Orofacial Cleft 1

Description

Nonsyndromic cleft lip with or without cleft palate is a complex disease with a wide phenotypic spectrum ranging from notches of the vermilion and/or grooves in the philtrum to complete unilateral and bilateral clefts of the lip and palate (summary by Neiswanger et al., 2007).

Genetic Heterogeneity of Orofacial Cleft

Isolated cleft lip with or without cleft palate (CL/P) is genetically heterogeneous. The OFC1 locus has been mapped to chromosome 6p24. Other CL/P loci have been mapped to 2p13 (OFC2; 602966), 19q13 (OFC3; 600757), 4q (OFC4; 608371), 13q33.1-q34 (OFC9; 610361), 8q24.3 (OFC12; 612858), and 1p33 (OFC13; 613857).

OFC5 (608874) is caused by mutation in the MSX1 gene (142983) on 4p16; OFC6 (608864) is associated with variation in an enhancer of the IRF6 gene (607199) on 1q; OFC7 (see 225060) is associated with mutation in the NECTIN1 gene (600644) on 11q23; OFC8 (618149) is caused by mutation in the TP63 gene (603273) on 3q28; OFC10 (613705) is associated with haploinsufficiency of the SUMO1 gene (601912) on 2q33; OFC11 (600625) is caused by mutation in the BMP4 gene (112262) on 14q22; OFC14 (615892) is associated with a 273-kb deleted region on 1p31; and OFC15 (616788) is caused by mutation in the DLX4 gene (601911) on 17q21.

A common polymorphism in the MTR gene (156570.0008) has been associated with susceptibility to orofacial clefting. Cleft lip with or without cleft palate has been found in association with gastric cancer (see 137215) in individuals with mutation in the CDH1 gene (192090).

Clinical Features

Several studies had demonstrated an association between facial shape in parents and the presence of oral clefts in their offspring. It was assumed that facial shape was one predisposing component among many in a multifactorial model of inheritance. By cephalometric analysis of a large family with 5 generations of affected individuals, Ward et al. (1994) concluded that facial shape can be used to identify presumed carriers of a major gene associated with an increased risk for oral clefts. Discriminant function analysis indicated that at-risk individuals could be recognized through a combination of increased midfacial and nasal cavity widths, reduced facial height, and a flat facial profile. The use of this approach in providing critical information needed in the search for molecular markers that segregate with the genetic risk for clefting was emphasized.

Using quantitative MRI analysis, Nopoulos et al. (2002) compared the brain morphology of adult males with nonsyndromic cleft lip and/or cleft palate with matched healthy controls. They found that subjects with nonsyndromic cleft lip and/or palate had significant abnormalities in brain morphology, consisting of abnormally enlarged anterior regions of the cerebrum, and decreased volumes of the posterior cerebrum and cerebellum. Overall, the most severely affected region was the left temporal lobe.

Neiswanger et al. (2007) noted that the spectrum of severity in visible CL/P is broad, ranging from notches of the vermilion and/or grooves in the philtrum to complete unilateral and bilateral clefts of the lip and palate. Minimal or microform expressions of the CL/P phenotype, typically involving subtle defects of the lip, alveolar arch, and/or inferior nasal region, are at the mild end of the spectrum. Using high-resolution ultrasonography to examine the orbicularis oris muscle, Neiswanger et al. (2007) found that 10.3% of 525 noncleft relatives of patients with nonsyndromic cleft lip had discontinuity of the orbicularis oris muscle compared to 5.8% of 257 controls (p = 0.04). Male relatives had a significantly higher rate of discontinuity than male controls (12.0% vs 3.2%; p = 0.01); female relatives also had a higher rate of discontinuity than female controls, but the increase was not statistically significant. These data confirm the hypothesis that subepithelial defects in the orbicularis oris are a mild manifestation of the cleft lip phenotype.

