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. Author manuscript; available in PMC: 2010 Jul 7.
Published in final edited form as: Am J Med Genet A. 2010 Jul;152A(7):1701–1710. doi: 10.1002/ajmg.a.33482

Follow-Up Association Studies of Chromosome Region 9q and Nonsyndromic Cleft Lip/Palate

Ariadne Letra 1,3,§, Renato Menezes 1,3,§, Manika Govil 1,3, Renata F Fonseca 4, Toby McHenry 1,3, José M Granjeiro 5, Eduardo E Castilla 6, Iêda M Orioli 4, Mary L Marazita 1,3,7,8, Alexandre R Vieira 1,2,3,7,9
PMCID: PMC2898904  NIHMSID: NIHMS194215  PMID: 20583170

Abstract

Cleft lip/palate comprises a large fraction of all human birth defects, and is notable for its significant lifelong morbidity and complex etiology. Several studies have shown that genetic factors appear to play a significant role in the etiology of cleft lip/palate. Human chromosomal region 9q21 has been suggested in previous reports to contain putative cleft loci. Moreover, a specific region (9q22.3-34.1) was suggested to present a ∼45% probability of harboring a cleft susceptibility gene. Fine mapping of fifty SNPs across the 9q22.3-34.11 region was performed to test for association with cleft lip/palate in families from United States, Spain, Turkey, Guatemala, and China. We performed family-based analysis and found evidence of association of cleft lip/palate with STOM (rs306796) in Guatemalan families (P=0.004) and in all multiplex families pooled together (P=0.002). This same SNP also showed borderline association in the US families (P=0.04). Under a nominal value of 0.05, other SNPs also showed association with cleft lip/palate and cleft subgroups. SNPs in STOM and PTCH genes and nearby FOXE1 were further associated with cleft phenotypes in Guatemalan and Chinese families. Gene prioritization analysis revealed PTCH and STOM ranking among the top fourteen candidates for cleft lip/palate among 339 genes present in the region. Our results support the hypothesis that the 9q22.32-34.1 region harbors cleft susceptibility genes. Additional studies with other populations should focus on these loci to further investigate the participation of these genes in human clefting.

Keywords: cleft lip and palate, chromosome 9q, fine mapping, craniofacial defect

Introduction

Oral-facial clefts arise as a failure of facial embryonic processes to completely merge and/or fuse. They comprise a large fraction of all human birth defects, and may occur as part of single-gene Mendelian syndromes, as part of chromosomal abnormalities, or due to teratogen exposure [Murray, 2002]. Clefts of the lip and clefts of the palate are notable for their significant lifelong morbidity and complex etiology, and are considered different entities: cleft lip can occur with or without cleft palate (cleft lip/palate) while cleft palate may occur as an isolated defect [Fogh-Andersen, 1942]. Cleft lip/palate affects about 1/700 births with wide variability related to geographic origin and socioeconomic status. In general, Native American and Asian populations present the highest frequencies, sometimes at 1/500 or higher, followed by Caucasian, and African-derived populations showing the lowest frequencies around 1/2500 births [Mossey and Little, 2002].

Several studies have shown that genetic factors appear to play a significant role in the etiology of cleft lip/palate. In recent years, it has become evident that cleft lip/palate is heterogeneous, and different chromosome regions such as 1q, 2p, 4q, 6p, 14q, 17q, and 19q have been claimed to contain a clefting locus [Vieira, 2008]. Association of specific genes such as MSX1 [Lidral et al., 1998; Beaty et al., 2001; Jezewski et al., 2003; Vieira et al., 2003], IRF6 [Zucchero et al., 2004; Ghassibe et al., 2005; Park et al., 2007; Vieira et al., 2007; Jia et al., 2009; Jugessur et al., 2009]; PVRL1 [Sozen et al., 2001; Avila et al., 2006], and genes of the FGF signaling pathway [Riley et al., 2007; Menezes et al., 2008] have been reported as well. Recently, genome-wide association studies with German, American and European populations have revealed a new major susceptibility locus for cleft lip/palate located in a gene desert region on chromosome 8q24 [Birnbaum et al., 2009; Grant et al., 2009; Nikopensus et al., 2009].

