Several histone deacetylase inhibitors (HDACis) are known to increase Survival Motor Neuron 2 (SMN2) expression for the therapy of spinal muscular atrophy (SMA). We aimed to compare the effects of suberoylanilide hydroxamic acid (SAHA) and Dacinostat, a novel HDACi, on SMN2 expression and to elucidate their acetylation effects on the methylation of the SMN2. Cell-based assays using type I and type II SMA fibroblasts examined changes in transcript expressions, methylation levels and protein expressions. In silico methods analyzed the intermolecular interactions between each compound and HDAC2/HDAC7. SMN2 mRNA transcript levels and SMN protein levels showed notable increases in both cell types, except for Dacinostat exposure on type II cells. However, combined compound exposures showed less pronounced increase in SMN2 transcript and SMN protein level. Acetylation effects of SAHA and Dacinostat promoted demethylation of the SMN2 promoter. The in silico analyses revealed identical binding sites for both compounds in HDACs, which could explain the limited effects of the combined exposure. With the exception on the effect of Dacinostat in Type II cells, we have shown that SAHA and Dacinostat increased SMN2 transcript and protein levels and promoted demethylation of the SMN2 gene.
Recent progress in genetic analysis reveals that a significant proportion of cryptogenic epileptic encephalopathies are single-gene disorders. Mutations in numerous genes for early-onset epileptic encephalopathies have been rapidly identified, including in SPTAN1, which encodes α-II spectrin. The aim of this review is to delineate SPTAN1 encephalopathy as a distinct clinical syndrome. To date, a total of seven epileptic patients with four different in-frame SPTAN1 mutations have been identified. The major clinical features of SPTAN1 mutations include epileptic encephalopathy with hypsarrhythmia, no visual attention, acquired microcephaly, spastic quadriplegia and severe intellectual disability. Brainstem and cerebellar atrophy and cerebral hypomyelination, as observed by magnetic resonance imaging, are specific hallmarks of this condition. A milder variant is characterized by generalized epilepsy with pontocerebellar atrophy. Only in-frame SPTAN1 mutations in the last two spectrin repeats in the C-terminal region lead to dominant negative effects and these specific phenotypes. The last two spectrin repeats are required for α/β spectrin heterodimer associations and the mutations can alter heterodimer formation between the two spectrins. From these data we suggest that SPTAN1 encephalopathy is a distinct clinical syndrome owing to specific SPTAN1 mutations. It is important that this syndrome is recognized by pediatric neurologists to enable proper diagnostic work-up for patients.
Glycine encephalopathy (GCE) or nonketotic hyperglycinemia is an inborn error of glycine metabolism, inherited in an autosomal recessive manner due to a defect in any one of the four enzymes aminomethyltransferase (AMT), glycine decarboxylase (GLDC), glycine cleavage system protein-H (GCSH) and dehydrolipoamide dehydrogenase in the glycine cleavage system. This defect leads to glycine accumulation in body tissues, including the brain, and causes various neurological symptoms such as encephalopathy, hypotonia, apnea, intractable seizures and possible death. We screened 14 patients from 13 families with clinical and biochemical features suggestive of GCE for mutation in AMT, GLDC and GCSH genes by direct sequencing and genomic rearrangement of GLDC gene using a multiplex ligation-dependant probe amplification. We identified mutations in all 14 patients. Seven patients (50%) have biallelic mutations in GLDC gene, six patients (43%) have biallelic mutations in AMT gene and one patient (7%) has mutation identified in only one allele in GLDC gene. Majority of the mutations in GLDC and AMT were missense mutations and family specific. Interestingly, two mutations p.Arg265His in AMT gene and p.His651Arg in GLDC gene occurred in the Penan sub-population. No mutation was found in GCSH gene. We concluded that mutations in both GLDC and AMT genes are the main cause of GCE in Malaysian population.
An imbalance in folate metabolism can adversely affect DNA synthesis and methylation systems which can lead to susceptibility to non-Hodgkin lymphoma (NHL). Whether single nucleotide polymorphisms (SNPs) and their haplotypes in the methylenetetrahydrofolate reductase (MTHFR) are associated with NHL, remain inconclusive. We investigated the association between MTHFR C677T and A1298C SNPs and NHL risk in a population which is made up of Malay, Chinese and Indian ethnic subgroups. A total of 372 NHL patients and 722 controls were genotyped using the Sequenom MassARRAY platform. Our results of the pooled subjects failed to demonstrate significant association between the MTHFR C677T and A1298C SNPs with NHL and its subtypes. The results were in agreement with the previous meta-analyses. In the Indian ethnic subgroup however, single locus analysis of MTHFR A1298C appears to confer risk to NHL (Odds ratio (OR) 1.91, 95% confidence interval (95% CI) 1.22-3.00, P=0.006). The risk is almost doubled in homozygous carrier of MTHFR 1298CC (OR 4.03, 95% CI 1.56-10.43, P=0.004). Haplotype analysis revealed higher frequency of CC in the Indian NHL patients compared with controls (OR 1.86, 95% CI 1.18-2.93, P=0.007). There is lack of evidence to suggest an association between MTHFR C677T and A1298C with the risk of NHL in the Malays and Chinese. In the Indians however, the MTHFR A1298C confers risk to NHL. This study suggests ethnicity modifies the relationship between polymorphisms in the folate-metabolizing gene and NHL.
