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Nonsyndromic orofacial clefts (NSOFCs) represent a large proportion (70%-80%) of all OFCs. They can be broadly categorized into nonsyndromic cleft lip with or without cleft palate (NSCL/P) and nonsyndromic cleft palate only (NSCPO). Although NSCL/P and NSCPO are considered etiologically distinct, recent evidence suggests the presence of shared genetic risks. Thus, we investigated the genetic overlap between NSCL/P and NSCPO using African genome-wide association study (GWAS) data on NSOFCs. These data consist of 814 NSCL/P, 205 NSCPO cases, and 2159 unrelated controls. We generated common single-nucleotide variants (SNVs) association summary statistics separately for each phenotype (NSCL/P and NSCPO) under an additive genetic model. Subsequently, we employed the pleiotropic analysis under the composite null (PLACO) method to test for genetic overlap. Our analysis identified two loci with genome-wide significance (rs181737795 [p = 2.58E-08] and rs2221169 [p = 4.5E-08]) and one locus with marginal significance (rs187523265 [p = 5.22E-08]). Using mouse transcriptomics data and information from genetic phenotype databases, we identified MDN1, MAP3k7, KMT2A, ARCN1, and VADC2 as top candidate genes for the associated SNVs. These findings enhance our understanding of genetic variants associated with NSOFCs and identify potential candidate genes for further exploration., (© 2024 The Authors. Genetic Epidemiology published by Wiley Periodicals LLC.)

Nonsyndromic orofacial clefts (OFCs) are among the most common craniofacial birth defects worldwide, and known to exhibit phenotypic and genetic heterogeneity. Cleft lip plus cleft palate (CLP) and cleft lip only (CL) are commonly combined together as one phenotype (CL/P), separately from cleft palate alone. In comparison, our study analyzes CL and CLP separately. A sample of 2218 CL and CLP cases, 4537 unaffected relatives of cases, and 2673 pure controls with no family history of OFC were selected from the Pittsburgh Orofacial Cleft (Pitt-OFC) multiethnic study.genome-wide association studies were run for seven specific phenotypes created based on the cleft type(s) observed within these families, as well as the combined CL/P phenotype. Five novel genome-wide significant associations, 3q29 (rs62284390), 5p13.2 (rs609659), 7q22.1 (rs6465810), 19p13.3 (rs628271), and 20q13.33 (rs2427238), and nine associations (p ≤ 1.0E-05) within previously confirmed OFC loci-PAX7, IRF6, FAM49A, DCAF4L2, 8q24.21, ARID3B, NTN1, TANC2 and the WNT9B:WNT3 gene cluster-were observed. We also found that single nucleotide polymorphisms within a subset of the associated loci, both previously known and novel, differ substantially in terms of their effects across cleft- or family-specific phenotypes, indicating not only etiologic differences between CL and CLP, but also genetic heterogeneity within each of the two OFC subtypes., (© 2022 Wiley Periodicals LLC.)

Orofacial clefts (OFCs) are common (1 in 700 births) congenital malformations that include a cleft lip (CL) and cleft lip and palate (CLP). These OFC subtypes are also heterogeneous themselves, with the CL occurring on the left, right, or both sides of the upper lip. Unilateral CL and CLP have a 2:1 bias towards left-sided clefts, suggesting a nonrandom process. Here, we performed a study of left- and right-sided clefts within the CL and CLP subtypes to better understand the genetic factors controlling cleft laterality. We conducted genome-wide modifier analyses by comparing cases that had right unilateral CL (RCL; N = 130), left unilateral CL (LCL; N = 216), right unilateral CLP (RCLP; N = 416), or left unilateral CLP (LCLP; N = 638), and identified a candidate region on 4q28, 400 kb downstream from FAT4, that approached genome-wide significance for LCL versus RCL (p = 8.4 × 10 -8 ). Consistent with its potential involvement as a genetic modifier of CL, we found that Fat4 exhibits a specific domain of expression in the mesenchyme of the medial nasal processes that form the median upper lip. Overall, these results suggest that the epidemiological similarities in left- to right-sided clefts in CL and CLP are not reflected in the genetic association results., (© 2021 Wiley Periodicals LLC.)

The chromodomain family member chromodomain 1 (CHD1) has been shown to have numerous critical molecular functions including transcriptional regulation, splicing, and DNA repair. Complete loss of function of this gene is not compatible with life. On the other hand, missense and copy number variants of CHD1 can result in intellectual disabilities and craniofacial malformations in human patients including cleft palate and Pilarowski-Bjornsson Syndrome. We have used the aquatic developmental model organism Xenopus laevis, to determine a specific role for Chd1 in such cranioafcial disorders. Protein and gene knockdown techniques in Xenopus, including antisense oligos and mosaic Crispr/Cas9-mediated mutagenesis, recapitulated the craniofacial defects observed in humans. Further analysis indicated that embryos deficient in Chd1 had defects in cranial neural crest development and jaw cartilage morphology. Additionally, flow cytometry and immunohistochemistry revealed that decreased Chd1 resulted in increased in apoptosis in the developing head. Together, these experiments demonstrate that Chd1 is critical for fundamental processes and cell survival in craniofacial development. We also presented evidence that Chd1 is regulated by retinoic acid signaling during craniofacial development. Expression levels of chd1 mRNA, specifically in the head, were increased by RAR agonist exposure and decreased upon antagonist treatment. Subphenotypic levels of an RAR antagonist and Chd1 morpholinos synergized to result in orofacial defects. Further, RAR DNA binding sequences (RAREs) were detected in chd1 regulatory regions by bioinformatic analysis. In summary, by combining human genetics and experiments in an aquatic model we now have a better understanding of the role of CHD1 in craniofacial disorders., (© 2020 Wiley Periodicals LLC.)