Human chromosomal region 9q21 has also been suggested in previous reports [Marazita et al., 2002; 2004; 2009], and in a mouse model [Juriloff et al., 2004] to contain putative cleft loci. A marker (D9S1122) located at 9q21.13 showed positive association with cleft lip/palate in Chinese multiplex families [Marazita et al., 2002]. Furthermore, a meta-analysis of a 10-cM genome scan of 388 extended multiplex families with cleft lip/palate from multiple populations revealed potential candidate genes in six chromosomal regions, including 9q21 (heterogeneity LOD score [HLOD] = 6.6). Positive association results (indicating close proximity to a clefting locus) were also found with four of the five selected candidate genes in the region [Marazita et al., 2004]. Moreover, the PPL (posterior probability of linkage) statistical test pointed towards a nearby region, from bands 9q22.3 to q34.1 (between 95Mb and 128Mb), as presenting ∼45% probability of harboring a causative gene for clefting (Govil et al., unpublished observations). The FOXE1 (forkhead box E1) gene, located in this region and implicated in syndromic cleft palate (Bamforth-Lazarus syndrome, OMIM 241850) has recently been reported to be associated with both isolated cleft lip with or without cleft palate and isolated cleft palate, and suggested as a key player in primary palatogenesis [Moreno et al., 2009].

To follow up on this data, we performed association studies with densely spaced markers spanning the 9q22.3-34.1 region to test for association with cleft lip/palate.

Materials and Methods

Subjects and Samples

Details of the population data sets are presented in Table I. Briefly, we assessed 291 multiplex cleft families (one or more members affected with an oral cleft) from the United States, Spain, Turkey, Guatemala and China. All cases had nonsyndromic cleft lip with or without cleft palate. Families were ascertained through probands, and additional relatives were recruited. Individuals presenting syndromic clefts, cleft palate only, or unknown cleft types, as well as control individuals presenting positive family history of clefting were excluded. This study was approved by local and the University of Pittsburgh institutional review boards. A signed informed consent sheet was obtained from all study participants after being explained the objectives and procedures of the study.

Table I.

Summary of families and individuals ascertained for the study.

Group Ancestral Origin No. of Families No. of Individuals Affected Individuals Unaffected Individuals Affected Individuals by Phenotype
CL1 CL/P2
Families
 North America US 89 529 145 384 39 106
 Central America Guatemala 77 514 93 421 20 73
 Europe Spain 36 136 43 93 10 33
Turkey 29 288 38 250 17 21
 East Asia China 60 180 60 120 14 46
 TOTAL 291 1647 379 1268 100 279
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1

CL, cleft lip;

2

CL/P, cleft lip with or without cleft palate.

Blood or saliva samples were collected to obtain genomic DNA. DNA samples from the populations from China, Turkey, and United States recruited until 2005 were obtained from blood. DNA samples from the population from Guatemala, Spain, and United States recruited after 2005 were obtained from saliva. DNA extraction was performed according to previously published protocols [Trevillato and Line, 2000]. There was no difference in the performance of the samples obtained from blood or saliva in regards to our genotyping approach described below.

SNP Selection and Genotyping

We used the International HapMap Project Database to guide our selection of single nucleotide polymorphisms (SNPs) to be genotyped. We based our selection using the approach devised by Carlson et al. [Carlson et al., 2004], selecting from a set of SNPs which maximally represented the linkage disequilibrium structure of the 9q22.3-34.1 region. Fifty SNPs spanning the 9q22.3-34.1 region were selected (Figure I). Preference was given to tag SNPs with a minor allele frequency of at least 10%, taking into consideration the various ethnicities represented in this study.

Figure I.

Figure I

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Overview of chromosome 9. The lighter box in the detail indicates the investigated region (9q22.32-34.11), spanning from 95Mb to 128Mb (Obtained from HapMap International Project, available at http://www.hapmap.org). Solid black bars below chromosome structure represent genes in region. Outlined gray bars below black bars indicate number of single nucleotide polymorphisms in the region.

Genotypes were generated using Taqman chemistry [Ranade et al., 2001] on an automatic sequence-detection instrument (ABI Prism 7900HT, Applied Biosystems, Foster City, CA). Assays and reagents were supplied by Applied Biosystems (Applied Biosystems, Foster City, CA). Details of the studied polymorphisms are presented in Table II.

Table II.

Details of the SNPs investigated in the study.

