H, Wilde AA, Probst V, Schott JJ, Dina C, Redon R. 2013. Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death. Nat Genet 45:1044–1049. Birnbaum RH, Schiefke F, Hemprich A, Potzsch S, Steegers-Theunissen RP, Potzsch B, Moebus S, Horsthemke B, Kramer FJ, Wienker TF, Mossey PA, Propping P, Cichon S, Hoffmann P, Knapp M, Nothen MM, Mangold E. 2009. Key susceptibility locus for nonsyndromic cleft lip with or without cleft palate on chromosome 8q24. Nat Genet 41:473–477. Cole P, Hatef DA, Kaufman Y, Magruder A, Bree A, Friedman E, Sindwani R, Hollier LH, Jr. 2009. Facial clefting and oroauditory pathway manifestations in ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome. Am J Med Genet A 149A:1910–1915.

AMERICAN JOURNAL OF MEDICAL GENETICS PART A Dixon MJ, Marazita ML, Beaty TH, Murray JC. 2011. Cleft lip and palate: Understanding genetic and environmental influences. Nat Rev Genet 12:167–178. Friedman MA, Miletta N, Roe C, Wang D, Morrow BE, Kates WR, Higgins AM, Shprintzen RJ. 2011. Cleft palate, retrognathia and congenital heart disease in velo-cardio-facial syndrome: A phenotype correlation study. Int J Pediatr Otorhinolaryngol 75:1167–1172. Gosain AK, Conley SF, Marks S, Larson DL. 1996. Submucous cleft palate: Diagnostic methods and outcomes of surgical treatment. Plast Reconstr Surg 97:1497–1509. Grant SF, Wang K, Zhang H, Glaberson W, Annaiah K, Kim CE, Bradfield JP, Glessner JT, Thomas KA, Garris M, Frackelton EC, Otieno FG, Chiavacci RM, Nah HD, Kirschner RE, Hakonarson H. 2009. A genome-wide association study identifies a locus for nonsyndromic cleft lip with or without cleft palate on 8q24. J Pediatr 155:909–913. Jugessur A, Shi M, Gjessing HK, Lie RT, Wilcox AJ, Weinberg CR, Christensen K, Boyles AL, Daack-Hirsch S, Trung TN, Bille C, Lidral AC, Murray JC. 2009. Genetic determinants of facial clefting: Analysis of 357 candidate genes using two national cleft studies from Scandinavia. PLoS ONE 4:e5385. Kondo HH, Pober AC, Moreno BR, Arcos-Burgos L, Valencia M, Houdayer C, Bahuau C, Moretti-Ferreira M, Richieri-Costa D, Dixon A, Murray MJ. 2002. Mutations in IRF6 cause van der woude and popliteal pterygium syndromes. Nat Genet 32:285–289. Leslie EJ, Marazita ML. 2013. Genetics of cleft lip and cleft palate. Am J Med Genet C Semin Med Genet 163C:246–258. Ludwig KU, Mangold E, Herms S, Nowak S, Reutter H, Paul A, Becker J, Herberz R, AlChawa T, Nasser E, Bohmer AC, Mattheisen M, Alblas MA, Barth S, Kluck N, Lauster C, Braumann B, Reich RH, Hemprich A, Potzsch S, Blaumeiser B, Daratsianos N, Kreusch T, Murray JC, Marazita ML, Ruczinski I, Scott AF, Beaty TH, Kramer FJ, Wienker TF, SteegersTheunissen RP, Rubini M, Mossey PA, Hoffmann P, Lange C, Cichon S, Propping P, Knapp M, Nothen MM. 2012. Genome-wide meta-analyses of nonsyndromic cleft lip with or without cleft palate identify six new risk loci. Nat Genet 44:968–971. Mangold E, Ludwig KU, Birnbaum S, Baluardo C, Ferrian M, Herms S, Reutter H, de Assis NA, Chawa TA, Mattheisen M, Steffens M, Barth S, Kluck N, Paul A, Becker J, Lauster C, Schmidt G, Braumann B, Scheer M, Reich RH, Hemprich A, Potzsch S, Blaumeiser B, Moebus S, Krawczak M, Schreiber S, Meitinger T, Wichmann HE, Steegers-Theunissen RP, Kramer FJ, Cichon S, Propping P, Wienker TF, Knapp M, Rubini M, Mossey PA, Hoffmann P, Nothen MM. 2010. Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate. Nat Genet 42:24–26. Mitchell LE, Beaty TH, Lidral AC, Munger RG, Murray JC, Saal HM, Wyszynski DF, International Consortium for Oral Clefts Genetics. 2002. Guidelines for the design and analysis of studies on nonsyndromic cleft lip and cleft palate in humans: Summary report from a workshop of the international consortium for oral clefts genetics. Cleft Palate Craniofac J 39:93–100. Peyrard-Janvid M, Leslie EJ, Kousa YA, Smith TL, Dunnwald M, Magnusson M, Lentz BA, Unneberg P, Fransson I, Koillinen HK, Rautio J, Pegelow M, Karsten A, Basel-Vanagaite L, Gordon W, Andersen B, Svensson T, Murray JC, Cornell RA, Kere J, Schutte BC. 2014. Dominant mutations in GRHL3 cause van der woude syndrome and disrupt oral periderm development. Am J Hum Genet 94:23–32. Reiter R, Brosch S, Ludeke M, Fischbein E, Haase S, Pickhard A, Assum G, Schwandt A, Vogel W, Hogel J, Maier C. 2012. Genetic and environmental risk factors for submucous cleft palate. Eur J Oral Sci 120:97–103. Reiter R, Brosch S, Ludeke M, Fischbein E, Rinckleb A, Haase S, Schwandt A, Pickhard A, Maier C, Hogel J, Vogel W. 2014. Do orofacial clefts represent different genetic entities. Cleft Palate Craniofac J.

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Reiter R, Brosch S, Wefel H, Schlomer G, Haase S. 2011. The submucous cleft palate: Diagnosis and therapy. Int J Pediatr Otorhinolaryngol 75: 85–88.

in healthy middle-aged subjects: Rationale and design of the Heinz Nixdorf RECALL Study Risk Factors, Evaluation of Coronary Calcium and Lifestyle. Am Heart J 144:212–218.

Schmermund A, Mo¨hlenkamp S, Stang A, Gro¨nemeyer D, Seibel R, Hirche H, Mann K, Siffert W, Lauterbach K, Siegrist J, Jo¨ckel KH, Erbe l R. 2002. Assessment of clinically silent atherosclerotic disease and established and novel risk factors for predicting myocardial infarction and cardiac death

Uslu VV, Petretich M, Ruf S, Langenfeld K, Fonseca NA, Marioni JC, Spitz F. 2014. Long-range enhancers regulating myc expression are required for normal facial morphogenesis. Nat Genet 46:753–758.

A post GWAS association study of SNPs associated with cleft lip with or without cleft palate in submucous cleft palate.

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