Complex segregation analysis of nonsyndromic cleft lip and palate.

This study was undertaken to examine the inheritance pattern of nonsyndromic cleft lip with or without cleft palate (CL/P). Complex segregation analysis using the unified model as in POINTER and the regressive model as in REGD programs were applied to analyze a midwestern U.S. Caucasian population of 79 families ascertained through a proband with CL/F. In REGD, the dominant or codominant Mendelian major locus models of inheritance were the most parsimonious fit. In POINTER, besides the Mendelian major locus model, the multifactorial threshold (MF/T) model and the mixed model were also consistent with the observed data. However, the high heritability parameter of .93 (SD .063) in the MF/T model suggests that any random exogenous factors are unlikely to be the underlying mechanisms, and the mixed model indicates that this high heritability is accounted for by a major dominant locus component. These findings indicate that the best explanation for the etiology of CL/P in this study population is a putative major locus associated with markedly decreased penetrance. Molecular studies may provide further insight into the genetic mechanism underlying CL/P.     

Genetics of cleft lip and cleft palate in China.

During the past 10 years, 60 cases of cleft lip with or without cleft palate [CL(P)] were recorded among 45,072 newborns at Shanghai International Peace Maternity and Infant Hospital, China. The incidence was 1.33 per 1,000 births. The family histories of 163 CL(P) patients were analyzed. The incidences of CL(P) in the first-, second-, and third-degree relatives of CL(P) patients were 11/246 (4.47%), 10/1,032 (0.97%), and 6/1,727 (0.35%), respectively. Of the 163 probands, three had a history of consanguinity of the parents (1.8%), in contrast to 0.77% in the general population. These data are suggestive of multifactorial inheritance. The heritability of CL(P) in our study calculated by Falconer's formula was 77.6%.     

Familial recurrence-pattern analysis of cleft lip with or without cleft palate.

Cleft lip with or without cleft palate (CL/P) is a common congenital malformation with an incidence in European white populations of about 1/1,000. The familial clustering of CL/P has been extensively characterized, and epidemiological studies have proposed monogenic models (with reduced penetrance), multifactorial/threshold models, and mixed major-gene/multifactorial models to explain its inheritance. The recognition of an association between two RFLPs at the transforming growth factor alpha (TGFA) locus and CL/P supports a major-gene component to the etiology of CL/P. Risch has shown that the recurrence risk ratio lambda R (risk to relatives, vs. population prevalence) is a useful pointer to the mode of inheritance. Here we further develop the use of lambda R to analyze recurrence-risk data for CL/P. Recurrence risks for first-, second-, and third-degree relatives equate well with oligogenic models with as few as four loci. A monogenic/additive model is strongly rejected. The limited available twin data are also consistent with this model. A "major gene" interacting epistatically with an oligogenic background is shown to be a plausible alternative. Power calculations for a linkage study to map the CL/P major-risk locus suggest that a sample of 50 affected sib pairs will be adequate, but linkage to minor-risk loci will require very much larger samples.     

Nonsyndromic cleft lip with or without cleft palate in west Bengal, India: evidence for an autosomal major locus.

Ninety extended families having one or more individuals affected with nonsyndromic cleft lip (CL) with or without cleft palate (CL/P) were ascertained in rural West Bengal, India. These families included 138 affected people, 64% of whom had CL alone and 66% of whom were male. Multiple-affected-member ("multiplex") pedigrees were less common than single-affected-member ("simplex") pedigrees, composing 34% of all extended pedigrees. There was no difference between multiplex and simplex pedigrees in the frequency of affected persons with CL alone, but multiplex pedigrees had a lower frequency of affected males (58%) than did simplex pedigrees (76%; P = .02). Complex segregation analysis using the POINTER computer program rejected both the hypothesis of no familial transmission (P < .0001) and the hypothesis that familiarity could be explained solely by a multifactorial/threshold model (P < .05). The hypothesis of major-locus inheritance alone could not be rejected. Among major-locus models examined, strictly recessive inheritance was rejected (P < .0001), 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. In conclusion, the results of complex segregation analysis were consistent with a dominant or codominant major-locus mode of inheritance of CL/P in these families.     

