Pichit Siriwan

A mutation of the p63 gene in non-syndromic cleft lip

Mutations in the p63 gene (TP63) underlie several monogenic malformation syndromes manifesting cleft lip with or without cleft palate (CL/P). We investigated whether p63 mutations also result in non-syndromic CL/P. Specifically, we performed mutation analysis of the 16 exons of the p63 gene for 100 Thai patients with non-syndromic CL/P. In total, 21 variant sites were identified. All were single nucleotide changes, with six in coding regions, including three novel non-synonymous changes: S90L, R313G, and D564H. The R313G was concluded to be pathogenic on the basis of its amino acid change, evolutionary conservation, its occurrence in a functionally important domain, its predicted damaging function, its de novo occurrence, and its absence in 500 control individuals. Our data strongly suggest, for the first time, a causative role of a heterozygous mutation in the p63 gene in non-syndromic CL/P, highlighting the wide phenotypic spectrum of p63 gene mutations.

Significant association between IRF6 820G→A and non-syndromic cleft lip with or without cleft palate in the Thai population

Background: Previous data have shown an association between DNA sequence variants in the IRF6 gene and an increased risk of non-syndromic cleft lip with or without cleft palate (CL/P) in some populations. Objective: To investigate Thai CL/P patients and relative for a 820G→A polymorphism. Subjects: 192 CL/P Thai patients, 177 of their mothers, 73 of their fathers, and 278 controls. Results: There were significant differences in the frequency distributions of both genotypes (p = 0.02) and alleles (p = 0.04) among probands as compared with the control group. The odds ratio calculated for the patients having the GG genotype compared with the other two genotypes (GA and AA) was 1.67 (95% confidence interval, 1.13 to 2.47). This pattern is consistent with a recessive effect of the G allele. No association between any of the parents’ genotypes and CL/P was found. The IRF6 820G→A was responsible for 16.7% of the genetic contribution to CL/P. Conclusions: The findings confirm that IRF6 820G→A is associated with CL/P.

Maternal 677CT/1298AC genotype of the MTHFR gene as a risk factor for cleft lip

Non-syndromic cleft lip with or without cleft palate (CL/P) is one of the most common congenital anomalies world wide. It has a prevalence of approximately 1/1000 among white populations1 and 1/600 among Thai newborns.2 Environmental and genetic factors have been implicated in CL/P and several different loci and genes have been associated with them.3 Maternal folic acid supplementation during early pregnancy may reduce the risk for oral clefts,4,5 but this is controversial.6 One of the mechanisms by which low folate levels predispose some subjects to oral clefts could be the presence of polymorphisms in the genes encoding enzymes of the folate pathway, such as 5,10-methylenetetrahydrofolate reductase ( MTHFR , MIM 236250). MTHFR catalyses the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and the carbon donor for the remethylation of homocysteine to methionine. Two polymorphisms, 677C>T and 1298A>C, in the MTHFR gene have been shown to have reduced MTHFR activity.7,8 The 677C>T transition, producing an alanine to valine amino acid substitution within the catalytic domain of the MTHFR enzyme,7 has been associated with many disorders and conditions including neural tube defects,9 vascular disease,7 migraine,10 smoking behaviour,11 and oral clefts.12,13 However, the last is controversial.14 Recent studies reported an association between the maternal polymorphism and the anomalies,15,16 but this again is not a consistent finding.17,18 No studies have investigated 1298A>C, the second most common polymorphism in MTHFR resulting in a glutamate to alanine substitution, in CL/P patients and their parents. We therefore carried out a case-control study to determine whether the two MTHFR polymorphisms in Thai patients with CL/P or their parents were associated with an increased risk of the anomaly. The study sample consisted of 109 CL/P patients, …