Neiswanger et al. (2009) analyzed lip prints from more than 450 individuals, including CL/P patients, their family members, and controls, from the United States, Argentina, and Hungary. Whether using a narrow or broad definition of lower-lip whorl, whorls were associated with CL/P. Under a narrow definition, the frequency of whorls in the US sample was significantly elevated in CL/P patients and their unaffected relatives compared to controls (p = 0.003 and 0.001, respectively), but whorl frequencies did not differ significantly between CL/P patients and their noncleft relatives. In the Argentinian sample, CL/P patients had significantly more whorls than their unaffected family members (p = 0.04, for narrow or broad definition of lower-lip whorl); unrelated Argentinian controls were not available. None of the participants from Hungary had a definite whorl on their lower lip. Neiswanger et al. (2009) suggested that whorl lip print patterns might be part of an expanded phenotypic spectrum of nonsyndromic CL/P.

Inheritance

Over 200 syndromes, including a number that are either chromosomal or mendelian in causation, have cleft lip and/or palate as feature(s) (Gorlin, 1982). It is clear from family studies that isolated cleft palate (119540) is genetically distinct from cleft lip with or without cleft palate. Curtis et al. (1961) estimated that the risk of recurrence of CL/P in subsequently born children is 4% if one child has it, 4% if one parent has it, 17% if one parent and one child have it, and 9% if two children have it. The syndrome of cleft lip with or without cleft palate in association with mucous pits of the lower lip is inherited as an autosomal dominant (119300).

Carter et al. (1982) followed up on the families of cases of CL/P operated on at The Hospital for Sick Children ('Great Ormond St.'), London, between 1920 and 1939, to obtain information on the proportion affected of children and grandchildren. The probands were those who had survived, were successfully traced, and found to have had at least 1 child. Patients of the 1920-1939 period traced through a child, either normal or affected, were excluded, as were patients with recognized syndromes. The proportion affected of children of probands was 3.15%, of sibs 2.79%, and of parents 1.18%. The lower proportion of parents affected was attributed to reduced reproductive fitness of patients born 2 generations ago. The proportion affected of nephews and nieces, aunts and uncles, and grandchildren was 0.47%, 0.59% and 0.8%, respectively. The proportion affected of first cousins was 0.27%. The birth frequency in England was estimated to be about 0.1%. The proportion of sibs affected increased with increasing severity of the malformation in the proband, when the proband was female, and when the proband had an affected parent or already had 1 affected sib. Carter et al. (1982) concluded that the most economical explanation of the findings is the multifactorial threshold model and that a single mutant gene in unlikely.

Chung et al. (1986) analyzed the genetics of CL/P on a comparative basis, in the Danish (Bixler et al., 1971; Melnick et al., 1980) and Japanese (Koguchi, 1975) data. Japanese are known to have a higher population incidence of CL/P and yet a lower recurrence risk among relatives than is true in Caucasian populations. Chung et al. (1986) concluded that the Danish data is best explained by a combination of major gene action and multifactorial inheritance. The major gene was thought to be recessive with a frequency of 0.035. Heritability was estimated as 0.97. On the contrary, the Japanese data could best be accounted for only by multifactorial inheritance with the heritability estimate of 0.77.

Following previous studies suggesting that symmetry for certain bilaterally represented features may be an indicator of genetic predisposition to CL/P, Crawford and Sofaer (1987) devised an asymmetry score which correctly classified 85% of familial cleft patients and unrelated noncleft controls. Applying the same stepwise logistic regression to sporadic cases, 26% fell into the range occupied by the majority of familial patients, suggesting that these had a high level of genetic predisposition.

Temple et al. (1989) described cleft lip and palate in 3 generations of each of 2 families; in 1 family, there was an instance of male-to-male transmission.

Hecht (1990) presented the pedigrees of 11 families with multigenerational involvement of cleft lip and palate. One family had affected persons in 3 successive generations. Hecht et al. (1991) performed complex segregation analysis of nonsyndromic CL/P in 79 families ascertained through a proband diagnosed at the Mayo clinic. In one analysis, the dominant or codominant mendelian major locus models of inheritance provided the most parsimonious fit. In another, the multifactorial threshold model and the mixed model were also consistent with the data. However, the high heritability (0.93) in the multifactorial threshold model suggested that any random exogenous factors were unlikely to be the underlying mechanism, and the mixed model indicated that this high heritability was accounted for by a major dominant locus component. Thus, the best explanation for the findings of the study was a putative major locus associated with markedly decreased penetrance.