Cleft lip with or without cleft palate (CL/P) is generally viewed as a complex trait with multiple genetic and environmental contributions. In 70% of cases, CL/P presents as an isolated feature and/or deemed nonsyndromic. In the remaining 30%, CL/P is associated with multisystem phenotypes or clinically recognizable syndromes, many with a monogenic basis. Here we report the identification, via exome sequencing, of likely pathogenic variants in two genes that encode interacting proteins previously only linked to orofacial clefting in mouse models. A variant in GDF11 (encoding growth differentiation factor 11), predicting a p.(Arg298Gln) substitution at the Furin protease cleavage site, was identified in one family that segregated with CL/P and both rib and vertebral hypersegmentation, mirroring that seen in Gdf11 knockout mice. In the second family in which CL/P was the only phenotype, a mutation in FST (encoding the GDF11 antagonist, Follistatin) was identified that is predicted to result in a p.(Cys56Tyr) substitution in the region that binds GDF11. Functional assays demonstrated a significant impact of the specific mutated amino acids on FST and GDF11 function and, together with embryonic expression data, provide strong evidence for the importance of GDF11 and Follistatin in the regulation of human orofacial development., (© 2019 Wiley Periodicals, Inc.)

Phenotypic heterogeneity is a hallmark of complex traits, and genetic studies of such traits may focus on them as a single diagnostic entity or by analyzing specific components. For example, in orofacial clefting (OFC), three subtypes-cleft lip (CL), cleft lip and palate (CLP), and cleft palate (CP) have been studied separately and in combination. To further dissect the genetic architecture of OFCs and how a given associated locus may be contributing to distinct subtypes of a trait we developed a framework for quantifying and interpreting evidence of subtype-specific or shared genetic effects in complex traits. We applied this technique to create a "cleft map" of the association of 30 genetic loci with three OFC subtypes. In addition to new associations, we found loci with subtype-specific effects (e.g., GRHL3 [CP], WNT5A [CLP]), as well as loci associated with two or all three subtypes. We cross-referenced these results with mouse craniofacial gene expression datasets, which identified additional promising candidate genes. However, we found no strong correlation between OFC subtypes and expression patterns. In aggregate, the cleft map revealed that neither subtype-specific nor shared genetic effects operate in isolation in OFC architecture. Our approach can be easily applied to any complex trait with distinct phenotypic subgroups., (© 2019 Wiley Periodicals, Inc.)

We previously demonstrated how sharing of rare variants (RVs) in distant affected relatives can be used to identify variants causing a complex and heterogeneous disease. This approach tested whether single RVs were shared by all sequenced affected family members. However, as with other study designs, joint analysis of several RVs (e.g., within genes) is sometimes required to obtain sufficient statistical power. Further, phenocopies can lead to false negatives for some causal RVs if complete sharing among affected is required. Here, we extend our methodology (Rare Variant Sharing, RVS) to address these issues. Specifically, we introduce gene-based analyses, a partial sharing test based on RV sharing probabilities for subsets of affected relatives and a haplotype-based RV definition. RVS also has the desirable feature of not requiring external estimates of variant frequency or control samples, provides functionality to assess and address violations of key assumptions, and is available as open source software for genome-wide analysis. Simulations including phenocopies, based on the families of an oral cleft study, revealed the partial and complete sharing versions of RVS achieved similar statistical power compared with alternative methods (RareIBD and the Gene-Based Segregation Test), and had superior power compared with the pedigree Variant Annotation, Analysis, and Search Tool (pVAAST) linkage statistic. In studies of multiplex cleft families, analysis of rare single nucleotide variants in the exome of 151 affected relatives from 54 families revealed no significant excess sharing in any one gene, but highlighted different patterns of sharing revealed by the complete and partial sharing tests., (© 2018 Wiley Periodicals, Inc.)

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect in humans and is notable for its apparent sexual dimorphism where approximately twice as many males are affected as females. The sources of this disparity are largely unknown, but interactions between genetic and sex effects are likely contributors. We examined gene-by-sex (G × S) interactions in a worldwide sample of 2,142 NSCL/P cases and 1,700 controls recruited from 13 countries. First, we performed genome-wide joint tests of the genetic (G) and G × S effects genome-wide using logistic regression assuming an additive genetic model and adjusting for 18 principal components of ancestry. We further interrogated loci with suggestive results from the joint test ( p < 1.00 × 10 -5 ) by examining the G × S effects from the same model. Out of the 133 loci with suggestive results ( p < 1.00 × 10 -5 ) for the joint test, we observed one genome-wide significant G × S effect in the 10q21 locus (rs72804706; p = 6.69 × 10 -9 ; OR = 2.62 CI [1.89, 3.62]) and 16 suggestive G × S effects. At the intergenic 10q21 locus, the risk of NSCL/P is estimated to increase with additional copies of the minor allele for females, but the opposite effect for males. Our observation that the impact of genetic variants on NSCL/P risk differs for males and females may further our understanding of the genetic architecture of NSCL/P and the sex differences underlying clefts and other birth defects., (© 2018 Wiley Periodicals, Inc.)

Orofacial clefts (OFCs) are common, complex birth defects with extremely heterogeneous phenotypic presentations. Two common subtypes-cleft lip alone (CL) and CL plus cleft palate (CLP)-are typically grouped into a single phenotype for genetic analysis (i.e., CL with or without cleft palate, CL/P). However, mounting evidence suggests there may be unique underlying pathophysiology and/or genetic modifiers influencing expression of these two phenotypes. To this end, we performed a genome-wide scan for genetic modifiers by directly comparing 450 CL cases with 1,692 CLP cases from 18 recruitment sites across 13 countries from North America, Central or South America, Asia, Europe, and Africa. We identified a region on 16q21 that is strongly associated with different cleft type (P = 5.611 × 10 -8 ). We also identified significant evidence of gene-gene interactions between this modifier locus and two recognized CL/P risk loci: 8q21 and 9q22 (FOXE1) (P = 0.012 and 0.023, respectively). Single nucleotide polymorphism (SNPs) in the 16q21 modifier locus demonstrated significant association with CL over CLP. The marker alleles on 16q21 that increased risk for CL were found at highest frequencies among individuals with a family history of CL (P = 0.003). Our results demonstrate the existence of modifiers for which type of OFC develops and suggest plausible elements responsible for phenotypic heterogeneity, further elucidating the complex genetic architecture of OFCs., (© 2017 WILEY PERIODICALS, INC.)

