Combination of Multiple Ligation-Dependent Probe Amplification and Illumina MiSeq Amplicon Sequencing for TSC1/TSC2 Gene Analyses in Patients with Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous disorder characterized by tumor growth in multiple organs and caused by mutations in either TSC1 or TSC2 genes. Because of their relatively large genomic sizes, absence of hotspots, and common type of mutations, mutation detection in TSC1 and TSC2 genes has been challenging. We devised a combination of multiple ligation-dependent probe amplification (MLPA) and amplicon sequencing (AS) to simplify the detection strategy, yet we come up with reasonably high detection rate. Thirty-four Malaysian patients diagnosed with TSC were referred to Human Genome Center, Universiti Sains Malaysia. We used a combination of MLPA to detect large copy number changes and AS to detect smaller mutations. TSC1 pathogenic or likely pathogenic mutations were found in 6 patients (18%) and TSC2 in 21 patients (62%), whereas 6 patients (18%) show no mutations and 1 patient (2%) showed only TSC2 missense variant with uncertain significance. Six of the mutations are novel. Our detection strategy costs 81% less and require 1 working week less than the conventional strategy. Confirmatory sequencing using Sanger method on a few representative mutations showed agreement with results of the AS. Combination of MLPA and Illumina MiSeq AS provides a simplified strategy and reasonably high detection rate for TSC1/TSC2 mutation, which suggested application of the strategies into clinical molecular diagnostics.

Transcript, methylation and molecular docking analyses of the effects of HDAC inhibitors, SAHA and Dacinostat, on SMN2 expression in fibroblasts of SMA patients.

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.

Rapamycin and its analogues (rapalogs) for Tuberous Sclerosis Complex-associated tumors: a systematic review on non-randomized studies using meta-analysis.

BACKGROUND: Rapamycin has gained significant attention for its potential activity in reducing the size of TSC-associated tumors, thus providing alternative to surgery. This study aimed at determining the efficacy of rapamycin and rapalogs for reducing the size of TSC-associated solid tumors in patients with Tuberous Sclerosis Complex (TSC). METHODS: Our data sources included electronic searches of the PubMed. We included into our meta-analysis any type of non-randomized study that reported the use of rapamycin and rapalogs for reducing the size of TSC-associated solid tumors in patients with TSC. Data was entered into Cochrane Review Manager Version 5.3 and analyzed. RESULTS: Four case reports and 4 clinical trials were included. Five patients from the case reports (all with SEGA) and 91 patients from the clinical trials (41 with SEGA, 63 with kidney angiomyolipoma and 5 with liver angiomyolipoma) were included into the analysis. Volume and diameter of SEGAs were significantly reduced by mean difference of 1.23 cc (95 % CI -2.32 to -0.13; p = 0.03) and 7.91 mm (95 % CI -11.82 to -4.01; p < 0.0001), respectively. Volume and mean of sum of longest diameter of kidney angiomyolipomas were significantly reduced by mean difference of 39.5 cc (95 % CI -48.85 to -30.15; p <0.00001) and 69.03 mm (95 % CI -158.05 to 12.65; p = 0.008), respectively. In liver angiomyolipomas, however, reduction in tumor size was not evident. Sum of longest diameter of liver angiomyolipomas in 4 patients were enlarged by 2.7 mm (95 % CI 28.42 to -23.02) by the end of treatment, though not significant (p = 0.84). CONCLUSIONS: Rapamycin and rapalogs showed efficacy towards reducing the size of SEGA and kidney angiomyolipoma but not liver angiomyolipomas. This finding is strengthening the conclusion of our Cochrane systematic review on the randomized trials.

Two novel gross deletions of TSC2 in Malaysian patients with tuberous sclerosis complex and TSC2/PKD1 contiguous deletion syndrome.

Tuberous sclerosis complex is an autosomal dominant neurocutaneous disorder affecting multiple organs. Tuberous sclerosis complex is caused by mutation in either one of the two disease-causing genes, TSC1 or TSC2, encoding for hamartin and tuberin, respectively. TSC2/PKD1 contiguous gene deletion syndrome is a very rare condition due to deletion involving both TSC2 and PKD1 genes. Tuberous sclerosis complex cannot be easily diagnosed since there is no pathognomonic feature, although there are consensus diagnostic criteria for that. Mutation analysis is useful and plays important roles. We report here two novel gross deletions of TSC2 gene in Malay patients with tuberous sclerosis complex and TSC2/PKD1 contiguous gene deletion syndrome, respectively.