In a reanalysis of recurrence patterns from several family studies of CL/P, Mitchell and Risch (1992) found that the recurrence patterns in first-degree relatives were compatible with expectations for either a multifactorial threshold trait or a generalized (precise mode of inheritance unspecified) single major locus trait. The use of multiple thresholds based on proband sex, defect bilaterality, or palate involvement did not help to discriminate between these models. They concluded, however, that the pattern of recurrence among MZ twins and more remote relatives is not consistent with the single-major-locus inheritance but is compatible with either a multifactorial threshold model or a model specifying multiple interacting loci. For such a model, no single locus could account for more than a 6-fold increase in risk to first-degree relatives. Between 1980 and 1987 in Shanghai, the birth incidence of nonsyndromic CL/P was 1.11/1,000, with a male/female ratio of 1.42 (Marazita et al., 1992).

Marazita et al. (1992) analyzed family data from almost 2,000 probands ascertained from among individuals operated on during the years 1956-1983 at surgical hospitals in Shanghai. They rejected the hypothesis of no familial transmission and of multifactorial inheritance alone. Of the major locus models, the autosomal recessive was significantly more likely. They concluded that the best-fitting, most parsimonious model for CL/P in Shanghai is that of an autosomal recessive major locus.

In West Bengal, India, Ray et al. (1993) ascertained 90 extended families having 1 or more individuals affected with CL/P. They concluded that the hypothesis of major-locus inheritance alone could not be rejected. Among major-locus models examined, strictly recessive inheritance was rejected, but codominant and dominant models were not. Neither the addition of a multifactorial component nor the addition of a proportion of sporadic cases to the major-locus model improved the fit of the data.

Population Genetics

Murray et al. (1997) reported the results of epidemiologic studies of CL/P ascertained from 6 sites within the Philippines between 1989 and 1996. The findings included a birth prevalence of 1.94 per 1,000 live births for CL/P. Recurrence rates in sibs for nonsyndromic CL/P were 23 per 1,000 for cleft lip with or without cleft palate and 14 per 1,000 for cleft palate only. The percentage of clefts associated with multiple congenital anomalies was 21% at birth and 6% for individuals examined during the screening process. These results indicated a high postnatal death rate. The data suggested a high incidence of cleft lip and palate in native-born Filipinos.

Mapping

Eiberg et al. (1987) selected 58 pedigrees with nonsyndromic orofacial cleft from among a comprehensive collection of Danish cases for apparent autosomal dominant inheritance. Linkage with 42 non-DNA polymorphic marker systems was investigated. Both CL/P and cleft palate only (CPO) were, for the purpose of linkage analysis, scored as if being due to an autosomal dominant gene with complete penetrance. Linkage was found with clotting factor XIIIA (134570); for males alone, the maximal lod score was 3.40 at theta = 0.00; for females alone, 0.30 at theta = 0.21; and for these combined, 3.66 at theta = 0.00 for males, and theta = 0.26 for females. The findings were taken to suggest that since F13A is located on the distal portion of 6p, a major locus for nonsyndromic orofacial cleft is also located in this region. Since both cleft lip with or without cleft palate and isolated cleft palate pedigrees contributed to the positive score, it is possible that the locus on 6p carries 2 cleft alleles. In a study of 12 autosomal dominant families with nonsyndromic cleft lip with or without cleft palate, Hecht et al. (1993) excluded linkage with HLA and F13A. Multipoint analysis showed no evidence of a clefting locus in a region spanning 54 cM on 6p in these CL/P families.

Davies et al. (1995) used YAC clones from contigs in 6p25-p23 to investigate 3 unrelated patients with cleft lip/palate who showed abnormalities of 6p. Case 1 had bilateral cleft lip and palate, and a balanced translocation reported as 46,XY,t(6,7)(p23;q36.1). Case 2 had multiple anomalies, including cleft lip and palate and was reported as 46,XX,del(6)(p23;pter). Case 3 had bilateral cleft lip and palate and carried a balanced translocation reported as t(6;9)(p23;q22.3). Davies et al. (1995) identified 2 YAC clones, both of which crossed the breakpoint in cases 1 and 3 and were deleted in case 2. These clones mapped to 6p24.3 and, therefore, suggested the presence of a locus for orofacial clefting in that region.

