Chalurmpon Srichomthong

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.

Germline and Somatic DICER1 Mutations in a Pituitary Blastoma Causing Infantile-Onset Cushing's Disease

CONTEXT Pituitary blastoma causing Cushings syndrome in infancy is very rare, and its molecular pathomechanism is not well understood. OBJECTIVE Our objective was to identify genetic changes of a pituitary blastoma causing infantile-onset Cushings syndrome in a Thai girl without a family history of cancers. METHODS Genomic DNA from both leukocytes and tumor tissues was used for whole-exome sequencing (WES) and Sanger sequencing of DICER1. The cDNA reverse-transcribed from RNA extracted from both leukocytes and tumor tissues was used for Sanger sequencing, quantitative real-time PCR (qRT-PCR), and pyrosequencing of DICER1. RESULTS WES of leukocytes identified a novel heterozygous c.3046delA (p.S1016VfsX1065) mutation in the DICER1 gene. WES of the tumor tissues detected the same frameshift germline mutation and another novel somatic missense c.5438A→T (p.E1813V) mutation. Both mutations were validated by Sanger sequencing. Quantitative real-time PCR revealed that the DICER1 mRNA levels of the tumor tissues were 54% compared with those of her leukocytes. Pyrosequencing showed that the deletion allele constituted 12% and 0% of the DICER1 cDNA of the probands leukocytes and tumor tissues, respectively. CONCLUSION Our study extends the phenotypic and mutational spectrum of DICER1 mutations to include infantile-onset Cushings disease and 2 novel mutations. Loss of function of both DICER1 alleles appears to be crucial to initiate tumor development.

MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia. We identified an X-linked recessive form of OI caused by defects in MBTPS2, which encodes site-2 metalloprotease (S2P). MBTPS2 missense mutations in two independent kindreds with moderate/severe OI cause substitutions at highly conserved S2P residues. Mutant S2P has normal stability, but impaired functioning in regulated intramembrane proteolysis (RIP) of OASIS, ATF6 and SREBP transcription factors, consistent with decreased proband secretion of type I collagen. Further, hydroxylation of the collagen lysine residue (K87) critical for crosslinking is reduced in proband bone tissue, consistent with decreased lysyl hydroxylase 1 in proband osteoblasts. Reduced collagen crosslinks presumptively undermine bone strength. Also, proband osteoblasts have broadly defective differentiation. These mutations provide evidence that RIP plays a fundamental role in normal bone development.

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.

