Mutational spectrum of dystrophinopathies in Singapore: Insights for genetic diagnosis and precision therapy.

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive disorders caused by mutations in the DMD gene. Emerging therapies targeting patients with specific mutations are now becoming a reality for many of these patients. Precise molecular diagnosis is essential to facilitate the identification of possible new treatments for patients in the local context. In this study, we screened 145 dystrophinopathic patients in Singapore and assessed their molecular status for eligibility to current emerging genetic therapies. Overall, 140 (96.5%) of all patients harbored pathogenic DMD mutations comprising 95 exonic deletions (65.5%), 14 exonic duplications (9.7%), and 31 pathogenic small mutations (21.4%). Nonsense and frameshift mutations constitute 83.9% of all the small mutations. We found 71% (103/145) of all Singaporean dystrophinopathy patients to be theoretically amenable for exon skipping, either through skipping of single (53.1%) or multiple exons (17.9%). This approach is applicable to 81.1% (77/95) of patients carrying deletions and 83.9% (26/31) of those with small mutations. Eteplirsen induced skipping of exon 51 is applicable to 12.4% of local patients. Nonsense read-through therapy was found to be applicable in another 12.4% of all patients. Mutation screening is crucial for providing insights into the underlying genetic signature of the disease in the local population and contributes toward existing information on DMD mutations in Asia and globally. This will guide future targeted drug development and clinical trial planning for this disease.

Spinal muscular atrophy: from gene discovery to clinical trials.

Spinal muscular atrophy (SMA) is a common neuromuscular disorder with autosomal recessive inheritance, resulting in the degeneration of motor neurons. The incidence of the disease has been estimated at 1 in 6000-10,000 newborns with a carrier frequency of 1 in 40-60. SMA is caused by mutations of the SMN1 gene, located on chromosome 5q13. The gene product, survival motor neuron (SMN) plays critical roles in a variety of cellular activities. SMN2, a homologue of SMN1, is retained in all SMA patients and generates low levels of SMN, but does not compensate for the mutated SMN1. Genetic analysis demonstrates the presence of homozygous deletion of SMN1 in most patients, and allows screening of heterozygous carriers in affected families. Considering high incidence of carrier frequency in SMA, population-wide newborn and carrier screening has been proposed. Although no effective treatment is currently available, some treatment strategies have already been developed based on the molecular pathophysiology of this disease. Current treatment strategies can be classified into three major groups: SMN2-targeting, SMN1-introduction, and non-SMN targeting. Here, we provide a comprehensive and up-to-date review integrating advances in molecular pathophysiology and diagnostic testing with therapeutic developments for this disease including promising candidates from recent clinical trials.

Interpreting parents' concerns about their children's development with the Parents Evaluation of Developmental Status: culture matters.

OBJECTIVE: This study explored the potential roles and utility of the Parents Evaluation of Developmental Status (PEDS) to screen children for developmental delays in a Southeast Asian clinical sample of preschool children. The PEDS is a 10-item questionnaire instrument used in pediatric settings for reporting parents' concerns for their children's development, learning, and behavior. Clinicians use it to make decisions about clinical pathways for high-, moderate-, and low-risk categories of concerns, but its utility in cross-cultural contexts has not been well documented. METHODS: Participants in this study were 1806 parents, teachers, and child care workers of preschool children in Singapore. Of these, 47.2% were English speaking, 21.2% were Mandarin Chinese speaking, and 31.6% were Malay speaking. PEDS was translated into Chinese and Malay for parents using these languages predominantly. RESULTS: Only parent results were analyzed. The reporting of significant parental concern was considerably higher than US norms and Australian pilot figures when western cutoff scores were applied. When cutoff scores were adjusted, similar patterns of reporting of high, medium, and low risk for disability could be captured. CONCLUSIONS: Parents' interpretation of the concept of "concern" varies across language and culture. Findings highlight the importance of evaluating a screening tool's use in local contexts before its widespread implementation to yield clinically meaningful results.

Detection of hemi/homozygotes through heteroduplex formation in high-resolution melting analysis.

Heteroduplex formation, required for the complete detection of hemi/homozygotes using high-resolution melting analysis, can be induced either by pre-PCR mixing of genomic DNAs or by post-PCR mixing of PCR products from unknown and reference samples. This study investigates the effects of both methods using two single nucleotide polymorphisms in X-linked DMD gene. The results show that both methods resulted in the same effect when mixing samples with the same gene copy number. Mixing samples with different gene copy numbers has not been previously explored and we show that post-PCR mixing is insensitive to gene copy number differences as compared to pre-PCR mixing.

Factors predictive of outcome in childhood stroke in an Asian population.

