Genomewide Scan for Nonsyndromic Cleft Lip and Palate in Multigenerational Indian Families Reveals Significant Evidence of Linkage at 13q33.1-34

Abstract

Nonsyndromic cleft lip with or without cleft palate (CL-P) is a common congenital anomaly with incidence ranging from 1 in 300 to 1 in 2,500 live births. We analyzed two Indian pedigrees (UR017 and UR019) with isolated, nonsyndromic CL-P, in which the anomaly segregates as an autosomal dominant trait. The phenotype was variable, ranging from unilateral to bilateral CL-P. A genomewide linkage scan that used ∼10,000 SNPs was performed. Nonparametric linkage (NPL) analysis identified 11 genomic regions (NPL>3.5; P<.005) that could potentially harbor CL-P susceptibility variations. Among those, the most significant evidence was for chromosome 13q33.1-34 at marker rs1830756 (NPL=5.57; P=.00024). This was also supported by parametric linkage; MOD score (LOD scores maximized over genetic model parameters) analysis favored an autosomal dominant model. The maximum LOD score was 4.45, and heterogeneity LOD was 4.45 (α=100%). Haplotype analysis with informative crossovers enabled the mapping of the CL-P locus to a region of ∼20.17 cM (7.42 Mb) between SNPs rs951095 and rs726455. Thus, we have identified a novel genomic region on 13q33.1-34 that harbors a high-risk variant for CL-P in these Indian families.

Nonsyndromic cleft lip with or without cleft palate (CL-P) is one of the most frequently occurring congenital malformations among live births. The prevalence varies widely, depending on the ethnicity and geographic location of the population, ranging from 1 in 300 to 1 in 2,500.^1,2 In the United States, it affects 1 in 700–1,000 newborns each year and is the fourth most common birth defect. In India, cleft lip/palate occurs in nearly 1 in 500 live births; the majority of these defects are not surgically corrected.³ Although Asians have the highest rate of orofacial clefts (OFCs) at birth, the majority of the genetic studies have been conducted with whites. There are two types of CL-P: syndromic and nonsyndromic. Nonsyndromic CL-P represents almost half of facial malformations and could be familial. More than 400 recognized syndromes may include a facial cleft as one of the manifestations. Some of the common syndromes and/or anomalies associated with clefting include Apert,⁴ Meckel,⁵ Treacher Collins,⁶ and van der Woude syndromes.⁷ Dental anomalies such as supernumerary, hypoplastic, or congenitally missing teeth and malocclusion are common in patients affected with CL-P.

The genes responsible for one form of X-linked (CPX [MIM 303400]) and one form of autosomal dominant (CPI [MIM 119530]) CL-P have been mapped to chromosomes Xq13-q21.31^8–12 and 2q32,¹³ respectively. Pathogenic mutations were identified in the TBX22 gene on Xq21.1. Genomic regions with evidence of linkage for nonsyndromic OFC were identified at 6p24.3 (OFC1 [MIM 119530]),^14,15 2p13 (OFC2 [MIM 602966]),¹⁶ 19q13 (OFC3 [MIM 600757]),¹⁷ 4q21-q31 (OFC4 [MIM 608371]),^18,19 4p16.1 (OFC5 [MIM 608874]),²⁰ and 1q32-q41 (OFC6 [MIM 608864]),^21,22 but not all responsible genes are yet identified. Chromosomal aberrations involving chromosomes 13 and 18 were reported to cause an increased incidence of clefts.^23–25 Genes associated with CL-P include MSX1, MSX2, PVRL1, IRF6, RARA, TGFA, TGFB3, TGFB2, MTHRF, GABRB3, FOXE1, GLI2, JAG2, LHX8, PHF8, SATB2, SKI, SPRY2, and TBX10.^20,26–29 However, none of these seem to play a major role in nonsyndromic CL-P, and they appear to be responsible for only a fraction of CL-P cases.²⁶

The ascertainment of occasional large multigenerational families segregating a “complex” trait such as CL-P is important, since it may reveal the existence of a single gene that, in these families, contributes significantly to the phenotype. There are only a few examples of such families with the CL-P phenotype used in linkage analysis who have revealed significant linkage to 4q,¹⁸ 17p,³⁰ and multiple loci.³¹ Here, we present genomewide linkage analysis of two multigenerational Indian families (UR017 and UR019) with CL-P, ascertained through a single proband with nonsyndromic CL-P. This analysis provided significant evidence of a susceptibility locus on a 7.42-Mb genomic region on chromosome 13q33.1-34.