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect in humans, affecting 1 in 700 live births. This malformation has a complex etiology where multiple genes and several environmental factors influence risk. At least a dozen different genes have been confirmed to be associated with risk of NSCL/P in previous studies. However, all the known genetic risk factors cannot fully explain the observed heritability of NSCL/P, and several authors have suggested gene-gene (G × G) interaction may be important in the etiology of this complex and heterogeneous malformation. We tested for G × G interactions using common single nucleotide polymorphic (SNP) markers from targeted sequencing in 13 regions identified by previous studies spanning 6.3 Mb of the genome in a study of 1,498 NSCL/P case-parent trios. We used the R-package trio to assess interactions between polymorphic markers in different genes, using a 1 degree of freedom (1df) test for screening, and a 4 degree of freedom (4df) test to assess statistical significance of epistatic interactions. To adjust for multiple comparisons, we performed permutation tests. The most significant interaction was observed between rs6029315 in MAFB and rs6681355 in IRF6 (4df P = 3.8 × 10 -8 ) in case-parent trios of European ancestry, which remained significant after correcting for multiple comparisons. However, no significant interaction was detected in trios of Asian ancestry., (© 2016 WILEY PERIODICALS, INC.)

By sequencing the exomes of distantly related individuals in multiplex families, rare mutational and structural changes to coding DNA can be characterized and their relationship to disease risk can be assessed. Recently, several rare single nucleotide variants (SNVs) were associated with an increased risk of nonsyndromic oral cleft, highlighting the importance of rare sequence variants in oral clefts and illustrating the strength of family-based study designs. However, the extent to which rare deletions in coding regions of the genome occur and contribute to risk of nonsyndromic clefts is not well understood. To identify putative structural variants underlying risk, we developed a pipeline for rare hemizygous deletions in families from whole exome sequencing and statistical inference based on rare variant sharing. Among 56 multiplex families with 115 individuals, we identified 53 regions with one or more rare hemizygous deletions. We found 45 of the 53 regions contained rare deletions occurring in only one family member. Members of the same family shared a rare deletion in only eight regions. We also devised a scalable global test for enrichment of shared rare deletions., (© 2016 WILEY PERIODICALS, INC.)

In a recent genome-wide association study (GWAS) from an international consortium, evidence of linkage and association in chr8q24 was much stronger among nonsyndromic cleft lip/palate (CL/P) case-parent trios of European ancestry than among trios of Asian ancestry. We examined marker information content and haplotype diversity across 13 recruitment sites (from Europe, United States, and Asia) separately, and conducted principal components analysis (PCA) on parents. As expected, PCA revealed large genetic distances between Europeans and Asians, and a north-south cline from Korea to Singapore in Asia, with Filipino parents forming a somewhat distinct Southeast Asian cluster. Hierarchical clustering of SNP heterozygosity revealed two major clades consistent with PCA results. All genotyped SNPs giving P < 10(-6) in the allelic transmission disequilibrium test (TDT) showed higher heterozygosity in Europeans than Asians. On average, European ancestry parents had higher haplotype diversity than Asians. Imputing additional variants across chr8q24 increased the strength of statistical evidence among Europeans and also revealed a significant signal among Asians (although it did not reach genome-wide significance). Tests for SNP-population interaction were negative, indicating the lack of strong signal for 8q24 in families of Asian ancestry was not due to any distinct genetic effect, but could simply reflect low power due to lower allele frequencies in Asians., (© 2012 Wiley Periodicals, Inc.)

A major concern for all copy number variation (CNV) detection algorithms is their reliability and repeatability. However, it is difficult to evaluate the reliability of CNV-calling strategies due to the lack of gold-standard data that would tell us which CNVs are real. We propose that if CNVs are called in duplicate samples, or inherited from parent to child, then these can be considered validated CNVs. We used two large family-based genome-wide association study (GWAS) datasets from the GENEVA consortium to look at concordance rates of CNV calls between duplicate samples, parent-child pairs, and unrelated pairs. Our goal was to make recommendations for ways to filter and use CNV calls in GWAS datasets that do not include family data. We used PennCNV as our primary CNV-calling algorithm, and tested CNV calls using different datasets and marker sets, and with various filters on CNVs and samples. Using the Illumina core HumanHap550 single nucleotide polymorphism (SNP) set, we saw duplicate concordance rates of approximately 55% and parent-child transmission rates of approximately 28% in our datasets. GC model adjustment and sample quality filtering had little effect on these reliability measures. Stratification on CNV size and DNA sample type did have some effect. Overall, our results show that it is probably not possible to find a CNV-calling strategy (including filtering and algorithm) that will give us a set of "reliable" CNV calls using current chip technologies. But if we understand the error process, we can still use CNV calls appropriately in genetic association studies., (© 2012 Wiley Periodicals, Inc.)

