Evidence for an association between markers on chromosome 19q and non-syndromic cleft lip with or without cleft palate in two groups of multiplex families

It has been reported that BCL3 on chromosome 19q, or a nearby gene, may play a role in the etiology of non-syndromic cleft lip with or without cleft palate (NSCL/P) in some families. We tested 30 USA and 11 Mexican multiplex NSCL/P families for four markers on chromosome 19q: D19S178, APOC2/AC1, APOC2/007, and BCL3. While likelihood-based linkage analysis failed to show significant evidence of linkage, the transmission disequilibrium test indicated highly significant deviation from independent assortment of allele 3 at the BCL3 marker in both data sets (USA:P = 0.001; Mexican: P = 0.018; both combined: P < 0.001) and for allele 13 of the D19S178 marker in the Mexican data set (P = 0.004). These results support an association, possibly due to linkage disequilibrium, between chromosome 19 markers and a putative NSCL/P locus. Received: 10 May 1996 / Revised: 31 July 1996     

Meta-analysis of 13 genome scans reveals multiple cleft lip/palate genes with novel loci on 9q21 and 2q32-35

Isolated or nonsyndromic cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex etiology. A 10-cM genome scan of 388 extended multiplex families with CL/P from seven diverse populations (2,551 genotyped individuals) revealed CL/P genes in six chromosomal regions, including a novel region at 9q21 (heterogeneity LOD score [HLOD]=6.6). In addition, meta-analyses with the addition of results from 186 more families (six populations; 1,033 genotyped individuals) showed genomewide significance for 10 more regions, including another novel region at 2q32-35 (P=.0004). These are the first genomewide significant linkage results ever reported for CL/P, and they represent an unprecedented demonstration of the power of linkage analysis to detect multiple genes simultaneously for a complex disorder.     

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.     

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).     

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.     

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.     

Strong evidence of linkage disequilibrium between polymorphisms at the IRF6 locus and nonsyndromic cleft lip with or without cleft palate, in an Italian population

Cleft lip with or without cleft palate (CL/P) is one of the most common birth defects, but its etiology is largely unknown. It is very likely that both genetic and environmental factors contribute to this malformation. Mutations in the gene for interferon regulatory factor 6 (IRF6) have been shown to be the cause of Van der Woude syndrome, a dominant disorder that has CL/P as a common feature. Recently, it has been reported that genetic polymorphisms at the IRF6 locus are associated with nonsyndromic CL/P, with stronger association in Asian and South American populations. We investigated four markers spanning the IRF6 locus, using the transmission/disequilibrium test. A sample of 219 Italian triads of patients and their parents were enrolled in the study. Strong evidence of linkage disequilibrium was found between markers and disease in both single-allele (P=.002 at marker rs2235375) and haplotype (P=.0005) analyses. These findings confirm the contribution of IRF6 in the etiology of nonsyndromic CL/P and strongly support its involvement in populations of European ancestry.     

Few associations of candidate genes with nonsyndromic orofacial clefts in the population of Lithuania

Nonsyndromic orofacial clefting (NS-OFC) is a common complex multifactorial trait with a considerable genetic component and a number of candidate genes suggested by various approaches. Twenty biallelic and microsatellite DNA markers in the strong candidate loci TGFA, TGFB3, GABRB3, RARA, and BCL3 were analysed for allelic association with the NS-OFC phenotype in 112 nuclear families (proband + both parents) from Lithuania by using the transmission disequilibrium test (TDT). Associations were found between the TGFA gene marker rs2166975 and nonsyndromic cleft palate only (CPO) phenotype (p = 0.045, df 1) as well as between the D2S292 marker and the cleft lip with or without cleft palate (CL/CP) phenotype in allele-wise TDT (P = 0.005, df 9) and genotype-wise TDT (P = 0.021, df 24). A weak association (P = 0.085, df 3) of the BCL3 marker (BCL3 gene) with the risk of CPO was also found. Thus our initial results support the contribution of allelic variation in the TGFA locus to the aetiology of CL/CP in the population of Lithuania but they do not point to TGFA as a major causal gene. Different roles of the TGFA and BCL3 genes in the susceptibility to NS-OFC phenotypes are suggested.     