The families studied include family UR017, who were from Andhra Pradesh, the Southern state of India, and had an apparent autosomal dominant mode of inheritance (fig. 1). The original five-generation pedigree consists of 96 members with 12 affected individuals (6 females and 6 males). In the present study, we included five affected and nine unaffected individuals.

Figure 1. .

Partial pedigrees of families UR017 and UR019 with CL-P. Affected individuals are shown with blackened symbols, and unaffected individuals are shown with unblackened symbols. Data are not available for deceased individuals shown with a question mark. Samples included in the analysis are numbered under their symbols in the pedigree.

Family UR019 is a six-generation family, also from Andhra Pradesh, with CL-P (fig. 1). The original pedigree is much larger than what is shown in figure 1. The family includes seven affected members (five males and two females). The mode of inheritance is apparently autosomal dominant. We analyzed three affected and two unaffected individuals from this family.

Each individual was evaluated by an experienced dysmorphologist and a clinical geneticist. Clinical and x-ray photographs were taken of selected individuals. All affected individuals had severe CL-P, and no syndromic anomalies were observed in either pedigree. Some of the affected individuals had dystrophic or congenitally missing teeth (due to severe clefting) and speech problems. The phenotype of a few deceased individuals is unknown, and they were considered “affected status unknown” in the linkage analysis. Blood samples were obtained from all available cooperative family members, with their informed consent. Apparently, these two families are unrelated, since we could not establish any link between them. They live in a small village with <15,000 inhabitants.

Blood DNA was purified, and the whole-genome genotyping scan was performed using the GeneChip Mapping 10K 2.0 SNP Array, which contains 11,555 SNPs. These are equally distributed in the genome, with a mean intermarker distance of 210 kb and an average heterozygosity of 0.38. The assay was performed using 250 ng of genomic DNA for each sample. For each sample, >99% of the SNPs were determined unequivocally. Scan images were processed with Affymetrix Micro Array Suite Software. Data were analyzed with GDAS v2 software. PedCheck was used for the detection of Mendelian errors.³²

In the parametric linkage analysis, the trait model (mode of inheritance, disease-allele frequency, and penetrance of genotypes) must be specified, which is a disadvantage when the true disease-model parameters are unknown.³³ Since the parameters of the disease model were uncertain, in the initial genome scan, we assessed the evidence of linkage with a nonparametric, penetrance-independent, affected-only, and allele-sharing model. On finding significant evidence of linkage by exceeding the predetermined threshold (P<.005) with an allele-sharing method, we fitted a range of parametric models to the data. The linkage package MERLIN, which can efficiently handle thousands of genotypes,³⁴ was used for nonparametric linkage (NPL) analysis. We calculated multipoint LOD scores maximized over genetic model parameters (MOD score analysis). For this analysis, we used the program Genehunter-Modscore,³⁵ which calculates MOD scores by varying the disease-allele frequency and penetrance. The genomic positions of SNPs are derived from the National Center for Biotechnology Information (NCBI) (Build 35.1).

A genomewide NPL scan revealed NPL>3.5 and P<.0005 for CL-P loci at 11 chromosomal regions: 1p32, 3q26, 4q28, 6p12, 7p21, 9p23, 10q25, 11p11, 13q33, 14q32, and 18q21 (table 1). Among them, the best evidence was found for chromosome 13q33.1-34. A maximum multipoint NPL was yielded for SNP marker rs1830756 (106,878,224 bp) on chromosome 13q33.1-34 (NPL=5.57 and P=.00024). These results are also supported by parametric linkage analysis (MOD score analysis). We used the “modcalc single” option, under which Genehunter-Modscore performs a separate maximization for each genetic position assumed for the putative disease locus. This procedure yields the MOD score in conjunction with the best-fitting penetrance and disease-allele frequency at each genetic position. Under the best-fitted autosomal dominant model (100% penetrance and disease-allele frequency 0.00001), the LOD score was 4.45 and heterogeneity LOD (HLOD) was 4.45 (α=1.0). The family-specific results, the combined parametric and nonparametric multipoint linkage results at the peak SNP (rs1830756), and adjacent SNPs are shown in table 2.

