Family-based genome-wide association study in Patagonia confirms the association of the DMD locus and cleft lip and palate.

The etiology of cleft lip with or without cleft palate (CL±P) is complex and heterogeneous, and multiple genetic and environmental factors are involved. Some candidate genes reported to be associated with oral clefts are located on the X chromosome. At least three genes causing X-linked syndromes [midline 1 (MID1), oral-facial-digital syndrome 1 (OFD1), and dystrophin (DMD)] were previously found to be associated with isolated CL±P. We attempted to confirm the role of X-linked genes in the etiology of isolated CL±P in a South American population through a family-based genome-wide scan. We studied 27 affected children and their mothers, from 26 families, in a Patagonian population with a high prevalence of CL±P. We conducted an exploratory analysis of the X chromosome to identify candidate regions associated with CL±P. Four genomic segments were identified, two of which showed a statistically significant association with CL±P. One is an 11-kb region of Xp21.1 containing the DMD gene, and the other is an intergenic region (8.7 kb; Xp11.4). Our results are consistent with recent data on the involvement of the DMD gene in the etiology of CL±P. The MID1 and OFD1 genes were not included in the four potential CL±P-associated X-chromosome genomic segments.

Interaction between IRF6 and TGFA genes contribute to the risk of nonsyndromic cleft lip/palate.

Previous evidence from tooth agenesis studies suggested IRF6 and TGFA interact. Since tooth agenesis is commonly found in individuals with cleft lip/palate (CL/P), we used four large cohorts to evaluate if IRF6 and TGFA interaction contributes to CL/P. Markers within and flanking IRF6 and TGFA genes were tested using Taqman or SYBR green chemistries for case-control analyses in 1,000 Brazilian individuals. We looked for evidence of gene-gene interaction between IRF6 and TGFA by testing if markers associated with CL/P were overtransmitted together in the case-control Brazilian dataset and in the additional family datasets. Genotypes for an additional 142 case-parent trios from South America drawn from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), 154 cases from Latvia, and 8,717 individuals from several cohorts were available for replication of tests for interaction. Tgfa and Irf6 expression at critical stages during palatogenesis was analyzed in wild type and Irf6 knockout mice. Markers in and near IRF6 and TGFA were associated with CL/P in the Brazilian cohort (p<10(-6)). IRF6 was also associated with cleft palate (CP) with impaction of permanent teeth (p<10(-6)). Statistical evidence of interaction between IRF6 and TGFA was found in all data sets (p = 0.013 for Brazilians; p = 0.046 for ECLAMC; p = 10(-6) for Latvians, and p = 0.003 for the 8,717 individuals). Tgfa was not expressed in the palatal tissues of Irf6 knockout mice. IRF6 and TGFA contribute to subsets of CL/P with specific dental anomalies. Moreover, this potential IRF6-TGFA interaction may account for as much as 1% to 10% of CL/P cases. The Irf6-knockout model further supports the evidence of IRF6-TGFA interaction found in humans.

CRISPLD2 variants including a C471T silent mutation may contribute to nonsyndromic cleft lip with or without cleft palate.