SNP No. SNP name Chromosome Region Base Position1 Mapping Gene (SNP Location) Mapping Gene Function Base Change
1 rs357564 9q22.32 97249415 PTCH PTCH coding-nonsynonymous AG
2 rs2236407 9q22.32 97277617 PTCH PTCH intron/next to exon AG
3 rs2297088 9q22.32 97282805 PTCH PTCH intron/next to exon AG
4 rs10512248 9q22.32 97299524 PTCH PTCH intron AC
5 rs894673 9q22.33 99652091 FOXE1 FOXE1 near gene 5′ UTR AT
6 rs3758249 9q22.33 99653961 FOXE1 FOXE1 near gene 5′ UTR AG
7 rs10984103 9q22.33 99679096 intergenic C9orf156 unknown AC
8 rs2900463 9q22.33 100017278 TBC1D2 TBC1D2 intron CT
9 rs2808557 9q22.33 100283185 GPR51 GPR51 intron/next to exon GT
10 rs1031111 9q22.33 101201619 intergenic intergenic unknown AG
11 rs1526267 9q31.1 101629566 NR4A3 NR4A3 5′ UTR AG
12 rs1226588 9q31.1 102380835 TMEFF1 TMEFF1 intron CT
13 rs1448576 9q31.1 105023498 340511 340511 unknown AG
14 rs1992917 9q31.1 105029464 340511 340511 unknown AG
15 rs1993434 9q31.1 105074129 intergenic intergenic unknown AG
16 rs2014343 9q31.3 112899132 intergenic intergenic unknown CG
17 rs4979032 9q31.3 113466796 intergenic intergenic intron AG
18 rs2762475 9q32 113928337 SUSD1 SUSD1 intron CT
19 rs1999263 9q32 114249494 HSDL2 HSDL2 intron AC
20 rs10739367 9q32 114406899 intergenic intergenic intron CT
21 rs1539339 9q32 114633550 SNX30 SNX30 intron/next to exon AC
22 rs818714 9q32 115223122 C9orf43 C9orf43 intron/next to exon CT
23 rs1008138 9q32 115849291 ZNF618 ZNF618 intron/next to exon GT
24 rs942519 9q32 116208854 DFNB31 DFNB31 coding-nonsynonymous AG
25 rs2992147 9q33.1 116889135 TNC TNC coding-synonymous CT
26 rs971037 9q33.1 117706363 C9orf27 C9orf27 intron/next to exon AT
27 rs1888636 9q33.1 117979155 PAPPA PAPPA intron AT
28 rs1418495 9q33.1 120130612 intergenic intergenic unknown AG
29 rs1335256 9q33.1 120366850 intergenic intergenic unknown CT
30 rs7859743 9q33.2 122196412 intergenic intergenic intron/next to exon AG
31 rs2300934 9q33.2 122848784 intergenic intergenic intron/next to exon AC
32 rs306796 9q33.2 123167156 STOM STOM intron CT
33 rs10739600 9q33.2 123663694 TTLL11 TTLL11 unknown AG
34 rs7853089 9q33.2 123972807 C9orf18 C9orf18 intron GT
35 rs1928623 9q33.2 124348830 OR1N2 OR1N2 unknown AC
36 rs2251495 9q33.2 124682508 MNAB MNAB intron/next to exon CT
37 rs2808416 9q33.2 125159980 CRB2 CRB2 intron AG
38 rs1928482 9q33.2 125472269 DENND1A DENND1A intron CT
39 rs2274782 9q33.3 126123895 intergenic intergenic intron/next to exon CT
40 rs10739650 9q33.3 126363597 NR6A1 NR6A1 intron CT
41 rs646527 9q33.3 126717053 GOLGA1 GOLGA1 intron CT
42 rs7853181 9q33.3 127262845 MAPKAP1 MAPKAP1 intron AG
43 rs10760403 9q33.3 127648316 PBX3 PBX3 intron GT
44 rs10987185 9q33.3 127981942 intergenic intergenic unknown CG
45 rs887659 9q33.3 128224130 C9orf28 C9orf28 intron/next to exon AG
46 rs3850585 9q33.3 128476093 LMX1B LMX1B intron CT
47 rs1890546 9q33.3 128991962 intergenic intergenic intron CT
48 rs7869023 9q33.3 129241499 RPL12 RPL12 intron AG
49 rs913989 9q34.1 129572809 SH2D3C SH2D3C intron/next to exon AC
50 rs10819354 9q34.1 129893311 SLC25 SLC25 intron, near 5′ UTR AG
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1

According to the UCSC Genome Browser March 2006 Assembly.

Statistical Analyses

The transmission disequilibrium test was used to assess association of alleles at each marker with cleft lip with or without cleft palate with the use of the Family Based Association Test (FBAT) software [Horvath et al., 2001]. We also performed haplotype analyses using the “hbat” function of the FBAT software to identify if sets of markers were in linkage disequilibrium with cleft lip/palate. We also analyzed the entire dataset according to the following cleft subgroups: CLO + CLCLP (families where all affecteds have cleft lip only plus families where at least one affected has cleft lip only and one affected has cleft lip and palate, excluding any family where an affected has cleft palate only); and CLO +CLP +CLCLP (families where all affecteds have cleft lip only plus families where all affecteds have cleft lip and palate plus families where at least one affected has cleft lip only and one affected has cleft lip and palate, excluding any family where an affected has cleft palate only). Populations were analyzed individually and as a pooled dataset. P-values below 0.001 (0.05 divided by 50 markers) were considered statistically significant.