SETBP1 mutations in two Thai patients with Schinzel–Giedion syndrome

To the Editor : Schinzel–Giedion syndrome (SGS; MIM #269 150) is an autosomal-dominant disorder characterized by profound developmental delay, severe growth failure, characteristic facial features, multiple congenital anomalies including skeletal, cardiac, genitourinary and renal malformations, as well as an increased prevalence of neoplasia (1). The gene responsible for SGS had been unidentified by traditional disease-gene identification approaches. Until very recently, using exome sequencing, Hoischen et al. successfully identified heterozygous de novo mutations in SETBP1 as genetic defects causing SGS (2). Of the 13 patients with SGS, 12 were found to have SETBP1 mutations. All were Caucasian (2). We identified two unrelated Thai patients who were referred to our Genetics Clinic at King Chulalongkorn Memorial Hospital at 1 month of age because of growth failure, developmental delay and multiple anomalies. Both were sporadic cases with no history of consanguinity in their families. Case 1 was a son of a G2 P1 26-yearold mother and a 29-year-old father. The patient’s elder sister was healthy. The pregnancy was complicated by maternal diabetes mellitus, adequately controlled by diet modification. He was born at term with a birth weight of 2650 g (third centile). The Apgar scores were 6 and 7 at 1 and 5 min, respectively. At age 1 month, he weighed 2800 g (<third centile) with head circumference (HC) of 36 cm (50th centile). Anterior fontanelle was 5 × 5 cm. He had coarse face, frontal bossing, prominent eyes, infraorbital grooves, midface retraction, upturned nose, low-set large pinnae with prominent ear lobes, macroglossia, micrognathia, stiff elbows, camptodactyly, micropenis, hypospadias, right indirect inguinal hernia, and spasticity (Fig. 1a,c). Radiographs showed wide occipital synchondrosis (Fig. S1), radioulnar synostosis and scoliosis (Fig. 1e). Thyroid function test showed free T4 (FT4) of 0.68 pg/dl (normal 0.8–1.8), T4 4.26 μg/dl (normal 6–12), thyroidstimulating hormone (TSH) 1.29 μU/ml (normal 0.3–4.1). Levothyroxine was given as the age of 1 month, which brought his FT4 and TSH levels back to normal. Karyotype was 46,XY. Computed tomography (CT) of the brain revealed ventriculomegaly. Renal ultrasound showed hydronephrosis bilaterally. Bronchoscopy revealed a very short epiglottis, vocal cord paralysis, and laryngomalacia. Visual evoke response was normal. Auditory brainstem response showed bilateral sensorineural hearing loss with a threshold of 50 and 70 dB on the left and right sides, respectively. He had recurrent pneumonia requiring tracheostomy intubation at age 2 months. A gastrostomy tube was placed at 4 months of age. He later developed intractable seizure. He had no neurodevelopment and passed away at the age of 6 years and 2 months. Case 2 was a son of a G1 P0 19-year-old mother and 18-year-old father. The pregnancy was uneventful. He was born at term by vaginal delivery with a birth weight of 3440 g (50th centile). The Apgar scores were 9 and 10 at 1 and 5 min, respectively. At the age of 1 month, his HC was 35 cm (25th centile) with anterior fontanelle 4 × 4 cm. He had prominent forehead, infraorbital grooves, midface retraction, prominent upturned nose, short philtrum, micropenis and hypertonia (Fig. 1b,d). Radiographs showed wide occipital synchondrosis (Fig. S2), short first metacarpal bones (Fig. 1f). Thyroid function test showed FT4 of 0.76 pg/dl (normal 0.8–1.8), TSH 1.77 μU/ml (normal 0.3–4.1). Karyotype was 46,XY. CT of the brain showed ventriculomegaly. Echocardiogram was normal. Renal ultrasound showed bilateral hydronephrosis. His head grew abnormally slow, with HC of 35.5 cm (10th centile) at 2 months of age. He developed intractable seizure at the age of 3 months, and his last follow-up was at the age of 11 months. The observation of a very short epiglottis and vocal cord paralysis in Case 1 and a possible hypothyroidism in both cases has never been previously reported in this syndrome. Although levothyroxine brought FT4 and TSH levels of

Disorders with similar clinical phenotypes reveal underlying genetic interaction: SATB2 acts as an activator of the UPF3B gene

Two syndromic cognitive impairment disorders have very similar craniofacial dysmorphisms. One is caused by mutations of SATB2, a transcription regulator and the other by heterozygous mutations leading to premature stop codons in UPF3B, encoding a member of the nonsense-mediated mRNA decay complex. Here we demonstrate that the products of these two causative genes function in the same pathway. We show that the SATB2 nonsense mutation in our patient leads to a truncated protein that localizes to the nucleus, forms a dimer with wild-type SATB2 and interferes with its normal activity. This suggests that the SATB2 nonsense mutation has a dominant negative effect. The patient’s leukocytes had significantly decreased UPF3B mRNA compared to controls. This effect was replicated both in vitro, where siRNA knockdown of SATB2 in HEK293 cells resulted in decreased UPF3B expression, and in vivo, where embryonic tissue of Satb2 knockout mice showed significantly decreased Upf3b expression. Furthermore, chromatin immunoprecipitation demonstrates that SATB2 binds to the UPF3B promoter, and a luciferase reporter assay confirmed that SATB2 expression significantly activates gene transcription using the UPF3B promoter. These findings indicate that SATB2 activates UPF3B expression through binding to its promoter. This study emphasizes the value of recognizing disorders with similar clinical phenotypes to explore underlying mechanisms of genetic interaction.