Mode of inheritance of nonsyndromic cleft lip with or without cleft palate: a reanalysis.

Nonsyndromic cleft lip with or without cleft palate (CL +/- P) is traditionally recognized as a multifactorial threshold trait (MFT). Recently, however, evidence for the involvement of a major gene in the etiology of CL +/- P has been reported. To assess the potential for major-gene involvement in the etiology of this trait, familial recurrence patterns from several family studies of CL +/- P were reanalyzed. The recurrence patterns in first-degree relatives of CL +/- P probands were found to be compatible with the expectations for either an MFT or a generalized single-major-locus (gSML) trait. The use of multiple thresholds based on proband sex, defect bilaterality, or palatal involvement did not help to discriminate between these models. However, the pattern of recurrence among MZ twins and more remote relatives of CL +/- P probands is not consistent with gSML inheritance but is compatible with either an MFT model or a model specifying multiple interacting loci. For such a model, no single locus can account for more than a sixfold increase in risk to first-degree relatives. These findings have important implications with regard to the feasibility of detecting linkage to loci conferring susceptibility to CL +/- P.     

Segregation analysis of cleft lip with or without cleft palate in the First Nations (Amerindian) people of British Columbia and review of isolated cleft palate etiologies

BACKGROUND: The First Nations (Amerindian) population of British Columbia, Canada, has the highest reported birth prevalence in the world of cleft lip with or without cleft palate (CL/P) at nearly 3 per 1000 births. In addition, a substantial proportion of cleft palate only (CPO) cases in this population has been reported to be X‐linked. The aims of this study were to perform complex segregation analysis to investigate the mode of inheritance of CL/P in the First Nations people of British Columbia and to review the etiology of the CPO cases. METHODS: All First Nations children born in British Columbia between 1952 and 1971 with an orofacial cleft were included in the study. Multiple sources of ascertainment were used, so that nearly 100% of live births were identified and included during this time. No stillbirths were found but would likely have been ascertained. Extended pedigrees were constructed from these probands and examination of immediate family members, e.g., parents and siblings, was done wherever possible. Complex segregation analysis included all family members. In addition, a CPO case review was conducted. RESULTS: Complex segregation analysis supports the hypothesis that the most likely mode of inheritance of CL/P in this population is a mixed model; that is, an autosomal major gene with polygenic component. The review of 26 CPO cases showed that a substantial proportion are syndromic. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

The role of major gene in clubfoot.

The roles of major gene and multifactorial inheritance in the etiology of clubfoot (talipes equinovarus) were studied using Caucasian clubfoot families ascertained in Indiana. The method of analysis used was complex segregation analysis under the mixed model, in which five genetic parameters were examined to test hypotheses on major gene by displacement (t), degree of dominance (d), gene frequency (q), transmission probability (tau2), and multifactorial inheritance by heritability (H). The analysis showed that the segregation pattern of clubfoot in these families is best explained by assuming the action of a major gene with additional contribution of multifactorial inheritance. The estimates of the parameters under the best-fitting model were d = .82, t = 4.69, q = .030, tau2 = .50, and H = .17.     

Linkage disequilibrium between GABRB3 gene and nonsyndromic familial cleft lip with or without cleft palate

The malformation of nonsyndromic cleft lip with or without cleft palate (CL/P) is a common congenital disease that affects approximately 1/1000 newborns in Caucasian populations. Genetic studies indicate that CL/P has the characteristics of a complex genetic trait. Linkage analysis and mouse-model knockout studies have suggested several candidate genes mapping in different chromosome regions for CL/P malformation. On these grounds, we have investigated, by linkage disequilibrium (LD) and parametric and nonparametric linkage analyses, five different candidate genes, including those for the beta3 subunit of the gamma-aminobutyric acid receptor (GABRB3), glutamic acid decarboxylase 1 (GAD1), retinoic acid receptor alpha (RARA), transforming growth factor beta3 (TGFB3), and msh ( Drosophila) homeobox homolog 1 (MSX1). Interestingly, a significant LD between GABRB3 and CL/P was obtained ( P-value=0.008 in the allele-wise analysis for multiallelic markers), suggesting that the GABRB3 gene is involved in this congenital disease. This new finding in humans is in agreement with previously reported data obtained with the murine model. Indeed, mouse studies indicate a role for gamma-aminobutyric acid (GABA) and its receptor in normal palate development. Exclusion of the GAD1 gene, which encodes the GABA-producing enzyme, in CL/P pathogenesis was obtained in our study. Moreover, we were unable to confirm the involvement of the MSX1 gene in nonsyndromic CL/P. Modest evidence of LD between marker alleles and CL/P was found at the RARA and TGFB3 loci suggesting a minor role for these genes in our family set of nonsyndromic CL/P.     