MSX1 mutations contribute to nonsyndromic cleft lip in a Thai population

AbstractPrevious studies observed that MSX1 mutations could contribute to nonsyndromic cleft lip with or without cleft palate (CL/P) in some populations. Of the proposed pathogenic mutations, the P147Q variant was predominant in Vietnamese and present in Filipino populations. We investigated whether MSX1 mutations also contribute to nonsyndromic CL/P in the Thai population. Specifically, we performed mutation analysis covering all the coding regions of the MSX1 gene for 100 Thai patients with nonsyndromic CL/P. A total of eight variant sites were identified. Six were in coding regions, including four nonsynonymous changes, 101C>G (A34G), 440C>A (P147Q), 799G>T (G267C), and 832C>T (P278S). The G267C and P278S variants were predicted to be “probably damaging” by PolyPhen, changed themselves as potential exonic splicing enhancers for serine/arginine-rich proteins, and were not present in 162 control individuals of Thai ethnic background. Unlike all of the previously reported potential missense mutations in MSX1, these two novel potential mutations were found in exon 2 on the C-terminal side of the homeodomain protein. Moreover, in contrast to previous reports, we found the P147Q variant in 8 out of 100 Thai controls and an association between the variant and CL/P in our population could not be detected, suggesting that it is not pathogenic. Our data support that MSX1 mutations are found in 2% of cases of CL/P and should be considered for genetic counseling implications, but suggest that the P147Q variant is not pathogenic.

TBX22 mutations are a frequent cause of non‐syndromic cleft palate in the Thai population

Mutations in the TBX22 gene underlie an X‐linked malformation syndrome with cleft palate (CP) and ankyloglossia. Its mutations also result in non‐syndromic CP in some populations. To investigate whether mutations in TBX22 play a part in the formation of non‐syndromic CP in the Thai population, we performed mutation analysis covering all the coding regions of the TBX22 gene in 53 unrelated Thai patients with non‐syndromic CP. We identified four potentially pathogenic mutations, 359G→A (R120Q), 452G→T (R151L), 1166C→A (P389Q), and 1252delG in four different patients. All mutations were not detected in at least 112 unaffected ethnic‐matched control chromosomes and had never been previously reported. R120Q and R151L, found in two sporadic cases, were located in the DNA binding T‐box domain. P389Q and 1252delG, found in two familial cases, were at the carboxy‐terminal region, which has never been described. Our study indicates that TBX22 mutations are responsible for a significant proportion of Thai non‐syndromic CP cases confirming its importance as a frequent cause of non‐syndromic CP across different populations.

PDGFRa mutations in humans with isolated cleft palate

Isolated cleft palate (CP) is common in humans and has complex genetic etiologies. Many genes have been found to contribute to CP, but the full spectrum of genes remains unknown. PCR-sequencing of the entire coding regions and the 3′ untranslated region (UTR) of the platelet-derived growth factor receptor alpha (PDGFRa) and the microRNA (miR), miR-140 identified seven novel single base-pair substitutions in the PDGFRa in 9/102 patients with CP (8.8%), compared with 5/500 ethnic-matched unaffected controls (1%) (the two-tailed P-value<0.0001). Of these seven, four were missense mutations in the coding regions and three in the 3′UTR. Frequencies of four changes (three in coding, one in 3′UTR) were statistically different from those of controls (P-value<0.05). The c.*34G>A was identified in 1/102 cases and 0/500 controls. This position is conserved in primates and located 10 bp away from a predicted binding site for the miR-140. Luciferase assay revealed that, in the presence of miR-140, the c.*34G>A significantly repressed luciferase activity compared with that of the wild type, suggesting functional significance of this variant. This is the first study providing evidence supporting a role of PDGFRa in human CP.