Gene Prioritization

We attempted to identify candidate genes present in the 9q22.3-34.1 region using the gene prioritization software Endeavour [Aerts et al., 2006; Tranchevent et al., 2008]. Endeavour is a web server that allows users to prioritize candidate genes with respect to their biological processes or diseases of interest. It relies on the similarity between candidate genes and models built with a set of manually inputted reference genes. We used 10 genes to compose the reference set: IRF6, MSX1, PVRL1, TGFB3, CLPTM1, TGFA, FGFR1, RARA, BCL3, and TBX22. We did not use PTCH or FOXE1 as reference genes in the target region as they would be automatically removed by the program due to redundancy between reference and test sets of genes.

Results

Fine Mapping Association Studies

We genotyped fifty SNPs spanning 339 genes present in chromosomal region 9q22.3-q34.1. Detailed information on allele frequencies per population and additional replication analyses of specific markers are available as Supplemental Material. We performed family-based analysis to assess genotype and allelic associations between the investigated SNPs and cleft lip/palate. After correcting for multiple testing, we found borderline association of cleft lip/palate with a SNP in STOM (rs306796, 9q33.2) in Guatemalan families (P=0.004) and all families pooled together (P=0.002). This same SNP (rs306796) also showed a trend for association in the US population (P=0.04) (Table III). Of note, SNPs in or nearby STOM also showed modest association with cleft lip/palate in four of the five populations studied (P=0.04). Under a nominal value of 0.05, other SNPs also showed association with cleft lip/palate and cleft subgroups (Tables III and IV).

Table III.

Summary of results for association tests with markers in the chromosome 9q region and cleft lip/palate in the studied populations.

SNP Name UCSC base position1 Mapping Gene (SNP Location) Family-Based Analyses (P-values2)
Individual Populations Pooled Populations
USA Guatemala Spain Turkey China
rs357564 95,289,149 PTCH 0.40 0.19 0.49 0.74 0.50 0.09
rs2236407 95,317,351 PTCH 0.49 0.16 0.40 0.64 1.00 0.52
rs2297088 95,322,539 PTCH 0.66 0.64 0.71 0.64 0.89 0.87
rs10512248 95,339,258 PTCH 0.75 0.16 1.00 0.64 0.58 0.90
rs894673 97,691,825 FOXE1 0.85 0.93 0.48 0.82 0.19 0.58
rs3758249 97,693,695 FOXE1 0.60 0.93 0.43 0.65 0.26 0.71
rs10984103 97,718,830 C9orf156 0.89 0.86 0.27 0.67 0.24 0.52
rs2900463 98,057,012 TBC1D2 0.63 0.52 0.87 0.06 0.18 0.11
rs2808557 98,322,919 GPR51 0.93 0.47 0.78 0.53 0.18 0.62
rs1031111 99,241,353 intergenic 0.66 0.85 0.55 1.00 0.71 0.99
rs1526267 99,669,300 NR4A3 0.49 0.65 0.05 0.12 0.48 0.79
rs1226588 100,420,569 TMEFF1 0.06 0.88 0.74 0.37 0.66 0.25
rs1448576 103,063,232 340511 0.56 0.65 1.00 0.68 0.04 0.13
rs1992917 103,069,198 340511 0.68 0.89 0.86 0.70 0.28 0.84
rs1993434 103,113,863 intergenic 0.92 0.66 0.42 0.84 0.76 0.59
rs2014343 110,938,866 intergenic 0.31 0.01 0.49 0.03 0.28 0.01
rs4979032 111,506,520 intergenic 0.89 0.39 0.74 0.39 0.58 0.50
rs2762475 111,968,071 SUSD1 0.47 0.84 0.49 1.00 0.79 0.89
rs1999263 112,289,228 HSDL2 0.71 0.20 1.00 0.25 0.15 0.97
rs10739367 112,446,633 intergenic 0.97 0.26 0.59 0.67 0.65 0.87
rs1539339 112,673,284 SNX30 0.94 0.83 0.44 1.00 0.09 0.25
rs818714 113,262,855 C9orf43 0.12 0.53 0.39 0.64 0.86 0.16
rs1008138 113,889,024 ZNF618 0.46 0.14 0.12 0.80 0.63 0.25
rs942519 114,248,587 DFNB31 0.80 0.18 0.45 0.47 0.53 0.52
rs2992147 114,928,868 TNC 0.33 0.78 0.85 0.37 0.88 0.37
rs971037 115,746,096 C9orf27 0.81 0.78 0.52 0.09 0.56 0.56
rs1888636 116,018,888 PAPPA 0.06 0.61 0.43 0.81 0.89 0.39
rs1418495 118,170,345 intergenic 0.04 0.91 0.42 0.68 0.53 0.27
rs1335256 118,406,583 intergenic 0.69 0.63 0.71 0.82 0.39 0.65
rs7859743 120,236,145 intergenic 0.75 0.18 0.04 0.37 0.79 0.19
rs2300934 120,888,517 intergenic 0.52 0.04 0.89 0.39 0.04 0.91
rs306796 121,206,889 STOM 0.04 0.004 0.26 0.41 0.06 0.002
rs10739600 121,703,427 TTLL11 0.92 0.93 0.49 0.82 0.09 0.82
rs7853089 122,012,540 C9orf18 0.44 0.45 0.87 0.22 0.81 0.73
rs1928623 122,388,563 OR1N2 0.71 0.17 0.41 0.25 0.16 0.06
rs2251495 122,722,241 MNAB 0.18 0.92 1.00 0.32 0.89 0.73
rs2808416 123,199,713 CRB2 0.51 0.25 0.21 0.83 0.74 0.55
rs1928482 123,512,002 DENND1A 0.03 0.41 0.39 0.24 0.65 0.24
rs2274782 124,163,628 intergenic 0.32 0.44 0.39 0.13 0.28 0.95
rs10739650 124,403,330 NR6A1 0.21 0.15 0.56 0.20 0.37 0.94
rs646527 124,756,786 GOLGA1 0.67 0.40 0.95 0.32 0.22 0.78
rs7853181 125,302,578 MAPKAP1 0.56 0.32 0.49 0.58 0.69 0.95
rs10760403 125,688,049 PBX3 0.52 0.90 0.65 0.41 0.54 0.90
rs10987185 126,021,675 intergenic 0.42 0.42 0.71 0.81 0.34 0.64
rs887659 126,263,863 C9orf28 0.85 0.63 0.83 0.49 0.47 0.82
rs3850585 126,515,826 LMX1B 0.95 0.73 0.21 0.62 0.79 0.64
rs1890546 127,031,695 intergenic 0.47 0.22 0.20 0.43 0.22 0.64
rs7869023 127,281,232 RPL12 0.58 0.65 0.05 0.65 0.17 0.09
rs913989 127,612,542 SH2D3C 0.52 0.22 0.20 0.67 0.41 0.68
rs10819354 127,933,044 SLC25 0.27 0.37 0.13 0.49 0.05 0.87
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1