Table 1. .

Initial Genome-Scan Results, Indicating Suggestive Evidence at Various Chromosomes Obtained with Parametric and Nonparametric Linkage (P<.005)

	Position		Nonparametric		Parametric
SNP Marker	Cytogenetic	Physicala (bp)	NPL	P	LOD	HLOD	α
rs1417367	1p32.3	52,010,490	3.74	.0046	1.48	1.48	1.00
rs1402229	3q26.33	181,552,274	4.44	.0013	1.72	1.50	.52
rs1878989	4q28.1	125,204,845	3.93	.0039	1.61	1.61	1.00
rs1372568	6p12.3	46,413,413	4.22	.0027	1.59	1.59	1.00
rs717698	7p21.3	8,167,488	3.55	.0066	.31	.71	.47
rs1343535	9p23	10,004,143	3.84	.0042	.28	.00	.00
rs765651	10q25.1	109,599,290	4.08	.0031	.12	.78	.48
rs2204184	11p11.12	50,418,021	4.08	.0031	.78	1.76	.50
rs1830756	13q33.3	106,878,224	5.57	.0002	4.44	4.44	1.00
rs719252	14q32.32	102,682,945	4.44	.0013	2.89	2.89	1.00
rs959655	18q21.1	48,132,862	3.64	.0066	.98	1.75	.50

^aDerived from the ^NCBI (Build 35.1).

Table 2. .

Family-Specific Results with Combined Parametric and Nonparametric Linkage Scores for the Peak Region at Chromosome 13q33.1-34^[Note]