Nonsyndromic cleft palate (CP) is a common birth defect with a complex and heterogeneous etiology involving both genetic and environmental risk factors. We conducted a genome-wide association study (GWAS) using 550 case-parent trios, ascertained through a CP case collected in an international consortium. Family-based association tests of single nucleotide polymorphisms (SNP) and three common maternal exposures (maternal smoking, alcohol consumption, and multivitamin supplementation) were used in a combined 2 df test for gene (G) and gene-environment (G × E) interaction simultaneously, plus a separate 1 df test for G × E interaction alone. Conditional logistic regression models were used to estimate effects on risk to exposed and unexposed children. While no SNP achieved genome-wide significance when considered alone, markers in several genes attained or approached genome-wide significance when G × E interaction was included. Among these, MLLT3 and SMC2 on chromosome 9 showed multiple SNPs resulting in an increased risk if the mother consumed alcohol during the peri-conceptual period (3 months prior to conception through the first trimester). TBK1 on chr. 12 and ZNF236 on chr. 18 showed multiple SNPs associated with higher risk of CP in the presence of maternal smoking. Additional evidence of reduced risk due to G × E interaction in the presence of multivitamin supplementation was observed for SNPs in BAALC on chr. 8. These results emphasize the need to consider G × E interaction when searching for genes influencing risk to complex and heterogeneous disorders, such as nonsyndromic CP., (© 2011 Wiley-Liss, Inc.)

An increased risk of facial clefts has been observed among mothers with lower intake of folic acid or vitamin A around conception. We hypothesized that the risk of clefts may be further moderated by genes involved in metabolizing folate or vitamin A. We included 425 case-parent triads in which the child had either cleft lip with or without cleft palate (CL/P) or cleft palate only (CPO), and no other major defects. We analyzed 108 SNPs and one insertion in 29 genes involved in folate/one-carbon metabolism and 68 SNPs from 16 genes involved in vitamin A metabolism. Using the Triad Multi-Marker (TRIMM) approach we performed SNP, gene, chromosomal region, and pathway-wide association tests of child or maternal genetic effects for both CL/P and CPO. We stratified these analyses on maternal intake of folic acid or vitamin A during the periconceptional period. As expected with this high number of statistical tests, there were many associations with P-values<0.05; although there were fewer than predicted by chance alone. The strongest association in our data (between fetal FOLH1 and CPO, P=0.0008) is not in agreement with epidemiologic evidence that folic acid reduces the risk of CL/P in these data, not CPO. Despite strong evidence for genetic causes of oral facial clefts and the protective effects of maternal vitamins, we found no convincing indication that polymorphisms in these vitamin metabolism genes play an etiologic role.

Mutations in the gene encoding interferon regulatory factor 6 (IRF6) underlie a common form of syndromic clefting known as Van der Woude syndrome. Lip pits and missing teeth are the only additional features distinguishing the syndrome from isolated clefts. Van der Woude syndrome, therefore, provides an excellent model for studying the isolated forms of clefting. From a population-based case-control study of facial clefts in Norway (1996-2001), we selected 377 cleft lip with or without cleft palate (CL/P), 196 cleft palate only (CPO), and 763 control infant-parent triads for analysis. We genotyped six single nucleotide polymorphisms within the IRF6 locus and estimated the relative risks (RR) conferred on the child by alleles and haplotypes of the child and of the mother. On the whole, there were strong statistical associations with CL/P but not CPO in our data. In single-marker analyses, mothers with a double-dose of the 'a'-allele at rs4844880 had an increased risk of having a child with CL/P (RR=1.85, 95% confidence interval: 1.04-3.25; P=0.036). An RR of 0.38 (95% confidence interval: 0.16-0.92; P=0.031) was obtained when the child carried a single-dose of the 'a'-allele at rs2235371 (the p.V274I polymorphism). The P-value for the overall test was <0.001. In haplotype analyses, several of the fetal and maternal haplotype relative risks were statistically significant individually but were not strong enough to show up on the overall test (P=0.113). Taken together, these findings further support a role for IRF6 variants in clefting of the lip and provide specific risk estimates in a Norwegian population., ((c) 2008 Wiley-Liss, Inc.)

Tie-2 stabilises pericyte-endothelial interactions during angiogenesis and is highly expressed on endothelium during several diseases, including arthritis, age-related macular degeneration and cancer. A vaccine that targets endothelium overexpressing Tie-2 may result in vessel damage and stimulate an inflammatory cascade resulting in disease regression. We have identified a region unique to Tie-2 (amino acids 1-196) that is homologous in humans and mice. Using computer algorithms, several HLA-A*0201 epitopes that are identical in mice and humans were predicted within this region; however, binding assays showed that the majority of these epitopes were of low affinity. Modification of the anchor residues of 4 epitopes enhanced HLA binding. These epitopes were incorporated by site-directed mutagenesis into a Tie-2 DNA construct. Immunisation of HLA*0201 transgenic mice with one of the modified Tie-2 constructs stimulated CTLs that recognised both wild-type and modified peptide-pulsed target cells. In contrast, no CTLs were generated in mice immunised with wild-type Tie-2 construct, demonstrating that the modified epitope was necessary in the generation of CTLs. Moreover, CTLs from mice immunised with the modified construct killed HLA-A*0201 endothelial cells overexpressing Tie-2. Our study demonstrates that it is possible to break tolerance to the endothelial antigen Tie-2, suggesting that it may be feasible to design a vaccine to activate CTLs to kill endothelial cells overexpressing Tie-2., (Copyright 2004 Wiley-Liss, Inc.)