Autosomal dominant nonsyndromic cleft lip and palate: significant evidence of linkage at 18q21.1

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common congenital facial defects, with an incidence of 1 in 700-1,000 live births among individuals of European descent. Several linkage and association studies of NSCL/P have suggested numerous candidate genes and genomic regions. A genomewide linkage analysis of a large multigenerational family (UR410) with NSCL/P was performed using a single-nucleotide-polymorphism array. Nonparametric linkage (NPL) analysis provided significant evidence of linkage for marker rs728683 on chromosome 18q21.1 (NPL=43.33 and P=.000061; nonparametric LOD=3.97 and P=.00001). Parametric linkage analysis with a dominant mode of inheritance and reduced penetrance resulted in a maximum LOD score of 3.61 at position 47.4 Mb on chromosome 18q21.1. Haplotype analysis with informative crossovers defined a 5.7-Mb genomic region spanned by proximal marker rs1824683 (42,403,918 bp) and distal marker rs768206 (48,132,862 bp). Thus, a novel genomic region on 18q21.1 was identified that most likely harbors a high-risk variant for NSCL/P in this family; we propose to name this locus "OFC11" (orofacial cleft 11).     

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.     

Identification of susceptibility loci for nonsyndromic cleft lip with or without cleft palate in a two stage genome scan of affected sib-pairs

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a complex disorder of multigenic origin involving between two and ten loci. Linkage and association studies of CL/P have implicated a number of candidate genes and regions but have often proved difficult to replicate. Here, we report the findings from a two-stage genome-wide scan of 92 affected sib-pairs to identify susceptibility loci to CL/P. An initial set of 400 microsatellite markers was used, with an average spacing of 10 cM throughout the genome. Eleven regions on eight chromosomes were found to have a P-value smaller than 0.05. These eight chromosomes were then further mapped with a second set of markers to increase the average map density to 5 cM. In seven out of eleven areas densely mapped, significance was markedly increased by decreasing the marker interval. Excessive allele sharing was found at 1p (NPL=2.35, P=0.009, MLS=1.51), 2p (NPL=1.77, P= 0.04, MLS=0.66), 6p (NPL=2.35, P=0.009, MLS=1.34), 8q (NPL=2.15, P=0.015, MLS= 1.51) 11 cen (NPL=2.70, P=0.003, MLS=2.10), 12q (NPL=2.08, P=0.02, MLS= 1.5), 16p (NPL=2.1, P=0.018, MLS=0.97) and Xcen-q (NPL=2.40, P=0.008, MLS=2.68). Although none reached the level required for significant susceptibility loci, two of these areas have previously been implicated in CL/P, viz. 2p13, an area harbouring the TGFA gene, and 6p23-24. We also demonstrate highly suggestive linkage to a susceptibility locus for nonsyndromic clefting on the X chromosome. Further studies are currently underway to replicate these findings in a larger cohort of affected sib-pairs.     

Nonsyndromic cleft lip and palate: no evidence of linkage to HLA or factor 13A.

Nonsyndromic cleft lip with or without cleft palate (CLP) is a common craniofacial anomaly, the etiology of which is not known. Population studies have shown that a large proportion of cases occur sporadically. Recently, segregation analyses applied to CLP families have demonstrated that an autosomal dominant/codominant gene(s) may cause clefting in cases. Associations of autosomal dominant CLP and nonsyndromic cleft palate (CP) with HLA and F13A genes on chromosome 6p have been suggested previously. Linkage to these two areas on chromosome 6p were tested in 12 autosomal dominant families with CLP. With a LOD score of -2 or less for exclusion, no evidence of linkage was found to four chromosome 6p markers. Multipoint analysis showed no evidence of a clefting locus in this region spanning 54 cM on chromosome 6p in these CLP families.     