Evidence, from family studies, for linkage disequilibrium between TGFA and a gene for nonsyndromic cleft lip with or without cleft palate.

The inheritance of alleles of the transforming growth factor alpha (TGFA) locus has been studied in families affected with cleft lip with or without cleft palate (CL/P), by using the transmission/disequilibrium test described by Spielman and colleagues. Only heterozygous parents with an affected child can be included in this test, but within such families a significantly greater frequency of C2 alleles were transmitted to affected children than would be expected by chance. There was no evidence that the total number of C2 alleles transmitted to affected and unaffected children differed significantly from random segregation. These data provide evidence from within families that a gene for susceptibility to CL/P is in significant linkage disequilibrium with the C2 allele of the TGFA locus.     

Racial differences in cephalometric measurements and incidence of cleft lip with or without cleft palate

A study was made to search for the morphological basis and, indirectly, the developmental basis for racial differences in risk of cleft lip with or without cleft palate CL(P) in Hawaii. A total of ten linear and three angular measurements read from anterior-posterior cephalographs were examined on 210 healthy adult Caucasian, Hawaiian, Japanese, Chinese, and Filipino subjects. Racial comparisons were made on these variables after adjusting for age and sex. Generally, the CL(P) high-risk group consisting of Japanese, Chinese, and Filipinos had smaller dimensions than Caucasians and Hawaiians in the variables (S-N, N-Ba) representing size of cranial base and the measurements of face height (N-A, N-ANS), palatal length (ANS-PNS), and mandibular length (Ar-Gn). Facial width relative to palatal length as measured by the ratio of bizygomatic diameter to palatal length showed a marked consistency with the racial differences in CL(P) risk. Possible significance of this finding is discussed in relation to development of the orofacial structure.     

Genome scan for loci involved in cleft lip with or without cleft palate,in Chinese multiplex families

Cleft lip with or without cleft palate (CL/P) is a common congenital anomaly. Birth prevalences range from 1/500 to 1/1,000 and are consistently higher in Asian populations than in populations of European descent. Therefore, it is of interest to determine whether the CL/P etiological factors in Asian populations differ from those in white populations. A sample of 36 multiplex families were ascertained through probands with CL/P who were from Shanghai. This is the first reported genome-scan study of CL/P in any Asian population. Genotyping of Weber Screening Set 9 (387 short tandem-repeat polymorphisms with average spacing approximately 9 cM [range 1-19 cM]) was performed by the Mammalian Genotyping Service of Marshfield Laboratory. Presented here are the results for the 366 autosomal markers. Linkage between each marker and CL/P was assessed by two-point and multipoint LOD scores, as well as with multipoint heterogeneity LOD scores (HLODs) plus model-free identity-by-descent statistics and the multipoint NPL statistic. In addition, association was assessed via the transmission/disequilibrium test. LOD-score and HLOD calculations were performed under a range of models of inheritance of CL/P. The following regions had positive multipoint results (HLOD > or =1.0 and/or NPL P< or =.05): chromosomes 1 (90-110 cM), 2 (220-250 cM), 3 (130-150 cM), 4 (140-170 cM), 6 (70-100 cM), 18 (110 cM), and 21 (30-50 cM). The most significant multipoint linkage results (HLOD > or =2.0; alpha=0.37) were for chromosomes 3q and 4q. Associations with P< or =.05 were found for loci on chromosomes 3, 5-7, 9, 11, 12, 16, 20, and 21. The most significant association result (P=.009) was found with D16S769 (51 cM).     