Beta-thalassemia is one of the most prevalent inherited diseases and a public health problem in Malaysia. Malaysia is geographically divided into West and East Malaysia. In Sabah, a state in East Malaysia, there are over 1000 estimated cases of β-thalassemia major patients. Accurate population frequency data of the molecular basis of β-thalassemia major are needed for planning its control in the high-risk population of Sabah. Characterization of β-globin gene defects was done in 252 transfusion dependent β-thalassemia patients incorporating few PCR techniques. The study demonstrates that β-thalassemia mutations inherited are ethnically dependent. It is important to note that 86.9% of transfusion-dependent β-thalassemia major patients in Sabah were of the indigenous population and homozygous for a single mutation. The Filipino β(0)-deletion was a unique mutation found in the indigenous population of Sabah. Mutations common in West Malaysia were found in 11 (4.3%) patients. Four rare mutations (Hb Monroe, CD 8/9, CD 123/124/125 and IVS I-2) were also found. This study is informative on the population genetics of β-thalassemia major in Sabah.
Hypercholesterolemia is caused by different interactions of lifestyle and genetic determinants. At the genetic level, it can be attributed to the interactions of multiple polymorphisms, or as in the example of familial hypercholesterolemia (FH), it can be the result of a single mutation. A large number of genetic markers, mostly single nucleotide polymorphisms (SNP) or mutations in three genes, implicated in autosomal dominant hypercholesterolemia (ADH), viz APOB (apolipoprotein B), LDLR (low density lipoprotein receptor) and PCSK9 (proprotein convertase subtilisin/kexin type-9), have been identified and characterized. However, such studies have been insufficiently undertaken specifically in Malaysia and Southeast Asia in general. The main objective of this study was to identify ADH variants, specifically ADH-causing mutations and hypercholesterolemia-associated polymorphisms in multiethnic Malaysian population. We aimed to evaluate published SNPs in ADH causing genes, in this population and to report any unusual trends. We examined a large number of selected SNPs from previous studies of APOB, LDLR, PCSK9 and other genes, in clinically diagnosed ADH patients (n=141) and healthy control subjects (n=111). Selection of SNPs was initiated by searching within genes reported to be associated with ADH from known databases. The important finding was 137 mono-allelic markers (44.1%) and 173 polymorphic markers (55.8%) in both subject groups. By comparing to publicly available data, out of the 137 mono-allelic markers, 23 markers showed significant differences in allele frequency among Malaysians, European Whites, Han Chinese, Yoruba and Gujarati Indians. Our data can serve as reference for others in related fields of study during the planning of their experiments.
Lafora progressive myoclonus epilepsy, also known as Lafora disease (LD), is the most severe and fatal form of progressive myoclonus epilepsy with its typical onset during the late childhood or early adolescence. LD is characterized by recurrent epileptic seizures and progressive decline in intellectual function. LD can be caused by defects in any of the two known genes and the clinical features of these two genetic groups are almost identical. The past one decade has witnessed considerable success in identifying the LD genes, their mutations, the cellular functions of gene products and on molecular basis of LD. Here, we briefly review the current literature on the phenotype variations, on possible presence of genetic modifiers, and candidate modifiers as targets for therapeutic interventions in LD.
Oral clefts are clinically and genetically heterogeneous disorders that are influenced by both genetic and environmental factors. The present family-based association study investigated the role of the MSX1 and TGFB3 genes in the etiology of non-syndromic oral cleft in a Malay population. No transmission distortion was found in the transmission disequilibrium analysis for either MSX1-CA or TGFB3-CA intragenic markers, whereas TGFB3-CA exhibited a trend to excess maternal transmission. In sequencing the MSX1 coding regions in 124 patients with oral cleft, five variants were found, including three known variants (A34G, G110G and P147Q) and two novel variants (M37L and G267A). The P147Q and M37L variants were not observed in 200 control chromosomes, whereas G267A was found in one control sample, indicating a very rare polymorphic variant. Furthermore, the G110G variant displayed a significant association between patients with non-syndromic cleft lip, with or without cleft palate, and normal controls (P=0.001, odds ratio=2.241, 95% confidence interval, 1.357-3.700). Therefore, these genetic variants may contribute, along with other genetic and environmental factors, to this condition.