OBJECTIVE: To assess the association between nonsyndromic (NS) cleft lip with or without cleft palate (CL(P)) and single-nucleotide polymorphisms (SNPs) within the CRISPLD2 gene (cysteine-rich secretory protein LCCL domain containing 2). DESIGN: Four SNPs within the CRISPLD2 gene domain (rs1546124, rs8061351, rs2326398, rs4783099) were genotyped to test for association via family-based association methods. PARTICIPANTS: A total of 5826 individuals from 1331 families in which one or more family member is affected with CL(P). RESULTS: Evidence of association was seen for SNP rs1546124 in U.S. (p  =  .02) and Brazilian (p  =  .04) Caucasian cohorts. We also found association of SNP rs1546124 with cleft palate alone (CP) in South Americans (Guatemala and ECLAMC) and combined Hispanics (Guatemala, ECLAMC, and Texas Hispanics; p  =  .03 for both comparisons) and with both cleft lip with cleft palate (CLP; p  =  .04) and CL(P) (p  =  .02) in North Americans. Strong evidence of association was found for SNP rs2326398 with CP in Asian populations (p  =  .003) and with CL(P) in Hispanics (p  =  .03) and also with bilateral CL(P) in Brazilians (p  =  .004). In Brazilians, SNP rs8061351 showed association with cleft subgroups incomplete CL(P) (p  =  .004) and unilateral incomplete CL(P) (p  =  .003). Prediction of SNP functionality revealed that the C allele in the C471T silent mutation (overrepresented in cases with CL(P) presents two putative exonic splicing enhancer motifs and creates a binding site AP-2 alpha, a transcription factor involved in craniofacial development. CONCLUSIONS: Our results support the hypothesis that variants in the CRISPLD2 gene may be involved in the etiology of NS CL(P).

Follow-up association studies of chromosome region 9q and nonsyndromic cleft lip/palate.

Cleft lip/palate comprises a large fraction of all human birth defects, and is notable for its significant lifelong morbidity and complex etiology. Several studies have shown that genetic factors appear to play a significant role in the etiology of cleft lip/palate. Human chromosomal region 9q21 has been suggested in previous reports to contain putative cleft loci. Moreover, a specific region (9q22.3-34.1) was suggested to present a approximately 45% probability of harboring a cleft susceptibility gene. Fine mapping of 50 SNPs across the 9q22.3-34.11 region was performed to test for association with cleft lip/palate in families from United States, Spain, Turkey, Guatemala, and China. We performed family-based analyses and found evidence of association of cleft lip/palate with STOM (rs306796) in Guatemalan families (P = 0.004) and in all multiplex families pooled together (P = 0.002). This same SNP also showed borderline association in the US families (P = 0.04). Under a nominal value of 0.05, other SNPs also showed association with cleft lip/palate and cleft subgroups. SNPs in STOM and PTCH genes and nearby FOXE1 were further associated with cleft phenotypes in Guatemalan and Chinese families. Gene prioritization analysis revealed PTCH and STOM ranking among the top fourteen candidates for cleft lip/palate among 339 genes present in the region. Our results support the hypothesis that the 9q22.32-34.1 region harbors cleft susceptibility genes. Additional studies with other populations should focus on these loci to further investigate the participation of these genes in human clefting.

Single median maxillary central incisor: new data and mutation review.

BACKGROUND: Single median maxillary central incisor (SMMCI) is a rare anomaly that may occur alone or associated with other conditions, frequently as part of the holoprosencephaly (HPE) spectrum. However, it has been suggested that SMMCI alone, or associated with some midline defects, may be considered a different entity from HPE (OMIM: 147250). Families with SMMCI, without HPE cases, are difficult to counsel for the risk of HPE in future generations because the same midline defects described as part of the "SMMCI syndrome" can also be part of the HPE spectrum. METHODS: We screened five cases of SMMCI for mutations in three HPE genes, SHH, TGIF, and SIX3. RESULTS: A missense mutation c.686C>T was found in the gene SIX3 of one patient, which did not differ from the accepted 20% of known HPE gene mutations among all HPE cases. Our results and an extensive literature review of gene mutations in patients with SMMCI showed that 27/28 of them were in HPE genes: SHH (n = 21), SIX3 (n = 3), TGIF (n = 1), GLI2 (n = 1), and PTCH (n = 1), and only one in the SALL4 gene. CONCLUSIONS: The clinical findings in patients with SMMCI without HPE in families with mutations in HPE genes cannot be distinguished from the findings reported in the SMMCI syndrome. Therefore, persons with SMMCI and their relatives should be carefully investigated for related midline disorders, especially of the HPE spectrum, and all known HPE genes screened.