Whole Genome and Exome Sequencing of Monozygotic Twins with Trisomy 21, Discordant for a Congenital Heart Defect and Epilepsy

Congenital heart defects (CHD) occur in 40% of patients with trisomy 21, while the other 60% have a structurally normal heart. This suggests that the increased dosage of genes on chromosome 21 is a risk factor for abnormal heart development. Interaction of genes on chromosome 21 or their gene products with certain alleles of genes on other chromosomes could contribute to CHD. Here, we identified a pair of monozygotic twins with trisomy 21 but discordant for a ventricular septal defect and epilepsy. Twin-zygosity was confirmed by microsatellite genotyping. We hypothesized that some genetic differences from post-twinning mutations caused the discordant phenotypes. Thus, next generation sequencing (NGS) technologies were applied to sequence both whole genome and exome of their leukocytes. The post-analyses of the sequencing data revealed 21 putative discordant exonic variants between the twins from either genome or exome data. However, of the 15 variants chosen for validation with conventional Sanger sequencing, these candidate variants showed no differences in both twins. The fact that no discordant DNA variants were found suggests that sequence differences of DNA from leukocytes of monozygotic twins might be extremely rare. It also emphasizes the limitation of the current NGS technology in identifying causative genes for discordant phenotypes in monozygotic twins.

ZRS 406A>G mutation in patients with tibial hypoplasia, polydactyly and triphalangeal first fingers

Werner mesomelic syndrome (WMS), an autosomal dominant disorder characterized by hypoplastic tibiae, triphalangeal thumbs and polydactyly, is caused by a specific point mutation at the position 404 in zone of polarizing activity regulatory sequence (ZRS). Here we identified two additional families with WMS. All three patients in three generations of Family 1 were found to harbor the same heterozygous 406A>G mutation in ZRS. The fourth patient from Family 2 was a sporadic case with the known 404 point mutation. The novel 406A>G mutation expands mutational spectrum in ZRS causing WMS, provides evidence for a functionally important nucleotide position 406 of ZRS in humans and has implications for genetic counseling.

Absent expression of the osteoblast-specific maternally imprinted genes, DLX5 and DLX6, causes split hand/split foot malformation type I

Background Split hand/split foot malformation (SHFM) type 1 is characterised by missing central digital rays with clefts of the hands and/or feet, which was linked to chromosome 7q21.3. While double knockout of Dlx5 and Dlx6 resulted in limb defects in mice, the majority of patients with SHFM1 had only heterozygous chromosomal abnormalities. Objective To investigate the clinical and molecular features of a large family with SHFM1. Methods Blood samples of family members were investigated by linkage analysis, array comparative genomic hybridisation, exome sequencing and PCR-Sanger sequencing. Cultures from bone specimens obtained from the proband and an unrelated unaffected individual were established and subjected to quantitative real-time PCR, reverse-transcribed PCR, Western blot and imprinting analysis. Results We report a large pedigree of SHFM1 with 10 members having a heterozygous 103 kb deletion, the smallest one ever reported to be associated with SHFM1. Of these 10, two had no limb anomalies, making a penetrance of 80%. The deletion encompassed exons 15 and 17 of DYNC1I1, which are known enhancers of two downstream genes, DLX5 and DLX6. Surprisingly, DLX5 and DLX6 RNA and proteins in our probands cultured osteoblasts, instead of 50% decrease, were absent. Allelic expression studies in cultured osteoblasts of the unaffected individual showed that DSS1, DLX6 and DLX5 expressed only paternal alleles. These lines of evidence indicate that DSS1, DLX6 and DLX5 were maternally imprinted in osteoblasts. Conclusions SHFM1 in our family is caused by a heterozygous paternal deletion of enhancers of the osteoblast-specific maternally imprinted DLX6 and DLX5 genes, leading to the absence of their proteins.

A common and two novel GBA mutations in Thai patients with Gaucher disease

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the glucocerebrosidase (GBA) gene, leading to a deficiency of lysosomal β-glucosidase and accumulation of glycosphingolipids in macrophages. We studied five Thai families with GD (four with GD type 1 and one with GD type 2). Using long-template PCR, PCR using specific primers for the functional gene, direct sequencing of all coding regions of GBA and restriction enzyme digestions, all 10 mutant alleles were successfully identified. The common c.1448T>C (p.L483P or L444P) mutation was identified in 60% of mutant alleles. Of the two patients homozygous for the p.L483P (L444P) mutation, one died from hepatic failure at age 16 years and the other died from sepsis at age 12 years. This p.L483P (L444P) mutation was found in four different haplotypes, suggesting that it was a recurrent mutation, not caused by a founder effect. Two novel mutations, a missense (c.1204T>C, p.Y402H), and a termination codon mutation (c.1609T>C, p.X537A) were found. Studies to determine the molecular pathomechanism of the p.X537A mutation, the first of its kind in this gene, showed that it decreased the amount of protein being expressed and the enzymatic activity, while it was still correctly localized.