We have previously reported a threefold risk of cleft palate only (CPO) among children homozygous for the less common allele A2 at the TaqI marker site of the transforming growth factor alpha gene (TGFA) (Jugessur et al. [2003a] Genet. Epidemiol. 24:230-239). Here we assess possible interaction between the child's TGFA TaqI A2A2 genotype and maternal cigarette smoking, alcohol consumption, use of multivitamins and folic acid. This was done by comparing the strength of genetic associations between strata of exposed and unexposed case-parent triads. We also looked for possible gene-gene interaction with the polymorphic variant C677T of the folic acid-metabolizing gene MTHFR. We analyzed a total of 88 complete CPO triads selected from a population-based study of orofacial clefts in Norway (May 1996-1998). No evidence of interaction was observed with either smoking or alcohol use. The risk associated with two copies of the A2 allele at TGFA TaqI was strong among children whose mothers did not use folic acid (relative risk=4.5, 95% confidence interval=1.3-15.7), and was only marginal among children whose mothers reported using folic acid (RR=1.4, 95% CI=0.2-12.7). Although the interaction between the child's genotypes at TGFA TaqI and MTHFR-C677T was not statistically significant, the effect of the TGFA TaqI A2A2 genotype appeared to be stronger among children with either one or two copies of the T-allele at C677T (RR=4.0, 95% CI=1.1-13.9) compared to children homozygous for the C-allele (RR=1.7, 95% CI=0.2-15.7). In conclusion, we find little evidence of interaction between the child's genotypes at TGFA TaqI and various exposures for cleft palate, with the possible exception of folic acid intake., (Copyright 2003 Wiley-Liss, Inc.)

We selected 262 case-parent triads from a population-based study of orofacial clefts in Norway, and examined variants of developmental genes TGFA, TGFB3, and MSX1 in the etiology of orofacial clefts. One hundred seventy-four triads of cleft lip cases (CL+/-P) and 88 triads of cleft palate only cases (CPO) were analyzed. There was little evidence for an association of any of these genes with CL+/-P. The strongest association was a 1.7-fold risk with two copies of the TGFB3-CA variant (95% CI=0.9-3.0). Among CPO cases, there was a 3-fold risk with two copies of the TGFA TaqI A2 allele, and no increase with one copy. Assuming this to be a recessive effect, we estimated a 3.2-fold risk among babies homozygous for the variant (95% CI=1.1-9.2). Furthermore, there was strong evidence of gene-gene interaction. While there was only a weak association of the MSX1-CA variant with CPO, the risk was 9.7-fold (95% CI=2.9-32) among children homozygous for both the MSX1-CA A4 allele and the TGFA A2 allele. No association of CPO with the TGFA variant was seen among the other MSX1-CA genotypes. In conclusion, no strong associations were found between CL+/-P and variants at these three genes. There was a possible recessive effect of the TGFA TaqI variant on the risk of CPO, with a 3-fold risk among children homozygous for the variant. The effect of this TGFA genotype was even stronger among children homozygous for the MSX1-CA A4 allele, raising the possibility of interaction between these two genes., (Copyright 2003 Wiley-Liss, Inc.)

Background: The etiology of monozygotic twinning is not known. Some investigators have implicated abnormal X-inactivation, which could also be related to the increased female:male ratio in higher order multiple gestations in general, and in monozygotic and conjoined twins (CTS) in particular. CTS are rare, and even more unusual when part of a triplet pregnancy., Methods: DNA polymorphism analysis using 13 markers in the buccal cells of the triplets and the lymphocytes of the parents were used to evaluate zygosity. We investigated the X-inactivation pattern of the triplets by analyzing methylation at the androgen receptor gene., Results: We found a female triplet gestation consisting of CTS and a normal singleton. The thoracopagus CTS were joined from the clavicles to the umbilicus. Congenital heart disease was suspected antenatally, but the precise delineation of the heart defects required extensive postnatal evaluation. There was a single placental mass with a thin dividing membrane. Cesarean section was carried out at 32 weeks after the onset of labor. Histologically, the placenta was diamniotic monochorionic. The normal singleton did well after delivery; the CTS died at 35 days from cardiopulmonary collapse. The babies were monozygotic (>99.99% probability). Each baby in this triplet set exhibited a random and symmetric X-inactivation pattern. The degree of X-inactivation skewing fell in the range of 50-65%., Conclusion: Genetic or environmental factors other than abnormal X-inactivation must be involved in causing monozygous multiple gestation or CTS. Despite prenatal diagnosis, shared myocardium or cardiac anomalies in CTS often determine the prognosis., (Copyright 2002 Wiley-Liss, Inc.)

The purpose of this study was to evaluate chromosomal regions previously linked to pathological myopia for linkage to juvenile myopia in a sample of myopic children and their families. Of 125 families with a myopic child participating in the Orinda longitudinal study of myopia, 53 submitted 221 buccal swab samples for genetic analysis. Myopia in proband children was defined as -0.75 D or more myopia in both meridians on cycloplegic autorefraction (1% tropicamide). Affected status in parents and siblings was obtained by survey. DNA was extracted from buccal mucosal cells, amplified by polymerase chain reaction (PCR), and then analyzed with seven markers for chromosome 12 and five markers for chromosome 18 in the regions previously associated with pathological myopia. LOD scores were not significant for any marker tested. The largest positive LOD score was 0.15 for GATA30F04. Model-free methods using a SimIBD approach suggested a possible linkage at one marker, GATA6H09 (P = 0.003), but these results were not supported by transmission disequilibrium test (TDT) analysis. The statistical power to detect LOD scores of > or =1.0, assuming homogeneity, was estimated at 93.2%. We found no confirmatory evidence of linkage between juvenile myopia and regions of chromosomes 12 and 18 previously associated with pathological myopia., (Copyright 2002 Wiley-Liss, Inc.)

Background: Advances in quantitative analysis and molecular genotyping have provided unprecedented opportunities to add biological sampling and genetic information to epidemiologic studies. The purpose of this article is to describe the incorporation of DNA sample collection into the National Birth Defects Prevention Study (NBDPS), an ongoing case-control study in an eight-state consortium with a primary goal to identify risk factors for birth defects., Methods: Babies with birth defects are identified through birth defects surveillance systems in the eight participating centers. Cases are infants with one or more of over 30 major birth defects. Controls are infants without defects from the same geographic area. Epidemiologic information is collected through an hour-long interview with mothers of both cases and controls. We added the collection of buccal cytobrush DNA samples for case-infants, control-infants, and their parents to this study., Results: We describe here the methods by which the samples have been collected and processed, establishment of a centralized resource for DNA banking, and quality control, database management, access, informed consent, and confidentiality issues., Conclusions: Biological sampling and genetic analyses are important components to epidemiologic studies of birth defects aimed at identifying risk factors. The DNA specimens collected in this study can be used for detection of mutations, study of polymorphic variants that confer differential susceptibility to teratogens, and examination of interactions among genetic risk factors. Information on the methods used and issues faced by the NBDPS may be of value to others considering the addition of DNA sampling to epidemiologic studies.