A Novel Mutation in EFNB1, Probably With a Dominant Negative Effect, Underlying Craniofrontonasal Syndrome

Craniofrontonasal syndrome (CFNS) is an X-linked disorder whose main clinical manifestations include coronal craniosynostosis and frontonasal dysplasia. Very recently, CFNS was shown to be caused by mutations in EFNB1 encoding ephrin-B1, and 20 mutations have been described. We report a Thai woman with CFNS, in whom a novel mutation was discovered: c.685_686insG, in exon 5 of EFNB1. It is the first insertion and the most 3′ point mutation in EFNB1 reported to date. The mutation is expected to result in a truncated ephrin-B1 of 230 amino acids, composed of a nearly complete extracellular part of ephrin-B1 with no transmembrane and cytoplasmic domains. This truncated protein might become a soluble form of the ligand, which previously was shown to be able to bind to receptors, but fail to cluster and to activate them—in other words, acting as a dominant negative protein. Nonetheless, further studies to detect the protein are needed to substantiate the hypothesis.

De novo missense mutation, S541Y, in the p63 gene underlying Rapp–Hodgkin ectodermal dysplasia syndrome

Rapp–Hodgkin syndrome (RHS) is an autosomal dominant disorder characterized by ectodermal dysplasia and cleft lip/cleft palate. Very recently, mutations in p63 have been identified as a cause of RHS; to date five such mutations have been identified. We describe a Thai girl with RHS. She had short stature, ectodermal dysplasia, epiphora, cleft lip, cleft palate, and normal development. Mutation analysis for the entire coding region of p63 identified a novel and de novo mutation, 1622C→A (S541Y), in the SAM domain, predicting an abnormal α tail of the p63α protein isotypes. This observation supports that majority of patients with RHS are caused by mutations affecting the tail of p63α, a region that also contains most of the pathogenic mutations in ankyloblepharon‐ectodermal dysplasia‐clefting (AEC) syndrome.

Study of the poliovirus receptor related-1 gene in Thai patients with non-syndromic cleft lip with or without cleft palate

Non-syndromic cleft lip with or without cleft palate (CL/P) has a complex etiology with several genetic and environmental factors playing a role. The poliovirus receptor related-1 gene (PVRL1) has been shown to underlie a syndromic form of CL/P and, in some populations, contribute to non-syndromic CL/P. To investigate whether mutations in PVRL1 play a part in the formation of non-syndromic CL/P in the Thai population, 100CL/P patients were analyzed for mutations in PVRL1 by polymerase chain reaction amplification and direct sequencing of all the coding regions of its alpha isoform. Of this series of patients, one was found to be heterozygous for 1183G>A in exon 6, expected to result in the substitution of a valine by a methionine at position 395 (V395M). This mutation was not found in 200 unaffected Thai control individuals. The valine position is conserved across all known mammalian PVRL1 sequences. In conclusion, a novel non-synonymous PVRL1 mutation was found in a Thai patient with non-syndromic CL/P, suggesting a possible etiologic role of PVRL1 in non-syndromic CL/P across different populations.

Three novel mutations of the IRF6 gene with one associated with an unusual feature in Van der Woude syndrome.

Van der Woude syndrome (VWS) is a dominantly inherited disorder characterized by cleft lip with or without cleft palate and lip pits. It remains the most common syndromic form of oral clefts. Mutations in the interferon regulatory factor 6 (IRF6) gene have been identified in patients with VWS. We reported three unrelated families with lower lip anomalies. Two had lower lip pits, a cardinal sign of VWS, but the other had a heart‐shaped mass on lower lip without pits, oral clefts, or hypodontia. This isolated anomaly has not been previously observed in VWS. We performed mutation analysis by PCR‐sequencing the entire coding region of the IRF6 gene. Three potentially pathogenic mutations, c.145C>T (p.Q49X), c.171T>G (p.F57L), and 1306C>G (p.L436V) were successfully identified. All the missense mutations were not detected in 100 unaffected ethnic‐matched control chromosomes and have never been previously reported. The p.Q49X and p.F57L mutations were located in the highly conserved DNA binding domain while the p.L436V was located at the carboxy‐terminal region. This study reported an undescribed clinical feature of VWS and three novel mutations, expanding the phenotypic spectrum of VWS and mutational spectrum of IRF6.