Histone deacetylase inhibitors as potential treatment for spinal muscular atrophy.

Histone acetylation plays an important role in regulation of transcription in eukaryotic cells by promoting a more relaxed chromatin structure necessary for transcriptional activation. Histone deacetylases (HDACs) remove acetyl groups and suppress gene expression. HDAC inhibitors (HDACIs) are a group of small molecules that promote gene transcription by chromatin remodeling and have been extensively studied as potential drugs for treating of spinal muscular atrophy. Various drugs in this class have been studied with regard to their efficacy in increasing the expression of survival of motor neuron (SMN) protein. In this review, we discuss the current literature on this topic and summarize the findings of the main studies in this field.

Mutation spectrum of dystrophin gene in malaysian patients with Duchenne/Becker muscular dystrophy.

We undertook the clinical feature examination and dystrophin analysis using multiplex ligation-dependent probe amplification (MLPA) and direct DNA sequencing of selected exons in a cohort of 35 Malaysian Duchenne/Becker muscular dystrophy (DMD/BMD) patients. We found 27 patients with deletions of one or more exons, 2 patients with one exon duplication, 2 patients with nucleotide deletion, and 4 patients with nonsense mutations (including 1 patient with two nonsense mutations in the same exon). Although most cases showed compliance to the reading frame rule, we found two unrelated DMD patients with an in-frame deletion of the gene. Two novel mutations have been detected in the Dystrophin gene and our results were compatible with other studies where the majority of the mutations (62.8%) are located in the distal hotspot. However, the frequency of the mutations in our patient varied as compared with those found in other populations.

Allele-specific PCR for a cost-effective & time-efficient diagnostic screening of spinal muscular atrophy.

BACKGROUND & OBJECTIVES: Genetic diagnosis of spinal muscular atrophy (SMA) is complicated by the presence of SMN2 gene as majority of SMA patients show absence or deletion of SMN1 gene. PCR may amplify both the genes non selectively in presence of high amount of DNA. We evaluated whether allele-specific PCR for diagnostic screening of SMA is reliable in the presence of high amount of genomic DNA, which is commonly used when performing diagnostic screening using restriction enzymes. METHODS: A total of 126 blood DNA samples were tested in amounts ranging 80-200 ng, referred for the genetic diagnosis of SMA using both conventional PCR-RFLP and allele-specific PCR. RESULTS: The results from both methods showed agreement. Further, allele-specific PCR was found to be a time-efficient and cost-effective method. INTERPRETATION & CONCLUSIONS: Our study demonstrated the accuracy of our allele-specific PCR and the results were comparable compatible with that of PCR-RFLP, indicating its practical application in SMA diagnostic screening.

Two closely spaced nonsense mutations in the DMD gene in a Malaysian family.

In Duchenne muscular dystrophy (DMD), identification of one nonsense mutation in the DMD gene has been considered an endpoint of genetic diagnosis. Here, we identified two closely spaced nonsense mutations in the DMD gene. In a Malaysian DMD patient two nonsense mutations (p.234S>X and p.249Q>X, respectively) were identified within exon 8. The proband's mother carried both mutations on one allele. Multiple mutations may explain the occasional discrepancies between genotype and phenotype in dystrophinopathy.

Screening of the LIX1 gene in Japanese and Malaysian patients with SMA and/or SMA-like disorder.

BACKGROUND: The majority of spinal muscular atrophy (SMA) patients showed homozygous deletion or other mutations of SMN1. However, the genetic etiology of a significant number of SMA patients has not been clarified. Recently, mutation in the gene underlying cat SMA, limb expression 1 (LIX1), has been reported. Similarity in clinical and pathological features of cat and human SMA may give an insight into possible similarity of the genetic etiology. PATIENTS AND METHODS: In this study, we screened for a mutation in LIX1 using direct DNA sequencing in our SMA and/or SMA-like patients who retained SMN1. A total of 33 patients were enrolled in this study, of which 22 were Japanese and 11 were Malaysians. All these patients possessed at least two copies of SMN1. RESULTS: We did not identify any pathogenic mutations in the coding regions or splice sites of LIX1 in the patients. In addition, we described a polymorphism within LIX1 intron 3, c.387+107A>T. We found that A-allele is significantly more frequent in SMA patients compared to normal individuals. CONCLUSION: Molecular genetic analysis of our SMA and/or SMA-like patients suggests that LIX1 is not associated with the development of their disorders. However, the number of patients analyzed in this study was very limited, and a larger study with bigger sample size is needed to confirm this result.

Biochemical profiling to predict disease severity in metachromatic leukodystrophy.