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., (Copyright 2002 Wiley-Liss, Inc.)

Van der Woude syndrome (VWS) is an orofacial clefting disorder with an autosomal dominant pattern of inheritance. In our efforts to clone the VWS gene, 900 kb of genomic sequence from the VWS candidate region at chromosome 1q32-q41 was analyzed for new DNA sequence variants. We observed that in clone CTA-321i20 a 7922 bp sequence is absent relative to the sequence present in PAC clone RP4-782d21 at positions 1669-9590, suggesting the presence of a deletion/insertion (del/ins) polymorphism. Embedded in this 7922 bp region was a TTCC short tandem repeat (STR). Genotype analysis showed that both the internal STR and the (del/ins) mutation were true polymorphisms. This is a novel example of intraallelic variation, a polymorphism within a polymorphism, and we suggest that it be termed a "Matroshka" polymorphism. Further genetic and DNA sequence analysis indicated that the ancestral state of the 1669-9590 del/ins polymorphism was the insertion allele and that the original deletion mutation probably occurred only once. A second class of novel DNA sequence variation was discovered on chromosome 5 that shared a 328 bp identical sequence with this region on chromosome 1. A single nucleotide polymorphism (SNP) was detected by SSCP using a pair of primers derived from the chromosome 1 sequence. Surprisingly, these primers also amplified the identical locus on chromosome 5, and the SNP was only located on chromosome 5. Since the probe unexpectedly detected alleles from another locus, we suggest that this type of sequence variant be termed an "ectopic" polymorphism. These two novel classes of DNA sequence polymorphisms have the potential to confound genetic and DNA sequence analysis and may also contribute to variation in disease phenotypes., (Copyright 2001 Wiley-Liss, Inc.)

In addition to craniofacial, auricular, ophthalmologic, and oral anomalies, the distinctive phenotype of the branchio-oculo-facial (BOF) syndrome (MIM 113620) includes skin defects in the neck or infra/supra-auricular region. These unusual areas of thin, erythematous wrinkled skin differ from the discrete cervical pits, cysts, and fistulas of the branchio-oto-renal (BOR) syndrome (MIM 113650). Although the BOF and BOR syndromes are sufficiently distinctive that they should not be confused, both can be associated with nasolacrimal duct stenosis, deafness, prehelical pits, malformed pinna, and renal anomalies. Furthermore, a reported father and son [Legius et al., 1990, Clin Genet 37:347-500] had features of both conditions. It was not clear whether they had an atypical presentation of either BOR or BOF syndrome, or represented a private syndrome. In light of these issues, we selected the BOR locus (EYA1) as a possible gene mutation for the BOF syndrome. In five BOF patients, there were no mutations detected in the EYA1 gene, suggesting that it is not allelic to the BOR syndrome., (Copyright 2000 Wiley-Liss, Inc.)

Van der Woude syndrome (VWS) is an autosomal dominant disorder comprising cleft lip and/or cleft palate and lip pits. We reported previously a family whose underlying mutation is a 500-800 kb deletion localized to chromosome bands 1q32-q41 [Sander et al., 1994: Hum Mol Genet 3:576-578]. Along with cleft lip/palate and lip pits, affected relatives exhibit developmental delays, suggesting that the function of a gene nearby may also be disrupted. To further localize the VWS gene we searched for other deletions that cause VWS. An allele loss assay was performed using a novel highly polymorphic marker, D1S3753. From a panel of 37 unrelated individuals, we detected an allele loss in one family, indicating the presence of a deletion. In this family, the phenotype in three generations of affected individuals was confined to the cardinal signs of VWS. Surprisingly, mapping of the new deletion showed that it extended 0.2-1 Mb beyond the proximal breakpoint for the deletion described previously. No deletions were detected in seven cases of popliteal pterygia syndrome, 76 cases of mixed syndromic forms of cleft lip and palate, and 178 cases of nonsyndromic cleft lip and palate. These observations suggest that genetic searches for microdeletions should be routine in screening patients for causes of VWS and may facilitate the positional cloning efforts of the VWS gene and of a nearby gene or genes that may be involved in brain development.

Neural tube defects (NTDs) are a common birth defect, seen in approximately 1/1,000 births in the United States. NTDs are considered a complex trait where several genes, interacting with environmental factors, create the phenotype. Using a Midwestern NTD population consisting of probands, parents, and siblings from Iowa, Minnesota, and Nebraska, we analyzed a range of candidate genes, including 5,10-methylenetetrahydrofolate reductase (MTHFR), folate receptors-alpha (FOLR1; hereafter abbreviated "FR-alpha") and -beta (FOLR2; hereafter, "FR-beta"), methionine synthase (hereinafter, "MS"), T, the human homolog of the murine Brachyury gene, and the paired-box homeotic gene 3 (PAX3), for association with NTDs. We were unable to demonstrate an association using a previously described Ala-->Val mutation in MTHFR and the majority of our NTD populations. However, we discovered a silent polymorphism in exon 6 of MTHFR which conserved a serine residue and which showed significant association with NTDs in our Iowa population. Analysis of exon 7 of MTHFR then demonstrated an Ala-->Glu mutation which was significantly associated with our Iowa NTD population; however, we could not replicate this result either in a combined Minnesota/ Nebraska or in a California NTD population. Using polymorphic markers for MS, FR-beta, T, and PAX3, we were unable to demonstrate linkage disequilibrium with our NTD populations. A mutation search of FR-alpha revealed one proband with a de novo silent mutation of the stop codon. This work provides a new panel of genetic variants for studies of folate metabolism and supports, in some NTD populations, an association between MTHFR and NTDs.