According to the UCSC Genome Browser March 2006 Assembly.

2

Family-Based Association Test.

Table IV.

Summary of results for association tests with markers in the chromosome 9q region and families with cleft lip only plus cleft lip and palate in Guatemala and China.

CLO + CL,CLP1
P-values
SNP Name SNP Location Guatemala China
rs357564 PTCH 0.41 --
rs2236407 PTCH 0.004 0.11
rs2297088 PTCH 0.01 0.11
rs10512248 PTCH 0.01 0.07
rs894673 FOXE1 0.07 0.05
rs3758249 FOXE1 0.13 0.05
rs10984103 C9orf156 0.07 0.16
rs2900463 TBC1D2 0.78 0.03
rs2808557 GPR51 0.16 0.16
rs1031111 intergenic 0.04 0.11
rs1526267 NR4A3 0.87 0.76
rs1226588 TMEFF1 0.05 0.25
rs1448576 340511 0.28 0.32
rs1992917 340511 0.94 0.05
rs1993434 intergenic 0.80 0.76
rs2014343 intergenic 0.05 0.81
rs4979032 intergenic 0.84 0.32
rs2762475 SUSD1 0.18 0.76
rs1999263 HSDL2 0.28 0.64
rs10739367 intergenic 0.32 0.44
rs1539339 SNX30 0.34 0.03
rs818714 C9orf43 0.26 0.56
rs1008138 ZNF618 0.13 0.29
rs942519 DFNB31 0.43 0.48
rs2992147 TNC 1.00 0.71
rs971037 C9orf27 0.41 0.83
rs1888636 PAPPA 0.49 0.71
rs1418495 intergenic 0.55 0.11
rs1335256 intergenic 0.83 0.37
rs7859743 intergenic 0.64 0.76
rs2300934 intergenic 0.03 0.53
rs306796 STOM 0.01 0.32
rs10739600 TTLL11 1.00 1.00
rs7853089 C9orf18 0.49 0.65
rs1928623 OR1N2 0.49 --
rs2251495 MNAB 0.41 0.48
rs2808416 CRB2 0.07 0.74
rs1928482 DENND1A 0.20 1.00
rs2274782 intergenic 0.74 0.76
rs10739650 NR6A1 0.65 1.00
rs646527 GOLGA1 1.00 0.29
rs7853181 MAPKAP1 0.94 0.74
rs10760403 PBX3 0.18 0.08
rs10987185 intergenic 0.65 0.48
rs887659 C9orf28 0.13 0.10
rs3850585 LMX1B 0.20 0.17
rs1890546 intergenic 0.22 1.00
rs7869023 RPL12 0.83 0.37
rs913989 SH2D3C 1.00 0.32
rs10819354 SLC25 0.40 0.74
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1

CLO + CL,CLP, families where all affecteds have cleft lip only plus families where at least one affected has cleft lip only and one affected has cleft lip and palate. Excludes any family where an affected has cleft palate only.