The enzyme 6-pyruvoyl-tetrahydropterin synthase (PTPS, gene symbol: PTS) is involved in the second step of the de novo biosynthesis of tetrahydrobiopterin (BH4), which is a vital cofactor of nitric oxide synthases and three types of aromatic amino acid hydroxylases; the latter are important enzymes in the production of neurotransmitters. We conducted a study of PTS mutations in East Asia, including Taiwan, Mainland China, Japan, South Korea, the Philippines, Thailand and Malaysia. A total of 43 mutations were identified, comprising 22 previously reported mutations and 21 new discovered mutations. Among these, the c.155A>G, c.259C>T, c. 272A>G, c.286G>A and c.84-291A>G mutations were the most common PTS mutations in East Asia, while the c.58T>C and c.243G>A mutations were, respectively, specific to Filipinos and Japanese originating from Okinawa. Further studies demonstrated that each of the mutations listed above was in linkage disequilibrium to a specific allele of polymorphic microsatellite marker, D11S1347. These results suggest the presence of founder effects that have affected these frequent mutations in East Asia populations. In this context, D11S1347 should become one of the most reliable polymorphic markers for use in prenatal diagnosis among PTPS deficient families, especially where mutations are yet to be identified.
Copy number variations can be identified using newer genotyping arrays with higher single nucleotide polymorphisms (SNPs) density and copy number probes accompanied by newer algorithms. McCarroll et al. (2008) applied these to the HapMap II samples and identified 1316 copy number polymorphisms (CNPs). In our study, we applied the same approach to 859 samples from three Singapore populations and seven HapMap III populations. Approximately 50% of the 1291 autosomal CNPs were found to be polymorphic only in populations of non-African ancestry. Pairwise comparisons among the 10 populations showed substantial differences in the CNPs frequencies. Additionally, 698 CNPs showed significant differences with false discovery rate (FDR)<0.01 among the 10 populations and these loci overlap with known disease-associated or pharmacogenetic-related genes such as CFHR3 and CFHR1 (age related macular degeneration), GSTTI (metabolism of various carcinogenic compounds and cancers) and UGT2B17 (prostate cancer and graft-versus-host disease). The correlations between CNPs and genome-wide association studies-SNPs were investigated and several loci, which were previously unreported, that may potentially be implicated in complex diseases and traits were found; for example, childhood acute lymphoblastic leukaemia, age-related macular degeneration, breast cancer, response to antipsychotic treatment, rheumatoid arthritis and type-1 diabetes. Additionally, we also found 5014 novel copy number loci that have not been reported previously by McCarroll et al. (2008) in the 10 populations.
Extraribosomal functions of human ribosomal proteins (RPs) include the regulation of cellular growth and differentiation, and are inferred from studies that linked congenital disorders and cancer to the deregulated expression of RP genes. We have previously shown the upregulation and downregulation of RP genes in tumors of colorectal and nasopharyngeal carcinomas (NPCs), respectively. Herein, we show that a subset of RP genes for the large ribosomal subunit is differentially expressed among cell lines derived from the human nasopharyngeal epithelium. Three such genes (RPL27, RPL37a and RPL41) were found to be significantly downregulated in all cell lines derived from NPC tissues compared with a nonmalignant nasopharyngeal epithelial cell line. The expression of RPL37a and RPL41 genes in human nasopharyngeal tissues has not been reported previously. Our findings support earlier suspicions on the existence of NPC-associated RP genes, and indicate their importance in human nasopharyngeal organogenesis.
The extensive nucleotide diversity in drug-related genes predisposes individuals to different drug responses and is a major problem in current clinical practice and drug development. Striking allelic frequency differences exist in these genes between populations. In this study, we genotyped 240 sites known to be polymorphic in the Japanese population in each of 270 unrelated healthy individuals comprising 90 each of Malaysian Malays, Indians, and Chinese. These sites are distributed in 109 genes that are drug related, such as genes encoding drug-metabolizing enzymes and drug transporters. Allele frequency and linkage disequilibrium distributions of these sites were determined and compared. They were also compared with similar data of 752 Japanese. Extensive similarities in allele frequency and linkage disequilibrium distributions were observed among Japanese, Malaysian Chinese, and Malays. However, significant differences were observed between Japanese and Malaysian Chinese with Malaysian Indians. These four populations were grouped into two genetic clusters of different ancestries. However, a higher correlation was found between Malaysian Malays and Indians, indicating the existence of extensive admixture between them. The results also imply the possible and rational use of existing single nucleotide polymorphism databases as references to assist future pharmacogenetic studies involving populations of similar ancestry.