Knowledge of the number of causative loci is necessary to estimate the power of mapping studies of complex diseases. Schliekelman and Slatkin (2002) reexamined a theory developed by Risch (1990) and its implications for estimating the number of causative loci. They showed that methods based on Risch's analysis could produce estimates that are inconsistent with the observed population prevalence of the disease. They showed how to incorporate disease prevalence and developed a maximum likelihood method for estimating the number of causative loci that uses the entire distribution of numbers of affected individuals in families. They stated that the method avoids the potential inconsistencies of the Risch method and has greater precision. They used cleft lip/palate and schizophrenia (181500) as examples to which they applied their method for estimating the number of loci underlying an inherited disease.

The division of clefts of the face into those that include the secondary palate only (the posterior or soft palate) or cleft palate only, and those that involve the primary palate and encompass clefts of the lip with or without the palate is valid, not only on genetic grounds, but also on embryologic grounds, since the primary and secondary palates form independently. Only in the van der Woude syndrome (119300) is a mixing of embryologic and genetic types, i.e., cleft palate only in some individuals and cleft lip with or without cleft palate in others, seen with any frequency (Burdick et al., 1985). Murray (1995) reviewed the genetic and exogenous factors in the causation of facial clefts that have been demonstrated or suspected. He concluded that 'the strongest evidence implicates a primary gene on 6p and a role of transforming growth factor alpha as a modifier of clefting status.'

Mitchell and Christensen (1996) linked data from 2 centralized data repositories in Denmark, the Danish Central Person Registry and the Danish Facial Cleft Database, and estimated the risks to first, second, and third-degree relatives of 3,073 CL/P probands born in Denmark from 1952 to 1987. Analyses of these data excluded single locus and additive multilocus inheritance and provided evidence that CL/P is most likely determined by the effects of multiple interacting loci. Under a multiplicative model, no single locus could account for more than a 3-fold increase in risk to first-degree relatives. These data provided further evidence that nonparametric linkage methods, for example, affected relative pair studies, are likely to represent a more realistic approach for identifying CL/P susceptibility loci, than are traditional pedigree-based methods. However, at least 100 and, more realistically, several hundred affected sib pairs are likely to be required to detect linkage to CL/P susceptibility loci.

The CL/P malformation has been associated with chromosomal aberrations involving 6p. As noted previously, Davies et al. (1995) investigated 3 unrelated cases of CL/P coincident with 6p region aberrations. They mapped the breakpoint to 6p24.3 near the HGP22 and AP2 genes, which are potentially involved in face formation. Linkage studies have yielded both positive and negative evidence concerning mapping of CL/P to this region or other regions of the short arm of chromosome 6. Scapoli et al. (1997) conducted a linkage study of 38 families using microsatellite markers that mapped to 6p24-p23. The admixture test, as implemented in the HOMOG program, was significant when tested against multipoint data; the lod score calculated, assuming heterogeneity, was 3.60 at 1 cM telomeric to D6S259. Taken together, these data were considered to demonstrate the presence of a locus for CL/P in the 6p23 chromosome region.

Genetic Heterogeneity

Prescott et al. (2000) reported the findings from a 2-stage genomewide scan of 92 affected sib pairs searching for susceptibility loci to CL/P. Eleven regions on 8 chromosomes were found to have a P value smaller than 0.05. These 8 chromosomes were then further mapped with a second set of markers to increase the average map density. In 7 of 11 areas densely mapped, significance was markedly increased by decreasing the marker interval. Although none reached the level required for significant susceptibility loci, 2 of these areas had previously been implicated in CL/P, viz., 2p13, an area harboring the TGFA gene (190170), and 6p24-p23. The authors also demonstrated highly suggestive linkage to a susceptibility locus for nonsyndromic clefting on the X chromosome.

