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: a comparison of Danish and Japanese data.

The genetic basis of cleft lip with or without cleft palate [CL(P)] remains unresolved. The controversy on the role of a major gene is confounded with possible population differences. This study examines the issue of population differences by comparing two contrasting populations: Caucasians and Japanese. Japanese are known to have higher population incidence of CL(P) and yet lower recurrence risks among relatives. The study subjects consist of 2,998 nuclear families of the Danish population and 627 families of the Japanese population. The uniformly coded data were subjected to complex segregation analysis based on the mixed model. The analysis has revealed that the Danish data can be best explained by a combination of major gene action and multifactorial inheritance. The best-fitting model is characterized by recessive gene with displacement effect (t) of 2.7 in the standardized unit and gene frequency of .035. The heritability is estimated as .97. The transmission probability of Aa----a for the major gene is consistent with 1/2. On the contrary, the Japanese data can be best accounted for only by multifactorial inheritance with the heritability estimate of .77. No major heterogeneity could be detected between subsets of the data within the populations as grouped by types of ascertainment or mating. It is thus concluded that the observed inconsistency between the two populations is explained by a significant role of major gene in the Caucasian population, but not in the Japanese population.     

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 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%.     

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.     

FGF signalling and SUMO modification: new players in the aetiology of cleft lip and/or palate

Owing to the complex aetiology and the variable penetrance of cleft lip and/or palate (CL/P), understanding the molecular basis has been challenging. Recent reports have identified two independent biochemical pathways that will help to elucidate the underlying pathology. Fibroblast growth factor signalling, previously known for its involvement in craniofacial development, is now implicated in the genetic basis of both syndromic and non-syndromic CL/P. At the same time, an important role in lip and palate development is beginning to emerge for small ubiquitin-like modifier modification, a widely used posttranslational regulatory mechanism. Both of these pathways might interact with environmental risk factors for CL/P. Here we review their contribution to normal and abnormal orofacial development.     

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.     

Effects of interracial crosses on cephalometric measurements

The effects of race and interracial crossing were examined on six cephalometric measurements among 9,912 schoolchildren in Hawaii. The measurements studied were face height, bizygomatic diameter, bigonial diameter, head breadth, head length, and cephalic index. Racial effects were studied in terms of general racial effect, maternal effect, and hybridity and recombination effects based on a model of diallel cross. Generally, Chinese, Japanese, Koreans, and Filipinos were characterized by longer lateral and smaller anterior-posterior dimensions relative to Caucasians. Maternal effects appeared to be present in the measures of lateral dimension. No clear effects of hybridity and recombination were seen except for bizygomatic diameter, which appears to behave as a partial dominant trait. The racial mean of bizygomatic diameter, or the ratio of this measure to head length, were found to have a relationship with the racial incidences of cleft lip with or without cleft palate.     

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.     

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.     

Incidence of cleft palate fistula: an institutional experience with two-stage palatal repair.

The purpose of this study was to determine the incidence of cleft palatal fistula in a series of nonsyndromic children treated at the authors' institution. This retrospective analysis of 103 patients with cleft palate treated by five surgeons between 1982 and 1995 includes 60 boys and 33 girls, whose median age was 18.4 months at the time of surgery. The median length of follow-up was 4.9 years after primary palatoplasty. Cleft palatal fistula was defined as a failure of healing or a breakdown in the primary surgical repair of the palate. Intentionally unrepaired fistulas of the primary and secondary palate were excluded. Extent of clefting was described according to the Veau classification. Statistical examination of multiple variables was performed using contingency table analysis, multivariate logistic regression, and the Wilcoxon rank sum test. The incidence of cleft palatal fistula in this series was 8.7 percent. All of these fistulas were clinically significant. The rate of fistula recurrence was 33 percent. The incidence of cleft palatal fistula when compared by Veau classification was statistically significant, with nine fistulas occurring in patients with Veau 3 and 4 clefts and no fistulas occurring in patients with Veau 1 and 2 clefts (p = 0.0441). No significant differences between patients with and without fistulas were identified with respect to operating surgeon, patient sex, patient age at palatoplasty, type of palatoplasty, and use of presurgical orthopedics or palatal expansion. All three recurrent fistulas occurred in the anterior palate, two in patients with Veau class 3 clefts and one in a patient with a Veau class 4 cleft. The low rate of clinically significant fistula was attributed to early delayed primary closure, with smaller secondary clefts allowing repair with a minimum of dissection and disruption of vascularity.     