When analyzing families according to cleft subgroups, we found association of markers in PTCH (rs2234607, P=0.004; rs2297088, P=0.01; and rs10512248, P=0.01) and in/nearby STOM (rs306796, P=0.01; and rs2300934, P=0.03) with the CLO+CLCLP subgroup in Guatemalan families. We also found borderline association of markers nearby FOXE1 in Chinese (rs2900463; P=0.03) and Guatemalan (rs1031111; P=0.04) families (Table IV). Results for the CLO +CLP +CLCLP groups did not differ from the association results in individual populations (data not shown).

We also performed haplotype analysis of the investigated SNPs by population (Table V) and by population according to cleft subgroup (Table VI). Altered transmission of haplotypes involving markers in/nearby STOM was evident in families from the US (rs1928623-rs2251495-rs2808416; P=0.02), Turkey (rs1928623-rs2251495-rs2808416-rs1928482; P=0.02), Guatemala (rs2300934-rs306796; P=0.02 and rs306796-rs10739600; P=0.03), and China (rs2300934-rs306796; P=0.04) (Table V). Altered transmission was also observed for PTCH rs357654-rs2236407-rs2297088 haplotype (P=0.03) and for were PTCH rs357654-rs2236407-rs2297088-rs10512248 haplotype (P=0.02) in Guatemalan families (Table V). When analyzed by cleft subgroup, interesting results were observed for haplotype markers involving PTCH (rs357654-rs2236407; P=0.01) and PTCH and FOXE1 (rs10512248-rs894673) in families from Guatemala (P=0.01) and China (P=0.03) presenting CLO + CLCLP (Table VI).

Table V.

Results of haplotype transmission analysis in families with cleft lip with or without cleft palate by population.

Marker Haplotype Genes in Haplotype Population HBAT P-value2
rs1928623-rs2251495-rs2808416 Intergenic-MNAB (∼1Mb of STOM)- CRB2 US 0.02
rs1888636-rs1418495-rs1335256-rs7859473 PAPPA-intergenic-intergenic-CDK5RAP2 US 0.02
rs7869023-rs913989 ZNF79-SH2D3C Spain 0.03
rs1928623-rs2251495-rs2808416-rs1928482 Intergenic-MNAB (∼1Mb of STOM)- CRB2-KIAA1608 Turkey 0.02
rs2014343-rs4979032 Intergenic-GNG10 Guatemala 0.04
rs357654-rs2236407-rs2297088 PTCH-PTCH-PTCH Guatemala 0.03
rs357654-rs2236407-rs2297088-rs10512248 PTCH-PTCH-PTCH-PTCH Guatemala 0.02
rs2300934-rs306796 C5-STOM Guatemala 0.03
rs306796-rs10739600 STOM-TTLL11 (∼500kb of STOM) Guatemala 0.03
rs306796-rs10739600-rs7853089-rs1928623 STOM-TTLL11 (∼500kb of STOM)-C9orf18 (∼800kb of STOM) Guatemala 0.02
rs2300934-rs306796 C5-STOM China 0.04
rs306796-rs10739600-rs7853089-rs1928623 STOM-TTLL11 (∼500kb of STOM)-C9orf18 (∼800kb of STOM) China 0.04
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1

Comprises analyses of US, Spain, and Turkey families pooled together.

2

Transmission distortion significant when p<0.05.

Table VI.

Results of haplotype transmission analysis according to cleft subgroup by population.

Marker Haplotype Genes in Haplotype Cleft Group Population HBAT P-value2
rs357654-rs2236407 PTCH-PTCH CLO + CLCLP1 Guatemala 0.01
rs10512248-rs894673 PTCH-FOXE1 CLO + CLCLP Guatemala 0.01
rs2297088-rs10512248-rs894673 PTCH-PTCH-FOXE1 CLO + CLCLP Guatemala 0.01
rs2236407-rs2297088-rs10512248-rs894673 PTCH-PTCH-PTCH-FOXE1 CLO + CLCLP Guatemala 0.01
rs10512248-rs894673 PTCH-FOXE1 CLO + CLCLP China 0.03
rs10512248-rs894673-rs3758249 PTCH-FOXE1-FOXE1 CLO + CLCLP China 0.03
rs10512248-rs894673-rs3758249-rs10984103 PTCH-FOXE1-FOXE1-C9orf156 CLO + CLCLP China 0.03
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1

CLO + CLCLP, families where all affecteds have cleft lip only plus families where at least one affected has cleft lip only and one affected has cleft lip and palate. Excludes any family where an affected has cleft palate only.

2

Transmission distortion significant when p<0.05.