A comprehensive linkage analysis of chromosome 21q22 supports prior evidence for a putative bipolar affective disorder locus.

Previously, we demonstrated evidence of linkage to bipolar affective disorder (BP) in a single large, multigenerational family with a LOD score of 3.41 at the PFKL locus on chromosome 21q22.3. Additional families showed little support for linkage to PFKL under homogeneity or heterogeneity, in that study. We have expanded on that analysis, with 31 microsatellite markers at an average marker spacing of </=2 cM, in the largest multigenerational BP pedigree series reported to date. A two-point heterogeneity (alpha=0.5) LOD score of 3.35 (P<.000156) was found at the D21S1260 locus, 5 cM proximal to PFKL. Polylocus analysis with a cluster of three neighboring markers was consistent with these results (PL-HetLOD = 3.25). In the design of this study, 373 individuals from 40 families (from a total set of 1,508 individuals in 57 families) were chosen, as a cost-effective approach to genotyping this large sample set. Linkage analyses were performed with an "affecteds-only" method. As such, our results are based solely on genetic information from affected individuals, without assumptions about the disease-locus genotypes of the unaffecteds. Furthermore, for ease of comparison, this study was performed with the same approach as a 10-cM genome scan for BP loci, the results of which will be reported elsewhere.     

The major locus for multifactorial nonsyndromic cleft lip maps to mouse Chromosome 11

Cleft lip with or without cleft palate, CL(P), a common human birth defect, has a genetically complex etiology. An animal model with a similarly complex genetic basis is established in the A/WySn mouse strain, in which 20% of newborn have CL(P). Using a newly created congenic strain, AEJ.A, and SSLP markers, we have mapped a major CL(P)-causing gene derived from the A/WySn strain. This locus, here named clf1 (cleft lip) maps to Chromosome (Chr) 11 to a region having linkage homology with human 17q21-24, supporting reports of association of human CL(P) with the retinoic acid receptor alpha (RARA) locus.     

A susceptibility locus for migraine with aura, on chromosome 4q24

Migraine is a complex neurovascular disorder with substantial evidence supporting a genetic contribution. Prior attempts to localize susceptibility loci for common forms of migraine have not produced conclusive evidence of linkage or association. To date, no genomewide screen for migraine has been published. We report results from a genomewide screen of 50 multigenerational, clinically well-defined Finnish families showing intergenerational transmission of migraine with aura (MA). The families were screened using 350 polymorphic microsatellite markers, with an average intermarker distance of 11 cM. Significant evidence of linkage was found between the MA phenotype and marker D4S1647 on 4q24. Using parametric two-point linkage analysis and assuming a dominant mode of inheritance, we found for this marker a maximum LOD score of 4.20 under locus homogeneity (P=.000006) or locus heterogeneity (P=.000011). Multipoint parametric (HLOD = 4.45; P=.0000058) and nonparametric (NPL(all) = 3.43; P=.0007) analyses support linkage in this region. Statistically significant linkage was not observed in any other chromosomal region.     

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.     

Dermatoglyphic fingerprint heterogeneity among individuals with nonsyndromic cleft lip with or without cleft palate and their unaffected relatives in China and the Philippines

Cleft lip with or without cleft palate (CL/P) is a common birth defect (birth prevalence ranging from 1/500 to 1/2,000) with a complex etiology. Traits potentially related to CL/P, such as dermatoglyphics, may reflect the genetic and epidemiologic heterogeneity observed in CL/P. Such phenotypic heterogeneity in dermatoglyphic patterns may account for some of the variability in previously reported associations of dermatoglyphics and CL/P. To test this hypothesis, we took dermatoglyphic prints from individuals with nonsyndromic CL/P (n = 460) and their unaffected relatives (n = 254) from the Philippines and China. For both samples three raters designated the patterns as arch, ulnar loop, radial loop, whorl, or "other." Chi-square analysis and standard ANOVA were used to investigate heterogeneity between Filipino and Chinese study subjects. The significant associations between particular pattern types and CL/P were not the same in both populations, demonstrating population-specific association of CL/P and dermatoglyphic pattern types. The ANOVA of pattern type included both CL/P cases and their relatives, with affection status, sex, and population group as variables. For each pattern type except arches, population was significant (p < 0.0001); for radial loops, affection status was additionally significant (p < 0.0001). When only CL/P cases were considered, population was again significant for the ulnar loop (p < 0.0001), whorl (p < 0.0001), and "other" (p = 0.0002) patterns. The ANOVAs demonstrate between-population heterogeneity in dermatoglyphic pattern types. These results support our hypothesis that population-specific associations and population heterogeneity in dermatoglyphic patterns exist for CL/P cases and their relatives.     

Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2): structure,gene mapping,polymorphisms, and candidate evaluation for human orofacial clefts

BACKGROUND: Nonsyndromic orofacial clefts have an estimated incidence of 1/1000 live births. Population genetic and embryologic studies suggest that cleft palate only (CPO) may be a distinct clinical entity from cleft lip with or without cleft palate (CL/P). Both CPO and CL/P are thought to be multifactorial in etiology, with evidence indicating that genetic, environmental, and developmental determinants may all play a role. The ARNT2 gene localizes to a conserved linkage group on mouse chromosome 7 that is syntenic with human chromosome 15q23-25. This chromosomal region was previously identified as a teratogen-induced clefting susceptibility locus in a genome-wide scan of AXB and BXA recombinant inbred mice. Arnt2 is expressed in the first branchial arch in mice. The teratogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) acts through the aryl hydrocarbon receptor (Ahr) pathway to produce dose-dependent CPO and thymic wasting in mice exposed in utero. Arnt2 and Ahr proteins dimerize in vitro. TCDD exposure is also associated with orofacial clefting in children of parents involved in agricultural work. METHODS: To determine whether ARNT2 influences human craniofacial development, we identified the human ARNT2 gene and conducted genomic structural analysis. Mutational screening was performed in infants with nonsyndromic CPO or CL/P who were identified by the Iowa Birth Defects Registry. RESULTS: A common amino acid polymorphism was detected but, no obvious disease-causing mutations were detected by SSCP analysis. The microsatellite marker, GATA89D04 (D15S823) was identified within intron 11 of the human ARNT2 gene, and linkage disequilibrium of nonsyndromic CPO and CL/P parent-infant trios was conducted. CONCLUSIONS: No association was demonstrated with CPO (n = 45) and CL/P (n = 37). Teratology 66:85-90, 2002.     

Association of MSX1 and TGFB3 with nonsyndromic clefting in humans.

Nonsyndromic cleft lip with or without cleft palate (CL/P) and nonsyndromic cleft palate only (CPO) are common congenital anomalies with significant medical, psychological, social, and economic ramifications. Both CL/P and CPO are examples of complex genetic traits. There exists sufficient evidence to hypothesize that disease loci for CL/P and CPO can be identified by a candidate-gene linkage-disequilibrium (LD) strategy. Candidate genes for clefting, including TGFA, BCL3, DLX2, MSX1, and TGFB3, were screened for LD with either CL/P or CPO in a predominantly Caucasian population, with both case-control- and nuclear-family-based approaches. Previously reported LD for TGFA with both CL/P and CPO could not be confirmed, except in CL/P patients with a positive family history. Also, in contrast to previous studies, no LD was found between BCL3 and either CL/P or CPO. Significant LD was found between CL/P and both MSX1 and TGFB3 and between CPO and MSX1, suggesting that these genes are involved in the pathogenesis of clefting. In addition, a mutation search in the genes DLX2, MSX1, and TGFB3 was performed in 69 CPO patients and in a subset of the CL/P patients. No common mutations were found in the coding regions of these genes; however, several rare variants of MSX1 and TGFB3 were found that may alter the latters' normal function. These results form the basis for future research, including (a) mutation searches in the MSX1 and TGFB3 genes in Caucasian CL/P patients and (b) extension of the search for MSX1 mutations in CPO patients to the noncoding regions.