Allelic polymorphism of MTHFR, MTR and MTRR genes in patients with cleft lip and/or palate and their mothers

The frequency of common MTHFR, MTR and MTRR genes polymorphisms was evaluated among patients with non-syndromic cleft lip and/or palate (CL/P), their mothers and healthy persons from West-Ukrainian region. MTHFR 677TT genotype was shown to increase more than three-fold risk of CL/P and for mothers the risk of having CL/P children may increase two-fold compared with homozygous carriers of MTHFR 677CC genotype (OR = 3.3, OR = 1.92, respectively). The heterozygous MTR 2756AG genotype was associated with 1.5-fold increased risk of CL/P compared with the AA genotype (OR = 1.48). The heterozygous genotype MTRR 66AG was associated with the 5.56-fold increased CL/P risk (OR = 5.56) and for mothers with 2.6-fold increased risk of delivering a CL/P offspring (OR = 2.6). The results showed that MTRR 66G allele is more prevalent than MTRR 66A (wild type) and the MTRR 66GG genotype frequency was significantly lower among CL/P patients and their mothers than in control group among Western Ukrainian inhabitants.     

The CRISPLD2 gene is involved in cleft lip and/or cleft palate in a Chinese population

BACKGROUND: Nonsyndromic cleft lip and/or cleft palate (NSCLP) are common congenital anomalies in humans, the etiologies of which are complex and associated with both genetic and environmental factors. Previous data suggested single nucleotide polymorphisms (SNPs) of rs1546124, rs4783099, and rs16974880 of the CRISPLD2 gene were associated with an increased risk of NSCLP; however, subsequent studies have yielded conflicting results. This study aims to evaluate the associations of the aforementioned polymorphisms with NSCLP in a Northwestern Chinese population. METHODS: Three CRISPLD2 SNPs were genotyped in a case‐control study (n = 907), including 444 NSCLP patients and 463 healthy individuals, using polymerase chain reaction–denaturing high‐performance liquid chromatography (PCR‐DHPLC). RESULTS: The genotype and allele frequencies of rs1546124 (odds ratio [OR], 2.30; 95% confidence interval [CI], 1.58–3.34; p = 1 × 10⁻⁵) and rs4783099 (OR, 0.73; 95% CI, 0.54–1.00; p = 0.05) were different in NSCLP patients compared with controls. Furthermore, the CC genotype at rs1546124 was associated with increased risk for cleft lip with or without cleft palate (CL/P; OR, 2.11; 95% CI, 1.41–3.15; p_correct = 1.5 × 10⁻⁴) and for cleft palate only (CPO; OR, 2.93; 95% CI, 1.69–5.07; p_correct = 5.4 × 10⁻⁴), whereas the T allele of rs4783099 was associated with decreased risk for CPO. Further gender stratification showed that the statistical association of these two loci is mainly in the male patients, and not in female patients. CONCLUSION: Our results suggest that the CRISPLD2 gene contributes to the etiology of NSCLP in the Northwestern Chinese population. SNP rs1546124 is significantly related to NSCLP, associated with both CL/P and CPO groups, and SNP rs4783099 is significantly associated with CPO. Birth Defects Research (Part A) 2011. © 2011 Wiley‐Liss, Inc.     

Ultrastructure of initial nasal process cell fusion in spontaneous and 6-aminonicotinamide-induced mouse embryo cleft lip

A search was made for cell ultrastructure differences in the initial fusion process of the medial and lateral nasal processes in mouse embryos of the following types: A/J with 12% cleft lip (CL), CL/Fr with 23% CL--both cleft-lip-predisposed strains, CL/Fr 6-aminonicotinamide (6AN)-treated (94% CL) and controls from the C57BL/6 strain (0% CL) and dancer stock (0% CL). No detectable differences were found between the A/J and CL/Fr strains and the controls in the epithelial cells showing initial contact and fusion. Epithelial surfaces not in contact in controls and where clefts were developing were smooth. Cells approaching or in contact had cell projections, intercellular junctions, desmosomes, and microfilaments demonstrating firm contact between the apposed epithelia. It has been postulated that spontaneous cleft lip was due to a predisposing face shape bringing about a failure of contact in some embryos and in others where contact was achieved fusion was normal. These data support this view. The situation, however, in 6AN-treated embryos is different. A few 6AN-treated embryos showed abnormal contact that appeared malpositioned and tenuous. The teratogen also caused increased cell death and a denser epithelium and mesenchyme. Thus 6AN-induced cleft lip could be due to the epithelial cell changes and/or to the reduction in size of the nasal processes.     

Regeneration in surgically produced cleft palate in rabbits. A microscopical study

In order to study the effects of surgical procedures on the regeneration process in cleft palate, an artificial cleft was made in 4-weeks old female New Zealand rabbits. The 47 animals were divided into 5 groups: group 1: controls, group 2: cleft made in the bony palate, using a dental burr (phi 3 mm), group 3: cleft as in group 2, oral mucosa stitched, group 4: cleft made without perforating the nasal mucosa; oral mucosa stitched ("Veau"), group 5: cleft made as in group 4; before stitching the oral mucosa pieces of autologous rib bone were introduced into the cleft ("Schuchardt"). At the ages of 4, 5, 6, 8, 12, 18 and 24 weeks, one or two animals per group were sacrificed. The palatal region was microscopically studied using 10 mu paraffin sections stained according to Herovici. It was found that it is difficult to make a reproducible cleft on the histological level. A large interindividual variation in local damage due to the surgical procedure was observed; this might contribute to the variation in growth results of the facial skeleton reported in literature. Signs of excessive bone resorption were found. The presence of bone in places where sutural tissue should be present, observed in some growing animals, will contribute to reduction of palatal width, medial collapse of the palate and asymmetry of the facial skeleton. There appeared to be no striking differences between the three methods of repair, used in group 3, 4 and 5 respectively.     