Nonsyndromic cleft lip with or without cleft palate: evidence of linkage to BCL3 in 17 multigenerational families.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a common craniofacial developmental defect. Recent segregation analyses have suggested that major genes play a role in the etiology of CL/P. Linkage to 22 candidate genes was tested in 11 multigenerational families with CL/P, and 21 of these candidates were excluded. APOC2, 19q13.1, which is linked to the proto-oncogene BCL3, gave suggestive evidence for linkage to CL/P. The study was expanded to include a total of 39 multigenerational CL/P families. Linkage was tested in all families, using an anonymous marker, D19S178, and intragenic markers in BCL3 and APOC2. Linkage was tested under two models, autosomal dominant with reduced penetrance and affecteds only. Homogeneity testing on the two-point data gave evidence of heterogeneity at APOC2 under the affecteds-only model. Both models showed evidence of heterogeneity, with 43% of families linked at zero recombination to BCL3 when marker data from BCL3 and APOC2 were included. A maximum multipoint LOD score of 7.00 at BCL3 was found among the 17 families that had posterior probabilities > = 50% in favor of linkage. The transmission disequilibrium test provided additional evidence for linkage with the 3 allele of BCL3 more often transmitted to affected children. These results suggest that BCL3, or a nearby gene, plays a role in the etiology of CL/P in some families.     

Association of genetic variation of the transforming growth factor-alpha gene with cleft lip and palate.

Complex segregation analysis of pedigrees having nonsyndromic cleft lip with or without cleft palate (CL/P) (Chung et al. 1986; Marazita et al. 1986) has shown that a major-locus model best explains the observed recurrence of CL/P in Caucasian families. To identify this major gene, we compared the frequencies of 12 RFLPs at five loci-epidermal growth factor, transforming growth factor-alpha, epidermal growth factor receptor, glucocorticoid receptor, and estrogen receptor-in both a group of 80 subjects with nonsyndromic CL/P and 102 controls. These candidate genes were selected because studies in rodents had suggested their possible involvement in palatogenesis. A significant association was observed between two RFLPs at the transforming-growth-factor-alpha (TGFA) locus and the occurrence of clefting (P = .0047 and P = .0052). This suggests that either the TGFA gene itself or DNA sequences in an adjacent region contribute to the development of a portion of cases of CL/P in humans and provides an opportunity to begin to examine the molecular events underlying lip and palate formation.     

Cleft lip with or without cleft palate in Shanghai, China: evidence for an autosomal major locus.

Orientals are at higher risk for cleft lip with or without cleft palate (CL +/- P) than Caucasians or blacks. We collected demographic and family data to study factors contributing to the etiology of CL +/- P in Shanghai. The birth incidence of nonsyndromic CL +/- P (Shanghai 1980-87) was 1.11/1,000, with a male/female ratio of 1.42. Almost 2,000 nonsyndromic CL +/- P probands were ascertained from individuals operated on during the years 1956-83 at surgical hospitals in Shanghai. Detailed family histories and medical examinations were obtained for the probands and all available family members. Genetic analyses of the probands' families were performed under the mixed model with major locus (ML) and multifactorial (MFT) components. The hypotheses of no familial transmission and of MFT alone could be rejected. Of the ML models, the autosomal recessive was significantly most likely and was assumed for testing three complex hypotheses: (1) ML and sporadics; (2) ML and MFT; (3) ML, MFT, and sporadics. None of the complex models were more likely than the ML alone model. In conclusion, the best-fitting, most parsimonious model for CL +/- P in Shanghai was that of an autosomal recessive major locus.     

Epidemiological and genetic study of 200 cases of oral cleft in the Emilia Romagna region of northern Italy

Epidemiological and genetic variables in clefts were analyzed during the years 1978-1986 in a case-control study of congenital malformations in the Emilia Romagna region of northern Italy. Among 150,168 newborns, 200 cases of cleft were detected, yielding a prevalence of 1.33 per 1,000. These clefts consisted of 112 (0.075%) cases of cleft lip with or without cleft palate (CL +/- P) and 88 cases (0.058%) of cleft palate (CP). Coexisting abnormalities were found in 32% of cases. The heritability coefficient of CL +/- P was 0.84. No cluster in time or space could be demonstrated. Epilepsy was the only maternal risk factor found to be correlated with clefts. A predominance of males was found among CL +/- P cases.     