Gene Prioritization

We found 339 genes within the 9q22.3-34.1 region with Endeavour (Supplementary Table I). The algorithm prioritized all genes within the 9q22.3-34.1 region in relation to cleft lip/palate. The highest priority gene according to Endeavour was TGFBR1. The associated genes, PTCH and STOM, ranked in tenth and fourteenth places in the priority output, respectively. FOXE1 ranked in 39th place. Detailed results are available as Supplemental Material.

Discussion

We investigated a region on chromosome 9q identified in previous studies as to harbor cleft susceptibility loci. Among the genes present in the region are PTCH and FOXE1, in which missense mutations and polymorphic variants have been described as having a role in cleft lip/palate [Marazita et al., 2004; 2009; Mansilla et al., 2006; Vieira et al., 2005; Moreno et al., 2009].

We performed fine mapping association studies with fifty SNPs across a 32.65 Mb region and multiple populations. Although using 50 SNPs to investigate 339 genes in a region may not seem a dense enough strategy, we used information on linkage disequilibrium of the markers to avoid choosing redundant markers. We found association of a polymorphism in STOM (rs306796) in the Guatemalan families (P=0.004) and in all families pooled together (P=0.002). STOM encodes stomatin, a membrane protein that was first isolated from human red blood cells [Zhang et al., 1999]. In mouse, the STOM homolog presents a wide pattern of expression, with high levels of mRNA in heart, liver, skeletal muscle, and testis but low levels in lung, brain, and spleen [Gallagher et al., 1995b]. Despite being associated with a variety of diseases such as cancer, kidney failure and anemia, precise functions of this protein remain unclear. In humans, the absence of stomatin is associated with a form of hemolytic anemia known as hereditary stomatocytosis [Yokoyama et al., 2006]. Lower expression of stomatin results in an increase in the basal rate of glucose transport [Zhang et al., 2001]. Interestingly, diabetic pregnant women are at an increased risk for having offspring with neural tube defects and oral-facial clefts [Spilson et al., 2001] and excessive exposure to glucose has been postulated to play a role in the pathogenesis of nonsyndromic cleft lip/palate [Krapels et al., 2004].

Suggestive evidence of association was also observed for several other markers among the populations implicating that the chromosome 9q region might contain cleft susceptibility genes (P<0.05). When analyzing the dataset according to cleft subgroups, we observed borderline association of PTCH variants (rs2236407, P=0.004; rs2297088, P=0.01; and rs10512248, P=0.01) in Guatemalan families with CLO + CLCLP (families where all affecteds have cleft lip only plus families where at least one affected has cleft lip only and one affected has cleft lip and palate). PTCH was initially suggested as a candidate for human clefting because of its mouse homolog located in the candidate region clf2 [Juriloff et al., 2001]. Although no causal mutation in Ptch coding sequence was found in mice, alterations in the regulatory sequence are not ruled out. Given the “normal” phenotype of noncleft A/∼ strain mice, a mild regulatory defect seems likely if Ptch is clf2 [Juriloff et al., 2004]. In humans, mutations in PTCH cause Gorlin syndrome with diverse developmental anomalies, often including rib and craniofacial abnormalities (and cleft palate) and a mixture of tumor types [Cohen, 1999]. In their study with PTCH and isolated cleft lip/palate, Mansilla et al. sequenced all 23 exons of the gene and found seven new normal variants spread along the entire gene and three missense mutations in cases with cleft lip/palate, one of which was not found in 1,188 control samples [Mansilla et al., 2006]. Although the authors did not find statistically significant evidence of linkage (multipoint HLOD peak=2.36), they reported over-transmission of the PTCH rs2297088–rs2236407 haplotype with borderline statistical significance (P=0.08) in Filipino families with two or more affected members. They further concluded that missense mutations in PTCH may be rare causes of isolated cleft lip/palate and yet unidentified variants near PTCH may act as modifiers of the cleft phenotype. Corroborating with these findings, we observed significant evidence of transmission distortion for the PTCH rs357654-rs2236407-rs2297088 (P=0.03) and rs357654-rs2236407-rs2297088-rs10512248 (P=0.02) haplotypes in Guatemalan families.

It is unknown whether the associated PTCH polymorphisms (rs2236407 and rs2297088) located in introns and therefore not in coding regions or splice sites might cause a functional change in the final protein. Notwithstanding, intronic polymorphisms have been demonstrated in association with other complex diseases, including the association of IRF6 with cleft lip/palate [Zucchero et al., 2004; Ghassibe et al., 2005; Park et al., 2007; Vieira et al., 2007; Jia et al., 2009; Jugessur et al., 2009] and thus should not be disregarded as potentially damaging. Of note, in a recent association study with genes in chromosome 9q, PTCH SNP rs2297088 showed the strongest signal for Filipino families, based on the lowest p-value (P-value=6.49E-03), indicating that PTCH may have a possible role in the etiology of cleft lip with or without cleft palate in some populations.