			Family UR019			Family UR017			Total (UR019 and UR017)
	Position		Parametric	Nonparametric		Parametric	Nonparametric		Parametric		Nonparametricb
SNP Marker	Cytogenetic	Physicala (bp)	LOD	Z Mean	P	LOD	Z Mean	P	LOD	HLOD	Z Mean	P
rs1923862	13q33.1	103,629,222	−3.63	.74	.52344	2.11	3.08	.03125	−1.52	1.51	2.70	.03912
rs728555	13q33.2	103,755,724	1.55	1.42	.01563	2.11	3.08	.03125	3.65	3.66	3.19	.02393
rs1590919	13q33.2	103,813,224	1.62	1.54	.01563	2.11	3.08	.03125	3.72	3.72	3.27	.02393
rs1590918	13q33.2	103,813,401	1.62	1.54	.01563	2.11	3.08	.03125	3.72	3.72	3.27	.02393
rs726039	13q33.2	103,947,454	1.71	1.72	.01563	2.11	3.08	.03125	3.81	3.81	3.40	.02393
rs930268	13q33.2	104,050,671	1.81	1.99	.01563	2.10	3.08	.03125	3.92	3.92	3.58	.02393
rs951095	13q33.2	104,276,645	1.95	2.43	.00781	2.10	3.08	.03125	4.05	4.05	3.89	.02130
rs952018	13q33.2	104,988,136	2.20	3.69	.00781	2.10	3.07	.03125	4.30	4.30	4.78	.00098
rs1933331	13q33.2	105,099,046	2.22	3.82	.00781	2.10	3.07	.03125	4.32	4.32	4.87	.00079
rs2149144	13q33.2	105,246,932	2.25	4.01	.00781	2.10	3.07	.03125	4.35	4.35	5.01	.00058
rs1927571	13q33.2	105,263,158	2.26	4.03	.00781	2.10	3.07	.03125	4.35	4.35	5.02	.00058
rs718831	13q33.3	105,841,769	2.33	4.63	.00781	2.10	3.08	.03125	4.43	4.43	5.45	.00024
rs1986664	13q33.3	106,099,421	2.34	4.73	.00781	2.10	3.08	.03125	4.44	4.44	5.53	.00024
rs2391376	13q33.3	106,199,369	2.34	4.76	.00781	2.10	3.08	.03125	4.44	4.44	5.55	.00024
rs288742	13q33.3	106,299,805	2.34	4.77	.00781	2.10	3.09	.03125	4.45	4.45	5.55	.00024
rs1927756	13q33.3	106,515,980	2.34	4.78	.00781	2.11	3.09	.03125	4.45	4.45	5.56	.00024
rs1323672	13q33.3	106,870,516	2.34	4.79	.00781	2.10	3.08	.03125	4.44	4.44	5.57	.00024
rs1830756	13q33.3	106,878,224	2.34	4.79	.00781	2.10	3.08	.03125	4.44	4.44	5.57	.00024
rs1935132	13q33.3	107,125,954	2.34	4.78	.00781	2.09	3.09	.03125	4.44	4.44	5.56	.00024
rs1935134	13q33.3	107,126,104	2.34	4.78	.00781	2.09	3.09	.03125	4.44	4.44	5.56	.00024
rs1935135	13q33.3	107,126,178	2.34	4.78	.00781	2.09	3.09	.03125	4.44	4.44	5.56	.00024
rs816958	13q33.3	107,320,896	2.34	4.75	.00781	2.10	3.09	.03125	4.44	4.44	5.54	.00024
rs725122	13q33.3	107,856,400	2.34	4.74	.00781	2.11	3.09	.03125	4.44	4.44	5.53	.00024
rs2391644	13q33.3	107,949,230	2.33	4.71	.00781	2.11	3.09	.03125	4.44	4.44	5.51	.00024
rs726449	13q33.3	107,949,490	2.33	4.71	.00781	2.11	3.09	.03125	4.44	4.44	5.51	.00024
rs984300	13q33.3	108,253,836	2.32	4.57	.00781	2.11	3.09	.03125	4.42	4.42	5.41	.00024
rs719737	13q34	109,148,350	2.27	4.20	.00781	2.10	3.07	.03125	4.37	4.37	5.14	.00031
rs496916	13q34	109,649,015	2.22	3.80	.00781	2.10	3.07	.03125	4.32	4.32	4.86	.00079
rs1961495	13q34	109,679,374	2.22	3.78	.00781	2.10	3.07	.03125	4.31	4.31	4.85	.00098
rs953386	13q34	109,741,693	2.21	3.76	.00781	2.10	3.07	.03125	4.31	4.31	4.83	.00098
rs1411040	13q34	109,741,922	2.21	3.76	.00781	2.10	3.07	.03125	4.31	4.31	4.83	.00098
rs2391882	13q34	110,377,552	2.16	3.36	.00781	2.10	3.08	.03125	4.25	4.25	4.55	.00171
rs1894756	13q34	110,816,774	2.00	2.52	.00781	2.11	3.08	.03125	4.10	4.10	3.96	.00894
rs953360	13q34	110,972,580	1.95	2.32	.00781	2.10	3.07	.03125	4.06	4.06	3.81	.02264
rs1411628	13q34	110,997,322	1.95	2.30	.01563	2.10	3.07	.03125	4.05	4.05	3.80	.02264
rs1113337	13q34	111,132,750	1.91	2.13	.01563	2.10	3.05	.03125	4.01	4.01	3.67	.02374
rs1113336	13q34	111,132,873	1.90	2.13	.01563	2.10	3.05	.03125	4.01	4.01	3.67	.02374
rs1112333	13q34	111,133,498	1.90	2.13	.01563	2.10	3.05	.03125	4.01	4.01	3.67	.02374
rs726455	13q34	111,699,501	−3.66	.50	.52344	−∞	2.93	.03125	−∞	.00	2.42	.67631

Note.— The model was used under full-penetrance dominant inheritance with 0.000001 disease-allele frequency.

^aDerived from the ^NCBI (Build 35.1).

^bα=1 for all markers.

A second interesting region was identified at 14q32 (NPL=4.44; P=.0013). Parametric analysis under a dominant model yielded a LOD score of 2.89 at marker rs2024863, which is ∼19 cM away from the NPL peak. Similar MOD score analyses among the other suggested genomic positions gave nonsignificant results with a range of 0.12–1.72.