Genetics of nonsyndromic cleft lip with or without cleft palate: is there a Mendelian sub-entity?

The mode of inheritance of nonsyndromic cleft lip with or without cleft palate (NSCLP) is still a matter of dispute. We performed segregation analysis on three data sets of families ascertained through an affected child with NSCLP. The first two data sets were selected in France and were pooled for a global analysis. No major gene effect could be evidenced in spite of a very large number of families (666 pedigrees including 719 nuclear families). The third data set was British and consisted of three-generation families including the offspring of probands. A major gene effect, as well as a strong residual multifactorial component, were highly significant and we could show that this evidence almost entirely came from the information on probands' offspring. We conclude that a mixture of monogenic and of multifactorial cases is probably the best explanation for the observations made in this study. Analyses performed in pedigrees with multiple cases closely related might allow reducing heterogeneity and help identifying those Mendelian sub-entities.     

Detection of major gene for Gilles de la Tourette syndrome

The families of 250 consecutive, unselected patients with Tourette syndrome (TS) were analyzed. If the parents had either motor or vocal tics, but not both, there was an increased risk of both TS and tics in the offspring. The mode of inheritance of the combined tic-Tourette trait was evaluated in both nuclear families and extended pedigrees. Complex segregation analysis was carried out allowing for possible contributions from both a major autosomal locus and multifactorial inheritance of variation in the background of each genotype. The most likely mode of inheritance was a major semidominant gene, Ts, with low heritability of the multifactorial background variation. This was true regardless of assumptions about the prevalence of the disorder. The hypothesis of strict multifactorial inheritance could not be rejected with nuclear family data alone. However, the hypothesis of no major gene effect was rejected using data on 3 generations for any estimate of lifetime risk less than 12 per 1,000 in the general population. A pure recessive major gene effect was also rejected. With a gene frequency of approximately .5%, the penetrance was estimated to be about 94% in abnormal Ts/Ts homozygotes, 50% in Ts/ts heterozygotes, and less than 0.3% in normal ts/ts homozygotes. More than two of every three cases are heterozygotes, and nearly all other cases are phenocopies or new mutations. This is the first demonstration by segregation analysis of a major gene in a human neuropsychiatric disorder with a frequency approaching 1% of the population.     

Analysis of candidate genes on chromosome 2 in oral cleft case-parent trios from three populations

Isolated oral clefts, including cleft lip with/without cleft palate (CL/P) and cleft palate (CP), have a complex and heterogeneous etiology. Case-parent trios from three populations were used to study genes spanning chromosome 2, where single nucleotide polymorphic (SNP) markers were analyzed individually and as haplotypes. Case-parent trios from three populations (74 from Maryland, 64 from Singapore and 95 from Taiwan) were genotyped for 962 SNPs in 104 genes on chromosome 2, including two well-recognized candidate genes: TGFA and SATB2. Individual SNPs and haplotypes (in sliding windows of 2–5 SNPs) were used to test for linkage and disequilibrium separately in CL/P and CP trios. A novel candidate gene (ZNF533) showed consistent evidence of linkage and disequilibrium in all three populations for both CL/P and CP. SNPs in key regions of ZNF533 showed considerable variability in estimated genotypic odds ratios and their significance, suggesting allelic heterogeneity. Haplotype frequencies for regions of ZNF533 were estimated and used to partition genetic variance into among-and within-population components. Wright’s fixation index, a measure of genetic diversity, showed little difference between Singapore and Taiwan compared with Maryland. The tensin-1 gene (TNS1) also showed evidence of linkage and disequilibrium among both CL/P and CP trios in all three populations, albeit at a lower level of significance. Additional genes (VAX2, GLI2, ZHFX1B on 2p; WNT6–WNT10A and COL4A3–COL4A4 on 2q) showed consistent evidence of linkage and disequilibrium only among CL/P trios in all three populations, and TGFA showed significant evidence in two of three populations.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.