In a study of 5 different candidate genes, Scapoli et al. (2002) found significant linkage disequilibrium between the GABRB3 gene (137192) on chromosome 15q11.2-q12 and nonsyndromic cleft lip with or without cleft palate. They noted that knockout of the Gabrb3 gene in mice causes clefting of the secondary palate only (Homanics et al., 1997). Tanabe et al. (2000) found no evidence that the GABRB3 gene is involved in clefting in Japanese cases.

In 36 Filipino families with 3 or more affected members, Schultz et al. (2004) examined 15 candidate regions for linkage to nonsyndromic cleft lip and palate. They concluded that their data combined with data from previous studies supported further investigation of 1p36, 2p13, 4p16, 6p23, 16q22, and 21q22.

Blanton et al. (2004) screened 6 candidate regions in 65 multiplex families with nonsyndromic cleft lip and palate and found that markers in 3 regions, 3p21, 10p13, and 16p13.3, yielded findings sufficiently significant to warrant closer investigation. In a separate study involving the same families, Blanton et al. (2004) analyzed 37 markers in 10 regions and demonstrated linkage and association in 4 chromosomal regions, 2q37, 11p12-p14, 12q13, and 16p13, in which linkage to nonsyndromic cleft lip/palate had been found in 2 other independent reports.

Marazita et al. (2004) reported a 10-cM genome scan of 388 extended multiplex families with CL/P from 7 diverse populations, revealing CL/P genes in 6 chromosomal regions, including a novel region at 9q21 (hlod = 6.6). In addition, metaanalyses with the addition of results from 186 more families (6 populations) showed genomewide significance for 10 more regions, including another novel region at 2q32-q35 (p = 0.0004); see OFC10 (601912).

Vieira et al. (2005) cited the estimate of the average birth prevalence of nonsyndromic or isolated cleft lip with or without cleft palate as 1 in 700, with a wide geographic distribution. They used direct sequencing as an approach to study candidate genes for CL/P. They reported results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. The genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For 7 genes with identified coding mutations that are potentially etiologic, they performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The cleft-related MSX1 mutation P147Q (142983.0007) was also studied in an additional 1,098 cleft cases. The aggregate data on these 7 genes suggested that the point mutations in them are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggested that point mutations in FOXE1 (602617), GLI2 (165230), MSX2 (123101), SKI (164780), SATB2 (608148), TBX10 (604648), and SPRY2 (602466) may be rare causes of isolated cleft lip with or without cleft palate. The linkage disequilibrium data supported a larger, as yet unspecified, role for variants in or near MSX2, JAG2 (602570), and SKI. Vieira et al. (2005) emphasized the need to test large numbers of controls to distinguish rare polymorphic variants.

In a case-control study of Brazilian families with CL/P, Gaspar et al. (2004) observed that with the presence of a maternal MTHFR 677T allele (607093.0003) there was an increased likelihood of offspring having the less common non-135-bp BCL3 allele (OR, 2.3, 95% CI, 1.1-4.8, P = 0.03). Gaspar et al. (2004) suggested that maternal MTHFR genotype plays a significant role in susceptibility to CL/P, but its teratogenic effect depends on the genotype of the offspring.

Park et al. (2006) described an approach combining statistical evidence on large numbers of single-nucleotide polymorphism (SNP) markers typed in case-parent trios with expression data to identify candidates for complex disorders. The disorder studied was nonsyndromic oral cleft. Sixty-four candidate genes were genotyped using the BeadArray approach in 58 case-parent trios. Thirteen candidate genes showed significant evidence of linkage in the presence of disequilibrium, and 10 of these were found to be expressed in relevant embryonic tissues (those involved in palate and lip development): SP100 (604585), MLPH (606526), HDAC4 (605314), LEF1 (153245), C6orf105, CD44 (107269), ALX4 (605420), ZNF202 (603430), CRHR1 (122561), and MAPT (157140). Three other genes showing statistical evidence were not expressed in the embryonic tissues examined in this study: ADH1C (103730), SCN3B (608214), and IMP5 (608284). Many of these genes had not previously been studied as candidates for oral cleft and warranted further investigation.