The combination of two silent mutations, c.1311C>T in exon 11 and IVS11 T93C (glucose-6-phosphate dehydrogenase (G6PD) 1311T/93C), with unknown mechanism, have been reported in G6PD-deficient individuals in Asian populations including Malaysian aboriginal group, Negrito. Here, we report the screening of G6PD gene in 103 Negrito volunteers using denaturing high-performance liquid chromatography (dHPLC) and direct sequencing. A total of 48 individuals (46.6%) were G6PD deficient, 83.3% of these carried G6PD 1311T/93C with enzyme activity ranging from 1.8 to 4.8 U gHb(-1). Three novel single-nucleotide polymorphisms (SNPs), rs112950723, rs111485003 and rs1050757, were found in the G6PD 3'-untranslated region (UTR). Strong association was observed between haplotype 1311T/93C and rs1050757G, which is located inside the 35 bp AG-rich region. In silico analysis revealed that the transition of A to G at position rs1050757 makes significant changes in the G6PD mRNA secondary structure. Moreover, putative micro (mi)RNA target sites were identified in 3'-UTR of G6PD gene, two of these in the region encompassing rs1050757. It could be speculated that rs1050757 have a potential functional effect on the downregulation of mRNA and consequently G6PD deficiency either by affecting mRNA stability and translation or mirRNA regulation process. This is the first report of biochemical association of an SNP in 3'-UTR of G6PD gene and the possible role of mRNA secondary structure.
We have previously demonstrated that compound heterozygous (SAO/G701D) and homozygous (G701D/G701D) mutations of the anion exchanger 1 (AE1) gene, encoding erythroid and kidney AE1 proteins, cause autosomal recessive distal renal tubular acidosis (AR dRTA) in Thai patients. It is thus of interest to examine the prevalence of these mutations in the Thai population. The SAO and G701D mutations were examined in 844 individuals from north, northeast, central, and south Thailand. Other reported mutations including R602H, DeltaV850, and A858D were also examined in some groups of subjects. The SAO mutation was common in the southern Thai population; its heterozygote frequency was 7/206 and estimated allele frequency 1.70%. However, this mutation was not observed in populations of three other regions of Thailand. In contrast, the G701D mutation was not found in the southern population but was observed in the northern, northeastern, and central populations, with heterozygote frequencies of 1/216, 3/205, and 1/217, and estimated allele frequencies of 0.23%, 0.73%, and 0.23%, respectively. The higher allele frequency of the G701D mutation in the northeastern Thai population corresponds to our previous finding that all Thai patients with AR dRTA attributable to homozygous G701D mutation originate from this population. This suggests that the G701D allele that is observed in this region might arise in northeastern Thailand. The presence of patients with compound heterozygous SAO/G701D in southern Thailand and Malaysia and their apparently absence in northeastern Thailand indicate that the G701D allele may have migrated to the southern peninsular region where SAO is common, resulting in pathogenic allelic interaction.
Southeast Asian ovalocytosis (SAO) is a red blood cell abnormality common in malaria-endemic regions and caused by a 27 nt deletion of the band 3 protein gene. Since band 3 protein, also known as anion exchanger 1, is expressed in renal distal tubules, the incidence of SAO was examined in distal renal tubular acidosis (dRTA) in Malays in Kelantan, Malaysia. Twenty-two patients with dRTA and 50 healthy volunteers were examined for complication of SAO by both morphological and genetic analyses. SAO was identified in 18 of the 22 dRTA patients (81.8%), but only two of the 50 controls (4%). The incidence of SAO was significantly high in those with dRTA (p<0.001), indicating a dysfunctional role for band 3 protein/anion exchanger 1 in the development of dRTA.