INTRODUCTION: While paediatric strokes are fairly uncommon, they are often associated with significant long-term disability. Diagnosis is often delayed because of the need to exclude conditions that mimic stroke. Understanding the outcomes related to stroke in children is important in the development of secondary prevention strategies. The aim of this study was to evaluate the epidemiology of childhood stroke in a tertiary paediatric unit in Singapore and to assess factors influencing outcome in these children. MATERIALS AND METHODS: A retrospective case-note review of all childhood strokes presenting to the Children's Medical Institute (CMI) at the National University Hospital (NUH), Singapore between October 1999 and May 2006. Data collected include demographic factors, clinical presentation, diagnosis, subsequent management and follow-up using specific outcome measures. RESULTS: Twenty-six children with a median age of 8.0 years at presentation were identified, comprising 15 ischaemic strokes (57.7%), 10 haemorrhagic strokes (38.5%) and 1 patient with both ischaemic and haemorrhagic lesions. The most common symptoms at presentation were seizures (15/26, 57.7%), lethargy (11/26, 42.3%), hemiparesis (10/26, 38.5%) and altered levels of consciousness (10/26, 38.5%). Vascular abnormalities accounted for 50% of strokes in our study population. The average length of follow-up was 33.2 months (range, 1 to 120) with only 11 children (11/26, 42.3%) achieving full recovery. Significant prognostic factors include altered consciousness and seizures at presentation, lesions in both cortical and subcortical locations, systemic disease aetiology, neurological deficits at discharge and seizures at the time of discharge. CONCLUSION: Long-term neurological, neuropsychological and functional impairment are common in survivors of paediatric strokes. Certain clinical features and lesion characteristics are useful indicators of prognosis in these children.

Should non-expert clinician examiners be used in objective structured assessment of communication skills among final year medical undergraduates?

BACKGROUND: Adoption of the objective structured clinical examination may be hindered by shortages of clinicians within a specialty. Clinicians from other specialties should be considered as alternative, non-expert examiners. AIMS: We assessed the inter-rater agreement between expert and non-expert clinician examiners in an integrated objective structured clinical examination for final year medical undergraduates. METHODS: Pairs of expert and non-expert clinician examiners used a rating checklist to assess students in 8 oral communication stations, representing commonly encountered scenarios from medicine, paediatrics, and surgery. These included breaking bad news, managing an angry relative, taking consent for lumbar puncture; and advising a mother on asthma and febrile fits, and an adult on medication use, lifestyle changes and post-suture care of a wound. 439 students participated in the OSCE (206 in 2005, 233 in 2006). RESULTS: There was good to very good agreement (intraclass coefficient: 0.57-0.79) between expert and non-expert clinician examiners, with 5 out of 8 stations having intraclass coefficients > or =0.70. Variation between paired examiners within stations contributed the lowest variance to student scores. CONCLUSION: These findings support the use of clinicians from other specialties, as 'non-expert' examiners, to assess communication skills, using a standardized checklist, thereby reducing the demand on clinicians' time.

Diagnostic strategy for the detection of dystrophin gene mutations in asian patients and carriers using immortalized cell lines.

Duchenne muscular dystrophy and Becker muscular dystrophy are X-linked recessive diseases of muscle degeneration caused by mutations in the dystrophin gene. More than half of our local Asian patients have point mutations that cannot be detected by conventional multiplex polymerase chain reaction deletion screening. This study aimed to develop mutational screening and carrier detection for Duchenne and Becker muscular dystrophy using protein truncation analysis from Epstein-Barr virus-transformed lymphocyte cell lines. Messenger ribonucleic acid was extracted from fresh lymphocytes and Epstein-Barr virus-transformed lymphocyte cell lines of 14 patients. Reverse transcriptase polymerase chain reaction was performed in 11 overlapping segments, followed by in vitro protein translation and truncation analysis. DNA sequencing was carried out for the corresponding complementary DNA regions, which showed aberrant truncated protein products. Carrier studies using this method were also performed for two families. Half of the patients had frame-shifting deletions, and the remaining seven patients showed point mutations, of which four were novel. These mutations were detected in messenger ribonucleic acid extracted from both fresh lymphocytes and Epstein-Barr virus-transformed lymphocyte cell lines. Carrier status was confirmed in one family and was found to be negative in the other family studied. Protein truncation analysis is an efficient method of screening truncating point mutations from immortalized lymphocyte cell lines from patients. This approach not only serves to prove the pathogenicity of both deletion- and nondeletion-type mutations; it is also effective for carrier detection. The use of such cell lines obviates the need for repeated blood and muscle sampling in patients and offers a perpetual source of messenger ribonucleic acid that can be used long after the patient's demise.

Simple instructions for partial sleep deprivation prior to pediatric EEG reduces the need for sedation.