D-penicillamine-induced cleft palate in mice

The teratogenic potential of the lathyrogen, D-penicillamine (DP), was assessed in pregnant mice, especially with respect to its ability to produce cleft palate. The dosage and the duration of treatment as they relate to the induction of cleft palate were also studied. Two different doses of DP were administered orally for either 5 or 4 consecutive days during the critical period of palatal closure. D-penicillamine (DP) at a dose level which does not have any apparent maternal toxic effects produced cleft palate in the offspring, and this teratogenic effect depended more upon the duration of treatment than the dosage administered. Inhibitory effects on the formation of bone matrix were observed at the base of the palatal shelf. It is suggested that DP is potentially an osteolathyrogenic agent. The mechanism of induction of cleft palate in DP-treated mice was explored by histological studies using light microscopy. Delayed elevation of the palatal shelves was observed and is considered to be the cause of the induction of cleft palate. No other external malformations could be detected in DP-treated fetuses.     

Evidence for an association between nonsyndromic cleft lip with or without cleft palate and a gene located on the long arm of chromosome 4.

Recent studies suggest that the familial aggregation of nonsyndromic cleft lip with or without cleft palate (CL +/- P) is likely to be attributable to the effects of several susceptibility loci, acting in a multiplicative fashion. Two potential CL +/- P susceptibility loci (CSL), transforming growth factor alpha (TGFA) and retinoic acid receptor (RARA), have been identified through association studies. In addition, recent evidence of linkage between CL +/- P and two markers (D4S175 and D4S192) in the region 4q25-4q31.3 raised the possibility that a CSL, with a larger effect than either TGFA or RARA, may reside within this region of the human genome. The present analyses were undertaken to determine whether D4S175 or D4S192 is significantly associated with CL +/- P in a sample of unrelated patients that have previously provided evidence of associations between CL +/- P and both TGFA and RARA. The results of these analyses provide further, tentative, evidence for the presence of a CSL locus on the long arm of chromosome 4 and help to refine the location of this locus in the region of D4S175 and D4S192.     

Pleiotropy method reveals genetic overlap between orofacial clefts at multiple novel loci from GWAS of multi-ethnic trios

Based on epidemiologic and embryologic patterns, nonsyndromic orofacial clefts– the most common craniofacial birth defects in humans– are commonly categorized into cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP), which are traditionally considered to be etiologically distinct. However, some evidence of shared genetic risk in IRF6, GRHL3 and ARHGAP29 regions exists; only FOXE1 has been recognized as significantly associated with both CL/P and CP in genome-wide association studies (GWAS). We used a new statistical approach, PLACO (pleiotropic analysis under composite null), on a combined multi-ethnic GWAS of 2,771 CL/P and 611 CP case-parent trios. At the genome-wide significance threshold of 5 × 10⁻⁸, PLACO identified 1 locus in 1q32.2 (IRF6) that appears to increase risk for one OFC subgroup but decrease risk for the other. At a suggestive significance threshold of 10⁻⁶, we found 5 more loci with compelling candidate genes having opposite effects on CL/P and CP: 1p36.13 (PAX7), 3q29 (DLG1), 4p13 (LIMCH1), 4q21.1 (SHROOM3) and 17q22 (NOG). Additionally, we replicated the recognized shared locus 9q22.33 (FOXE1), and identified 2 loci in 19p13.12 (RAB8A) and 20q12 (MAFB) that appear to influence risk of both CL/P and CP in the same direction. We found locus-specific effects may vary by racial/ethnic group at these regions of genetic overlap, and failed to find evidence of sex-specific differences. We confirmed shared etiology of the two OFC subtypes comprising CL/P, and additionally found suggestive evidence of differences in their pathogenesis at 2 loci of genetic overlap. Our novel findings include 6 new loci of genetic overlap between CL/P and CP; 3 new loci between pairwise OFC subtypes; and 4 loci not previously implicated in OFCs. Our in-silico validation showed PLACO is robust to subtype-specific effects, and can achieve massive power gains over existing approaches for identifying genetic overlap between disease subtypes. In summary, we found suggestive evidence for new genetic regions and confirmed some recognized OFC genes either exerting shared risk or with opposite effects on risk to OFC subtypes.     

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.