G6PD deficiency is the commonest enzyme deficiency found in humans. Current diagnostic methods lack sensitivity to detect all cases of G6PD deficiency. We evaluated the reverse dot blot flow-through hybridisation assay designed to detect simultaneously multiple known G6PD mutations in a group of Malaysian neonates. Archival DNA samples from 141 G6PD-deficient neonates were subjected to reverse dot blot flow-through hybridisation assay using the GenoArray Diagnostic Kit (Hybribio Limited, Hong Kong) and DNA sequencing. The method involved PCR amplification of 5 G6PD exons using biotinylated primers, hybridisation of amplicons to a membrane containing oligoprobes designed for G6PD mutations known to occur in the Malaysian population and colour detection by enzyme immunoassay. The assay detected 13 of the 14 G6PD mutations and genotyped 133 (94.3%) out of 141 (102 males, 39 females) cases. Among the 39 female G6PD-deficient neonates, there were 7 homozygous and 6 compound heterozygous cases. The commonest alleles were G6PD Viangchan 871G > A (21%) and G6PD Mahidol 487G > A(20%) followed by G6PD Mediterranean 563C > T, (14%), G6PD Vanua Lava 383T > C (12%), G6PD Canton 1376G > T (10%), G6PD Orissa 131C > G (6.3%) G6PD Coimbra 592C > T (5.6%) plus 6 other mutations. DNA sequencing of remaining cases revealed 6 cases of intron 11 nt 93C > T not previously reported in Malaysia and two novel mutations, one case each of nt 1361G > T and nt 1030G > A. We found the reverse dot blot assay easy to perform, rapid, accurate and reproducible, potentially becoming an improved diagnostic test for G6PD deficiency.
Coffin-Siris syndrome (CSS, MIM#135900) is a congenital disorder characterized by coarse facial features, intellectual disability, and hypoplasia of the fifth digit and nails. Pathogenic variants for CSS have been found in genes encoding proteins in the BAF (BRG1-associated factor) chromatin-remodeling complex. To date, more than 150 CSS patients with pathogenic variants in nine BAF-related genes have been reported. We previously reported 71 patients of whom 39 had pathogenic variants. Since then, we have recruited an additional 182 CSS-suspected patients. We performed comprehensive genetic analysis on these 182 patients and on the previously unresolved 32 patients, targeting pathogenic single nucleotide variants, short insertions/deletions and copy number variations (CNVs). We confirmed 78 pathogenic variations in 78 patients. Pathogenic variations in ARID1B, SMARCB1, SMARCA4, ARID1A, SOX11, SMARCE1, and PHF6 were identified in 48, 8, 7, 6, 4, 1, and 1 patients, respectively. In addition, we found three CNVs including SMARCA2. Of particular note, we found a partial deletion of SMARCB1 in one CSS patient and we thoroughly investigated the resulting abnormal transcripts.
We report the second case of early infantile epileptic encephalopathy (EIEE) arising from a homozygous truncating variant of NECAP1. The boy developed infantile-onset tonic-clonic and tonic seizures, then spasms in clusters. His electroencephalogram (EEG) showed a burst suppression pattern, leading to the diagnosis of Ohtahara syndrome. Whole-exome sequencing revealed the canonical splice-site variant (c.301 + 1 G > A) in NECAP1. In rodents, Necap1 protein is enriched in neuronal clathrin-coated vesicles and modulates synaptic vesicle recycling. cDNA analysis confirmed abnormal splicing that produced early truncating mRNA. There has been only one previous report of a mutation in NECAP1 in a family with EIEE; this was a nonsense mutation (p.R48*) that was cited as EIEE21. Decreased mRNA levels and the loss of the WXXF motif in both the families suggests that loss of NECAP1 function is a common pathomechanism for EIEE21. This study provided additional support that synaptic vesicle recycling plays a key role in epileptogenesis.
Casein kinase 2 (CK2) is a serine threonine kinase ubiquitously expressed in eukaryotic cells and involved in various cellular processes. In recent studies, de novo variants in CSNK2A1 and CSNK2B, which encode the subunits of CK2, have been identified in individuals with intellectual disability syndrome. In this study, we describe four patients with neurodevelopmental disorders possessing de novo variants in CSNK2A1 or CSNK2B. Using whole-exome sequencing, we detected two de novo variants in CSNK2A1 in two unrelated Japanese patients, a novel variant c.571C>T, p.(Arg191*) and a recurrent variant c.593A>G, p.(Lys198Arg), and two novel de novo variants in CSNK2B in Japanese and Malaysian patients, c.494A>G, p.(His165Arg) and c.533_534insGT, p.(Pro179Tyrfs*49), respectively. All four patients showed mild to profound intellectual disabilities, developmental delays, and various types of seizures. This and previous studies have found a total of 20 CSNK2A1 variants in 28 individuals with syndromic intellectual disability. The hotspot variant c.593A>G, p.(Lys198Arg) was found in eight of 28 patients. Meanwhile, only five CSNK2B variants were identified in five individuals with neurodevelopmental disorders. We reviewed the previous literature to verify the phenotypic spectrum of CSNK2A1- and CSNK2B-related syndromes.