OBJECTIVE: To study the effects of providing simple instructions for partial sleep deprivation on the necessity for sedation in children and adolescents undergoing electroencephalography (EEG). METHODS: Children and adolescents below 18 years undergoing non-urgent routine EEG were studied for the need for sedation during the EEG test. Two consecutive 3-year periods were reviewed. During the first 3 years no instructions for sleep deprivation were given, and during the second 3-year period, simple instructions were given to the patient or parents of young children to have less sleep prior to the EEG test. This was achieved by using the same sleep deprivation schedule irrespective of the age of the patient. RESULTS: In the first 3-year period between January 1996 and December 1998, 785 non-urgent routine EEG recordings were performed in which only 146 (19%) pediatric patients managed to fall asleep without the need for any sedation within 30 min of being ready for the sleep recording. When partial sleep deprivation was implemented in the 3-year period between January 2000 and December 2002, 449 (55%) out of 821 patients undergoing the test fell asleep in the laboratory without sedation, an overall increase of 36%. Analyzing the different age-specific groups, the maximal increase in the success for natural sleep following partial sleep deprivation was 44% for pediatric patients aged above 10 years. CONCLUSIONS: Simple instructions for partial sleep deprivation prior to the EEG reduced the need for sedation in children and adolescents undergoing the test.

The impact of factor XIIIa V34L polymorphism on plasma factor XIII activity in the Chinese and Asian Indians from Singapore.

Factor XIII (FXIII) is a plasma transglutaminase that is essential for normal haemostasis and fibrinolysis. A few polymorphic sites have been identified in the gene, one of them being a point mutation (V34L) in exon 2 of the FXIIIa subunit gene leading to an amino acid change of valine to leucine. We have examined the role of this polymorphism in relation to plasma FXIII activity in a total of 532 healthy individuals belonging to two ethnic groups in Singapore. The frequency of the L34 allele was significantly higher ( P<0.001) among the Asian Indians (0.08) when compared with the Chinese (0.005). No significant difference in frequency of the L34 allele was observed between Asian Indian CAD patients and controls. The mean FXIII levels were significantly higher ( P<0.0005) among the Asian Indians (148.4%+/-35.5) when compared with Chinese (111.2%+/-26.7). The L34 variant was associated with increased FXIII activity among Asian Indian females. This study shows that both racial and genetic components play a significant role in determining plasma FXIII activity. The effect of V34L polymorphism on FXIII activity in the Indian females is independent of the effects of the P564L and E651Q polymorphic sites in the FXIIIa gene.

ABCA1 gene polymorphisms and their associations with coronary artery disease and plasma lipids in males from three ethnic populations in Singapore.

Mutations in the ATP-binding cassette transporter ABCA1 underlie Tangier disease and familial hypoalphaliproteinemia (FHA), disorders that are characterised by reduced high-density lipoprotein-cholesterol (HDL-C) concentration and cholesterol efflux, and increased coronary artery disease (CAD). We explored if polymorphisms in the ABCA1 gene are associated with CAD and variations in plasma lipid levels, especially HDL-C, and whether the associations may depend on ethnicity. Male cases and controls from the Singapore Chinese, Malay and Indian populations were genotyped for five ABCA1 single nucleotide polymorphisms. Various single-locus frequency distribution differences between cases and controls were detected in different ethnic groups: the promoter -14C>T in Indians, exon 18 M883I in Malays, and 3'-untranslated (UTR) region 8994A>G in Chinese. For the Malay population, certain haplotypes carrying the I825- A (exon 17) and M883- G alleles were more frequent among cases than controls, whereas the converse was true for the alternative configuration of V825- G and I883- A, and this association was reinforced in multi-locus disequilibrium analysis that utilized genotypic data. In the healthy controls, associations were found for -14C>T genotypes with HDL-C in Chinese; 237indelG (5'UTR) with apolipoprotein A1 (apoA1) in Malays and total cholesterol (TC) in Indians; M883I with lipoprotein(a) [Lp(a)] in Malays and apolipoprotein B (apoB) in Chinese; and 8994A>G with Lp(a) in Malays, and TC, low-density lipoprotein-cholesterol (LDL-C) as well as apoB in Indians. While genotype-phenotype associations were not reproduced across populations and loci, V825I and M883I were clearly associated with CAD status in Malays with no effects on HDL-C or apoA1.

Comparative study on deletions of the dystrophin gene in three Asian populations.

The frequency and distribution of deletions of 19 deletion-prone exons clustered in two hot spots in the proximal and central regions of the dystrophin gene were compared in three populations from Singaporean, Japan, and Vietnam. DNA samples obtained from 105 Singaporean, 86 Japanese, and 34 Vietnamese Duchenne muscular dystrophy patients were examined by polymerase chain reaction amplification. Deletions of the examined exons were found in 51.2% of Japanese patients but in 40.0% or less of the Singaporeans and Vietnamese. About two thirds of the deletions were localized in the central region and the remaining deletions were clustered at the proximal region. The most commonly deleted exons at the central deletion hot spot were exon 50 in the Singaporean, exons 49 and 50 in the Japanese, and exon 51 in the Vietnamese population. At the proximal deletion hot spot, the most commonly deleted exons were exons 6 and 8 in the Singaporeans, exons 12 and 17 in the Japanese, and exons 8 and 12 in the Vietnamese. Two cases each from Singapore and Japan had large-scale gross mutations spanning both deletion hot spots. Our results suggest that, although the presence and frequency of the two deletion hot spots may be similar in the three Asian populations analyzed, the distribution and frequency of deletions among the different exons can vary as a result of population-specific intronic sequences that predispose individuals to preferential deletion breakpoints.