Shi et al. (2007) followed up on the recognized risk for orofacial clefts related to maternal smoking. Maternal or fetal pharmacogenetic variants were considered plausible modulators of this risk. They studied 5,427 DNA samples, including 1,244 from subjects in Denmark and Iowa with facial clefting and 4,183 from patients, sibs, or unrelated population controls. They examined 25 single-nucleotide polymorphisms in 16 genes in pathways for detoxification of components of cigarette smoke to look for evidence of gene-environment interactions. In genes identified as related to oral clefting, they studied gene expression profiles in fetal development in the relevant tissues and time intervals. Maternal smoking was a significant risk factor for clefting and showed dosage effects, in both the Danish and Iowan data. Suggestive effects of variants in the fetal NAT2 (612182) and CYP1A1 (108330) genes were observed in both the Iowan and the Danish participants. In an expanded case set, NAT2 continued to show significant overtransmission of an allele to the fetus, with a final P value of 0.00003. There was an interaction between maternal smoking and fetal inheritance of a GSTT1-null deletion (600436) seen in both the Danish and Iowan studies. Gene expression analysis demonstrated expression of GSTT1 in human embryonic craniofacial tissues during the relevant developmental interval. This study benefited from 2 large samples, involving independent populations, that provided substantial power and a framework for future studies that could identity a susceptible population for preventive health care.

Chiquet et al. (2008) presented evidence that variation in WNT genes may play a role in susceptibility to cleft lip with or without cleft palate. SNP analysis of 132 affected multiplex families and 354 simplex parent-child trios found an association between the phenotype and SNPs in the WNT3A gene (606359) on chromosome 1q42 (p = 0.006), WNT5A gene (164975) on chromosome 3p21-p14 (p = 0.002), and WNT11 gene (603699) on chromosome 11q13.5 (p = 0.0001).

In a family-based association study of 218 case/parent triads with nonsyndromic CL/P, Martinelli et al. (2007) found suggestive association with 2 SNPs adjacent to the MYH9 gene (160775) on chromosome 22q11.2, rs3752462 and rs2009930 (global p value = 0.001). Chiquet et al. (2009) genotyped 6,008 SNPs in 9 Caucasian NSCLP multiplex families and a single large African American NSCLP multiplex family and identified 14 chromosomal regions with lod scores greater than 1.5, including 22q12.2-q12.3 (lod score, 2.66) and 8q21.3-24.12 (lod score, 2.98), respectively. Analysis of the candidate MYH9 gene revealed linkage between rs1002246 in intron 10 and the entire dataset; however, the previously identified MYH9 SNP rs3752462 was only marginally significant, and no high-risk haplotype could be identified. Chiquet et al. (2009) noted that a CLP locus had previously been suggested on chromosome 8q (Prescott et al., 2000; Marazita et al., 2004); Chiquet et al. (2009) found no linkage to 2 candidate genes in the region, SDC2 (142460) and GDF6 (601147).

In a cohort of cleft lip and palate (CL/P) families from Colombia, United States, and the Philippines, Moreno et al. (2009) tested 397 SNPs spanning 9q22-q33 for association. Significant SNP and haplotype association signals narrowed the interval to a 200-kb region containing FOXE1 (602617), C9ORF156, and HEMGN (610715). Association results were replicated in CL/P families of European descent; when all populations were combined, the 2 most associated SNPs, rs3758249 (p = 5.01E-13) and rs4460498 (p = 6.51E-12), were located inside a 70-kb high linkage disequilibrium block containing FOXE1. Association signals for Caucasians and Asians clustered 5-prime and 3-prime of FOXE1, respectively. Isolated cleft palate (CP) was also associated, indicating that FOXE1 may play a role in 2 phenotypes thought to be genetically distinct. Foxe1 expression was found in the epithelium undergoing fusion between the medial nasal and maxillary processes. Mutation screens of FOXE1 identified 2 family-specific missense mutations (ile59 to ser and pro208 to arg) at highly conserved amino acids. Although predicted to be benign by a computer program, both mutations are near previously identified deleterious mutations. The authors concluded that FOXE1 may be a major gene for CL/P and CP.