Metachromatic leukodystrophy is a neurodegenerative disease that is characterized by a deficiency of arylsulfatase A, resulting in the accumulation of sulfatide and other lipids in the lysosomal network of affected cells. Accumulation of sulfatide in the nervous system leads to severe impairment of neurological function with a fatal outcome. Prognosis is often poor unless treatment is carried out before the onset of clinical symptoms. Pre-symptomatic detection of affected individuals may be possible with the introduction of newborn screening programs. The ability to accurately predict clinical phenotype and rate of disease progression in asymptomatic individuals will be essential to assist selection of the most appropriate treatment strategy. Biochemical profiling, incorporating the determination of residual enzyme protein/activity using immune-based assays, and metabolite profiling using electrospray ionization-tandem mass spectrometry, was performed on urine and cultured skin fibroblasts from a cohort of patients representing the clinical spectrum of metachromatic leukodystrophy and on unaffected controls. Residual enzyme protein/activity in fibroblasts was able to differentiate unaffected controls, arylsulfatase A pseudo-deficient individuals, pseudo-deficient compound heterozygotes and affected patients. Metachromatic leukodystrophy phenotypes were distinguished by quantification of sulfatide and other secondarily altered lipids in urine and skin fibroblasts; this enabled further differentiation of the late-infantile form of the disorder from the juvenile and adult forms. Prediction of the rate of disease progression for metachromatic leukodystrophy requires a combination of information on genotype, residual arylsulfatase A protein and activity and the measurement of sulfatide and other lipids in urine and cultured skin fibroblasts.

Combination of SMN2 copy number and NAIP deletion predicts disease severity in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene. The SMN2 gene is highly homologous to SMN1 and has been reported to be correlated with severity of the disease. The clinical presentation of SMA varies from severe to mild, with three clinical subtypes (type I, type II, and type III) that are assigned according to age of onset and severity of the disease. Here, we aim to investigate the potential association between the number of copies of SMN2 and the deletion in the NAIP gene with the clinical severity of SMA in patients of Malaysian origin. Forty-two SMA patients (14 of type I, 20 type II, and 8 type III) carrying deletions of the SMN1 gene were enrolled in this study. SMN2 copy number was determined by fluorescence-based quantitative polymerase chain reaction assay. Twenty-nine percent of type I patients carried one copy of SMN2, while the remaining 71% carried two copies. Among the type II and type III SMA patients, 29% of cases carried two copies of the gene, while 71% carried three or four copies of SMN2. Deletion analysis of NAIP showed that 50% of type I SMA patients had a homozygous deletion of exon 5 of this gene and that only 10% of type II SMA cases carried a homozygous deletion, while all type III patients carried intact copies of the NAIP gene. We conclude that there exists a close relationship between SMN2 copy number and SMA disease severity, suggesting that the determination of SMN2 copy number may be a good predictor of SMA disease type. Furthermore, NAIP gene deletion was found to be associated with SMA severity. In conclusion, combining the analysis of deletion of NAIP with the assessment of SMN2 copy number increases the value of this tool in predicting the severity of SMA.

A qualitative study on malnutrition in children from the perspectives of health workers in Tumpat, Kelantan

Underlying causes of most nutrition related problems are diverse, including biological, social, cultural, and economic factors. Qualitative approaches complement quantitative methods in identifying the underlying meanings and patterns of relationships involved in managing malnutrition. This study examined perceptions regarding malnutrition among health workers from 7 clinics (community and health clinics) in Tumpat, Kelantan. A total of 18 nurses and 2 doctors, who were involved in monitoring child health and nutrition, were included in the study. These health workers were interviewed using a semi-structured questionnaire adapted from Sastry’s framework on malnutrition (Sastry, 1996). The questionnaire included biological, behavioral and environmental factors that influence child health and nutrition. All the health workers perceived that mothers/caregivers play the main role in improving the health of malnourished children. The quality of childcare was rated as moderately satisfactory by the health workers. Most of the affected families who were given the Food Baskets did not fully use all the items for the malnourished child. Child feeding practice was based on the needs of the whole family rather than according to the target child’s needs. Most of the mothers preferred processed cereals than rice porridge because the former is easier to prepare for the child. Although they were from a low socioeconomic background, most of the mothers were not earning additional income for the family. The qualitative methodology provided information that can be used as a basis for the designing of quantitative questionnaires to assess malnutrition among children. The induction characteristic of qualitative methods was used to gain an understanding of the underlying reasons or phenomena such as behaviours that are directly observable.