Previous studies suggest that the relationship between genes and nonsyndromic cleft lip +/- cleft palate (CLP) or cleft palate only (CP) may be modified by the environment. Using data from a population-based case-control study, we examined allelic variants for three genes, i.e., transforming growth factor alpha (TGFA), transforming growth factor beta 3 (TGFB3), and Msh (Drosophila) homeobox homolog 1 (MSX1), and their interactions with two exposures during pregnancy (maternal cigarette smoking and alcohol consumption) as risk factors for CLP and CP. For each cleft phenotype, risk estimates associated with most allelic variants tended to be near unity. Risk estimates for maternal smoking (> or = 10 cigarettes/day) were significantly elevated for CP and were most elevated among infants with allelic variants at the TGFB3 or MSX1 sites. By comparison, risk estimates for maternal alcohol consumption (> or = 4 drinks/month) were significantly elevated for CLP and were most elevated among infants with allelic variants at the MSX1 site. Our results suggest that development of CLP and CP may be influenced independently by maternal exposures but more significantly by interaction of such exposures and specific allelic variants.

Accurate family histories of birth defects are critical for risk assessment and etiologic investigations. Typically, information about family history of birth defects is ascertained from interviews with birth mothers of index children; however, the quality of these interviews is rarely assessed. We evaluated family history information provided by case (n = 28) and control (n = 29) mothers who participated in a population-based, case-control study of orofacial clefts. Interview responses from mothers were compared to questionnaire reports collected by mail from first- and second-degree parental relatives. A total of 345 case and 380 control adult relatives completed questionnaires. These relatives also provided reports for 130 case and 169 control offspring. To examine the quality of birth defect reports, the sensitivity and specificity of birth mother responses were calculated. Sensitivity for presence (yes/no) of a birth defect was 31% for case mothers and 9% for control mothers. Specificity for case and control mothers was 98% and 97%, respectively. Interview responses from mothers who participate in family genealogy were more likely to be concordant with relative reports than were responses from mothers who do not participate in family genealogy. Case mother responses were more likely to be concordant than control mother responses. These results suggest that reliance on interview reports from birth mothers may lead to an underestimation of the occurrence of birth defects in relatives. Future investigations should explore methods to improve the quality of informant reports about family histories of birth defects. One alternative approach is discussed.

Fibroblast growth factor-8 (Fgf8) is a recently identified growth factor that stimulates the androgen-dependent growth of mouse mammary carcinoma cells. Evidence from mouse development also shows that Fgf8 may play an important role in growth and patterning of limbs, face, and the central nervous system. We describe here the human FGF8 genomic sequence and demonstrate conservation between the human and mouse sequences, including alternatively spliced exons in the mouse. Mapping of FGF8 by FISH using an FGF8-containing bacterial artificial chromosome and by genetic linkage using a SSCP variant identified in this study is also reported and refines the FGF8 map location to 10q24. Since FGF8 maps to the same chromosomal region as FGFR2, has indeed been shown to be a ligand for FGFR2, and has an expression pattern consistent with limb and craniofacial anomalies, we have screened two kindreds with Pfeiffer syndrome that were previously linked to markers from 10q24-25 and a large number of individuals with craniosynostosis and limb anomalies for mutations in the coding sequence of FGF8. While no such mutations were identified, a rare polymorphic variant, consisting of an 18-base-pair (six-amino-acid) duplication in exon 1c, is reported that apparently has no clinical effect. Our exclusionary data suggest that mutations in FGF8 would be, at best, an infrequent cause of such disorders.

Maternal alcohol use during pregnancy is a known cause of birth defects associated with the fetal alcohol syndrome, but its role in more common, isolated, craniofacial birth defects is not well understood. A population-based, case-control study of orofacial clefts was conducted in Iowa using births during 1987-1991. Cases were identified by the Iowa Birth Defects Registry and classified as having a cleft lip with or without cleft palate (CLP) or cleft palate only (CP) and whether the cleft was isolated or occurred with other birth defects. Controls were selected from normal Iowa births. Maternal alcohol use during pregnancy was classified according to self-reported drinks consumed per month. Results are based on 302 controls and the following numbers in each case group: 118 isolated CLP, 56 isolated CP, 51 CLP with multiple defects, and 62 CP with multiple defects. Compared to women who did not drink alcohol during pregnancy, the relative odds of isolated CLP rose with increasing level of maternal drinking as follows: 1-3 drinks per months, 1.5; 4-10 drinks per month, 3.1; more than 10 drinks per month, 4.7 (chi-square test for trend, P = 0.003). Adjustment for maternal smoking, vitamin use, education, and household income did not substantially alter these results. No significant association was found between alcohol use and isolated cleft palate or clefts in children with multiple birth defects. Alcohol use during pregnancy may be a cause of isolated cleft lip with or without cleft palate.

A critical step in the metastatic spread of tumour cells is the interaction of circulating tumour cells with the vascular endothelium. We have investigated the role of CD44 and its variants in the adhesion of a human melanoma cell line (RPMI-7951) and a breast adenocarcinoma cell line (MDA-MB-231) to quiescent human umbilical vein endothelial cells (HUVEC) in vitro. Both tumour cell lines express CD44H, CD44A and CD44v9, while HUVEC express only CD44H. Pre-treatment of endothelial cell monolayers with a blocking monoclonal antibody against CD44H (MAb 5A4) reduced the adhesion of RPMI-7951 cells but not that of MDA-MB-231. In contrast, pre-treatment of both tumour cell lines with the same antibody had no effect on adhesion. Digestion of the CD44 ligand hyaluronic acid (HA) on RPMI-7951 cells significantly reduced adhesion to endothelial monolayers, while digestion of HUVEC HA had no effect. We conclude that CD44H expressed on the surface of quiescent endothelial monolayers mediates in part the adhesion of the metastatic melanoma cell line RPMI-7951 but not that of a breast adenocarcinoma line. It does so by acting as a receptor for HA on the tumour cell surface. Tumour cell CD44H and variants CD44A and CD44v9 do not appear to be involved in adhesion to endothelial cells.