Beaty et al. (2010) conducted a genomewide association study of nonsyndromic CL/P involving 1,908 case-parent trios from Europe, the United States, China, Taiwan, Singapore, Korea, and the Philippines. SNPs near 2 genes not theretofore associated with CL/P, MAFB (608968, most significant SNP rs13041247, OR per minor allele = 0.704, 95% CI, 0.635-0.778, p = 1.4 x 10(-11)) and ABCA4 (601691, most significant SNP rs560426, OR = 1.432, 95% CI, 1.292-1.587, p = 5.01 x 10(-12)) attained genomewide significance. Beaty et al. (2010) also confirmed 2 previously identified regions, at chromosome 8q24 (see OFC12, 612858) and IRF6 (607199) (see OFC6, 608864). Stratifying trios into European and Asian ancestry groups revealed differences in statistical significance, although estimated effect sizes remained similar. Replication studies from several populations showed confirming evidence, with families of European ancestry giving stronger evidence for markers in 8q24, whereas Asian families showed stronger evidence for association with MAFB and ABCA4. Whole-mount in situ hybridization analysis of Mafb and immunodetection of expressed Mafb in mouse embryos detected Mafb mRNA and protein in both craniofacial ectoderm and neural crest-derived mesoderm between embryonic days 13.5 and 14.5; expression was strong in the epithelium around the palatal shelves and in the medial edge epithelium during palatal fusion. After fusion, Mafb expression was stronger in oral epithelium compared to mesenchymal tissue.

Ingersoll et al. (2010) analyzed the 2-Mb region around the MSX1 gene on chromosome 4p16.1 (see OFC5; 608874) in 297 CL/P case-parent trios and 84 CP case-parent trios from Maryland, Taiwan, Singapore, and Korea, and found evidence suggesting that several genes in this region in addition to MSX1 may influence the risk of oral clefts. Three genetic regions of particular interest included the STK32B gene, the EVC (604831)-EVC2 (607261)-CRMP1 (602462) region, and the STX18 (606046)-MSX1 region.

Ludwig et al. (2012) conducted the first metaanalyses for nonsyndromic cleft lip with or without cleft palate (NSCL/P) using data from the 2 largest genomewide association studies published to that time. They confirmed associations with all previously identified loci and identified 6 additional susceptibility regions (1p36, 2p21, 3p11.1, 8q21.3, 13q31.1, and 15q22). Analysis of phenotypic variability identified the first specific genetic risk factor for nonsyndromic cleft lip plus palate (NSCLP) at rs8001641 on chromosome 13q31.1; p(NSCLP) = 6.51 x 10(-11); homozygote relative risk = 2.41, 95% CI, 1.84-3.16.

Molecular Genetics

In 3 patients with cleft lip/palate who had balanced translocations involving 6p and had previously been studied by Davies et al. (1995), Davies et al. (2004) refined the breakpoint regions and identified 2 positional candidate genes, TFAP2A (107580) and OFCC1 (614287). Breakpoints in the 3 patients were within 375 to 930 kb of the 5-prime end of the TFAP2A gene, suggesting that they might interfere with regulatory elements. In addition, breakpoint mapping in 1 of the patients indicated that the translocation disrupted the OFCC1 gene in intron 3, potentially abolishing full-length transcription of the gene from the translocated chromosome; in the other 2 patients, the breakpoints were relatively close to the 3-prime end of the gene, suggesting the possibility of position effects.

Animal Model

It is noteworthy that there is some homology of synteny between human 6p and mouse chromosome 13. Furthermore, Wakasugi et al. (1988) demonstrated an autosomal dominant mutation of facial development in a transgenic mouse. The facial malformation was characterized by a short snout and a twisted upper jaw. The malformation of the nasal and premaxillary bone was considered to be secondary to a developmental defect in the first branchial arch. In the attempt to establish a mouse model of familial amyloid polyneuropathy, they microinjected the cloned human mutant transthyretin gene (176300) into fertilized eggs. They demonstrated that the insertion occurred in chromosome 13 of the mouse. These results were thought to indicate that the malformation was due to the insertional disruption of a host gene; however, the possibility that this mutation was caused by an inappropriate expression of the injected gene remained to be investigated.