A previous study from our group has shown that point mutations in FOXE1 may be rare causes for isolated cleft lip/palate [Vieira et al., 2005]. Following studies have shown positive association and linkage between FOXE1 and cleft lip/palate [Marazita et al., 2004; 2009], including a recent publication which identified FOXE1 in strong association with both isolated cleft lip with or without cleft palate and isolated cleft palate [Moreno et al., 2009]. We did not find significant association of FOXE1 with cleft phenotypes in the populations tested although suggestive association was observed for variants in/nearby FOXE1 in Chinese and Guatemalan families with CLO + CLCLP (P=-0.03 and P=0.04, respectively). FOXE1 belongs to a family of fork-head domain-genes involved in embryonic pattern formation, which have been identified as factors that bind to regulatory elements in mammalian genes expressed in terminally differentiated cells [Kaestner et al., 1993]. Foxe1 null mice exhibit cleft palate and either a sublingual or completely absent thyroid gland [De Felice et al., 1998]. In humans, mutations in FOXE1 result in the Bamforth syndrome, characterized by thyroid agenesis, cleft palate, spiky hair, and choanal atresia [Clifton-Bligh et al., 1998]. It is possible that variants in this gene may be in linkage disequilibrium with variants in other genes that jointly increase the susceptibility to cleft lip/palate. For example, we found overtransmission of PTCH-FOXE1 haplotypes in families Guatemalan and Chinese families presenting CLO + CLCLP (P=0.01 and P=0.03, respectively).

Using gene prioritization software, we identified 339 genes in the 9q22.3-34.11 candidate region, which were ranked based on similar roles or participation in similar biological processes with genes known to be associated with cleft lip/palate. Other investigators have used this gene prioritization approach as well, in studies of obesity and type II diabetes genes [Elbers et al., 2007; Sookoian et al., 2009], idiopathic pulmonary fibrosis [Tzoulevekis et al., 2007] and even cleft lip/palate [Osoegawa et al., 2008]. This approach however has the limitation of not allowing any control on the quality of gene annotation and quantity and quality of information across genes in different databases. It is difficult to predict how much these aspects influenced our results and the results of others but at minimal, this information adds to the bulk of the results and help interpreting the association data. In comparison with a reference set of 11 established candidate genes for human clefting, PTCH and STOM ranked in tenth and fourteenth places, respectively, among the 339 genes present in the investigated region, thus at the top five percent ranking as candidates for cleft lip/palate. These observations taken together with the association findings, suggest that those genes may be considered plausible candidates for cleft lip/palate. Meanwhile, we checked for conservation of the associated SNPs, and found that the wild-type nucleotide is conserved in the following species: rhesus monkey, dog and horse for rs2297088 in PTCH, and dog and horse for rs306796 in STOM.

In summary, we performed fine mapping analysis of chromosome 9q22.32-34.1 region previously suggested to harbor cleft susceptibility genes. Our association results support a role for PTCH as contributor for cleft lip/palate and suggest STOM as a possible new candidate gene. Haplotype and gene prioritization analyses confirmed the individual association findings with PTCH and STOM, ranking in the top five percent of the highest priority candidate genes for cleft lip/palate. We failed to replicate previous findings suggesting FOXE1 contributes to nonsyndromic cleft lip and palate. Additional studies with other populations should focus on these loci to further investigate the participation of these genes in human clefting.

Supplementary Material

Supplementary material 1
Supplementary table 2

Acknowledgments

We gratefully acknowledge individuals and families for their valuable collaboration. Thanks to research coordinators and staff at each collection site. Wendy Carricato helped with sample organization. Rebecca DeSensi and Kathleen Deeley provided technical assistance. Maria Adela Mansilla and Lina Maria Moreno at the University of Iowa helped with SNP marker selection. This work was supported by the National Institutes of Health grants: K99-DE018954 (to AL); K99-DE018413 (to RM); K99-DE018085 (to MG); R01-DE016148 and P50-DE016215 (to MLM); R21-DE16718 (to ARV); FAPERJ grant E-26/152.831/2006, CNPq grants 308885/2006-0 and 401467/2004-0 (to IMO); and CAPES, Brazil (to RFF). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Dental and Craniofacial Research or the National Institutes of Health. This paper is partly based on a thesis submitted to the graduate faculty, Federal University of Rio de Janeiro, in partial fulfillment of the requirements for the PhD degree (for RFF).

Footnotes

Web Resources: Accession numbers and URLs for data presented herein are as follows:

Applied Biosystems, http://www.appliedbiosystems.com/index.cfm

Family Based Association Test, http://www.biostat.harvard.edu/∼fbat

International HapMap Project, http://hapmap.org

Endeavour, http://www.esat.kuleuven.be/endeavour

Supplementary information is available at the American Journal of Medical Genetics website.

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Supplementary Materials

Supplementary material 1
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Supplementary table 2
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