Atypical cytogenetic abnormalities were detected in peripheral primitive neuroectodermal tumors (PPNET) of the extremity in two children. One had an osseous tumor with a balanced reciprocal translocation, t(5;9)(q22;q32), and had a complete response to therapy. The other had a non-osseous tumor with an interstitial deletion, del(18)(q12.2q21.2), was resistant to combination therapy, and at autopsy had evidence of possible clonal evolution with the karyotype 46,XX der(8)t(8;8)(p11.2;q13), inv(16)(p13.2q12),del(18)(q12.2q21.2). Neither tumor demonstrated the t(11;22)(q24;q12) typically found in Ewing's sarcoma and PPNET, suggesting heterogeneity of the cytogenetic aberrations seen in this rare childhood malignancy.

An 11-year-old boy with prior bone marrow and testicular relapses of his acute lymphoblastic leukemia (ALL) developed an isolated metatarsal bone relapse during complete hematologic remission 10 months after completion of chemotherapy. Although there was no radiographic or histologic evidence of additional occult leukemia, the polymerase chain reaction (PCR) technique detected a leukemic clone in both his bone marrow and metatarsal. A literature survey revealed only 10 reported cases of isolated bone relapse occurring during complete bone marrow remission in childhood ALL. Most of these patients had prior bone marrow or extramedullary relapses. The majority experienced subsequent relapses after their isolated bone recurrence. We report a case of isolated bone recurrence, review all previously reported cases, and suggest that PCR elucidation of clonal disease may provide a better understanding of these extremely rare extramedullary events.

Flavone acetic acid (FAA) is a potentially useful anti-tumour agent which has been reported to induce changes in tumour vasculature, in particular loss of bloodflow. This led us to examine whether endothelium could be a cellular target of FAA action, with resultant modulation of cell-surface coagulant properties leading to activation of coagulation and blockade of tumour blood flow. Incubation of endothelium with FAA led to the expression of functional tissue factor on the cell surface, in a time-dependent and dose-dependent (half-maximal at 0.6-0.7 mg/ml) manner. Induction of tissue-factor activity resulted from de novo translation of the tissue factor message. To explain the selectivity of FAA's action on tumour vasculature in vivo, we considered its interaction with tumour-derived factors. Starting with serum-free FO-I-melanoma cell-conditioned medium, a co-factor enhancing FAA-mediated induction of endothelial tissue factor (FO-I factor) was partially purified by sequential ion exchange and reverse phase chromatography, followed by preparative SDS-PAGE. The FO-I factor migrates with an apparent Mr of approx. 20 to 25,000 on non-reduced SDS-PAGE, is sensitive to protease K, and augments the effect of FAA on endothelial-cell-tissue factor. This activity is not found in supernatants from non-neoplastically transformed cell lines. These data lead us to hypothesize that FAA exerts its action, at least in part, by promoting activation of coagulation on the endothelial surface, and this effect is selective for the tumour bed by virtue of its interaction with a tumour-derived factor. The interaction of FAA with host factors may be important for optimizing its therapeutic efficacy for a particular tumour.

We have investigated the possibility of mutations in the calcitonin/calcitonin gene related peptide (CGRP) gene in children with Williams syndrome. Involvement of the calcitonin/CGRP gene in Williams syndrome is postulated on the basis that Williams syndrome children often have infantile hypercalcemia and deficient expression of calcitonin, a hormone that lowers serum calcium levels. To test the hypothesis that mutations in the calcitonin/CGRP gene might be responsible for the reduced calcitonin levels, we examined the calcitonin/CGRP gene structure in Williams syndrome children. Analysis of white blood cell DNA by Southern blot hybridizations in 5 individuals did not show any detectable large deletions or rearrangements in the calcitonin/CGRP gene locus. The possibility of small deletions or point mutations within the exon encoding the mature calcitonin hormone is unlikely based on ribonuclease protection assays with patient DNA amplified by the polymerase chain reaction (PCR) technique. These findings suggest that the calcitonin deficiency might be due either to mutations elsewhere in the gene or to defects in the cellular machinery needed for calcitonin synthesis and/or secretion.

Börjeson-Forssman-Lehmann syndrome (BFLS) is a form of X-linked mental retardation (XLMR) with characteristic minor physical anomalies. It has no biochemical or cytogenetic markers. Heterozygous females may be entirely normal or may have mild-to-moderate manifestations. We studied 41 individuals from one family with BFLS for linkage on the X chromosome. The highest lod scores were 2.32 with DXS10 and 2.24 with DXS51, both at a theta = 0.0. A single recombinant was found between HPRT and BFLS. These results suggest that the BFLS locus is on the distal portion of Xq. Previously reported linkage studies in families with XLMR have not shown linkage with DXS10. This study suggests that one of the several X chromosome loci whose dysfunction is associated with mental retardation is located on distal Xq.

We report on the incidence of Rh isoimmunization after genetic amniocentesis at our institution. In 115 Rh negative women who underwent amniocentesis and subsequently delivered Rh positive infants, there were 4 (3.4%) sensitizations before birth. This was significantly greater than the 1.5% rate of gestational sensitization found in pooled populations of women who did not undergo amniocentesis. We also noted a significant increase in the number of sensitizations that occurred before 28 weeks. The results were consistent with those of previous studies of this issue, and are discussed in relation to current policies for managing Rh negative women who have second trimester amniocentesis.