It has been demonstrated that applying linkage analyses that assume linkage equilibrium to dense markers may lead to bias,^36,37 especially in the analysis of SNP linkage maps in data sets in which some parental genotypes are missing. Therefore, we assessed the impact of linkage disequilibrium (LD) on linkage at chromosome 13. We used MERLIN to accommodate marker-to-marker LD in both parametric and nonparametric analyses, by organizing closely located adjacent markers into clusters. Although many empirical studies have shown that the extent and distribution of LD are extremely variable throughout the genome, in most cases, significant LD does not influence markers separated by >0.1 cM in outbred populations.^38–40 Accordingly, we used markers within 0.2 cM of each other in a cluster. Several clusters of two to six SNPs demonstrated LD. With the assumption of no LD within the cluster, MERLIN uses population haplotype frequencies while calculating linkage. At chromosome 13, the LOD score and NPL score are reduced to 3.46 and 3.03 (P=.002), respectively. However, this reduction in linkage scores might be due to both the effect of LD as well as the reduction of information content. Because of the clustering (hence, the reduction of markers), the information content was reduced from 89% to 74% at the peak region. Nonetheless, evidence of linkage, especially the parametric LOD score, at 13q33.1-34 is still very significant.

Haplotype analysis for the 13q-linked region was performed. A total of 45 informative SNP markers on 13q33.1-34 were used. Haplotype analysis (fig. 2) revealed informative recombination events in the affected individual V-7 (7815) of family UR019, with the candidate susceptibility locus confined to a region distal to rs951095 (map position 104,276,645 bp) and proximal to rs726455 (map position 111,699,501 bp), with a 20.17-cM genetic interval that corresponds to 7.42 Mb.

Figure 2. .

Partial pedigrees of UR017 and UR019, with genotypes and haplotypes of chromosome 13q. SNP markers are shown below selected individuals. Haplotypes associated with affected status are shown in red. Haplotype analysis indicated that the cosegregating segment of the CL-P locus is flanked proximally by rs951095 and distally by rs726455 on chromosome 13q33.1-34.

This interval could not be further narrowed because samples from additional individuals in these families were not available. The genomic interval between these two SNP markers contains 18 putative transcripts (Ensembl). Potential candidate genes in this region might include transcription factor DP1 (TFDP1 [MIM 189902]), inhibitor of growth 1 (ING1 [MIM 601566]), and a tumor suppressor, α-1 chain of collagen IV (COL4A1 [MIM 120130]). Mutation analysis for each transcript is required to first detect sequence variants and then to determine which of these are associated with the CL-P phenotype in families UR017 and UR019. In addition, all conserved noncoding elements need to be included in the mutation analysis.

Various parametric and nonparametric association and linkage studies of different populations provided evidence of several loci on various chromosomal regions contributing to CL-P.^{15–19,41–48} A number of additional candidate genes and regions also have been proposed through observations from chromosomal abnormalities in patients with CL-P.^49,50 A few reports strongly supported the involvement of the IRF6 gene in some families with nonsyndromic CL-P^21,22,51; however, in the present study, we did not observe any positive association. A previous linkage study conducted with 38 multiplex Indian CL-P families yielded weak evidence at multiple loci⁵²; however, none of these previous linkage studies gave evidence involving chromosome 13 for the CL-P phenotype.

CL-P is very common in patients associated with trisomy involving all or part of chromosome 13.²⁵ Increased incidence of chromosome 13–related anomalies are reported to be involved with CL-P.^53–64 Najafzadeh et al.⁶⁵ reported a newborn infant with CL-P and a 46,XX,13q+ karyotype derived from a paternal t(4;13)(q25;q32), with resulting del(13q) and dup(4q). A female with multiple congenital anomalies, including CL-P, and a karyotype of 46,XX,-13,+t(13q;13q) has been reported.⁶⁶ A fetus with various developmental anomalies, including cleft lip, who had duplication of 13q32→qter due to unbalanced segregation of t(4;13)(p16;q32) in her father, was also reported.⁶⁷

Multigenerational families with an autosomal dominant and an autosomal recessive inheritance of CL-P with reduced penetrance have been reported.^18,31 An autosomal dominant form of inheritance is likely in the two pedigrees reported here. Families UR017 and UR019 live in the same village of <15,000 inhabitants. It is possible that there was a common ancestor for these families; however, for several generations, no marriages were reported between these two. Therefore, we could not establish any demonstrable relationship between them. Haplotype analysis from the 13q-linked region of the affected individuals did not find extensive common haplotypes shared between these two families, although there were some patches of identical alleles observed. Identification of the susceptibility variation at 13q33.1-34 for nonsyndromic CL-P in an Asian population will foster a better understanding of the molecular pathophysiology of this developmental anomaly.