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We investigated the effectiveness of DNA-based next-generation sequencing (NGS) targeting different genomic region and coverage from five platforms in resolving the diagnostic odyssey of a patient with an unidentified neuromuscular disorder. There were advantages and limitations associated with the different platform approaches. On average, over 22,000 rare protein effecting single nucleotide variants (SNVs), 1955 structural variants (SVs), and 229 copy number variants (CNVs) were identified and analyzed. Seven candidate SNVs fulfilled filtration criteria but were likely to be non-causative due to their classification or disease phenotype. Assessment of an intronic event through IGV and PCR suggested a region of structural rearrangement in DMD gene, which mismatched to two other genes on chromosome X hinting towards a possible novel mechanism of gene inactivation. Our results show that NGS platforms detect candidate variants but some disease mechanisms may remain undetected and points for need for caution when applying NGS for diagnostic purposes.

Background Hirschsprung disease (HSCR) is a heterogeneous genetic disorder characterized by absence of ganglion cells along the intestines resulting in functional bowel obstruction. Mutations in neuregulin 1 (NRG1) gene have been implicated in some cases of intestinal aganglionosis. This study aims to investigate the contribution of the NRG1 gene to HSCR development in an Indonesian population. Methods We analyzed the entire coding region of the NRG1 gene in 54 histopathologically diagnosed HSCR patients. Results All patients were sporadic non-syndromic HSCR with 53/54 (98%) short-segment and 1/54 (2%) long-segment patients. NRG1 gene analysis identified one rare variant, c.397G > C (p.V133 L), and three common variants, rs7834206, rs3735774, and rs75155858. The p.V133 L variant was predicted to reside within a region of high mammalian conservation, overlapping with the promoter and enhancer histone marks of relevant tissues such as digestive and smooth muscle tissues and potentially altering the AP-4_2, BDP1_disc3, Egr-1_known1, Egr-1_known4, HEN1_2 transcription factor binding motifs. This p.V133 L variant was absent in 92 non-HSCR controls. Furthermore, the rs7834206 polymorphism was associated with HSCR by case–control analysis (p = 0.037). Conclusions This study is the first report of a NRG1 rare variant associated with HSCR patients of South-East Asian ancestry and provides further insights into the contribution of NRG1 in the molecular genetic pathogenesis of HSCR. Electronic supplementary material The online version of this article (10.1186/s12887-018-1265-x) contains supplementary material, which is available to authorized users.

Autism spectrum disorders (ASD) is a complex disorder in which both genes and environmental factors play important roles. ASD is characterized by a high concordance rate in monozygotic twins pointing to high heritability for this disorder. Notably, the sibling recurrence risk ratio (λs) is 22 for autism. Despite the high heritability clinical symptoms are markedly variable and multiple genetic factors including Mendelian mutations, copy number variations and common polymorphisms all contribute to the genetic load towards passing the clinical threshold to ASD. Indeed, multiple genetic loci have been with various degrees of certainty associated with this disorder. In addition to genetic factors environmental variables and epigenetic signatures are also important modifiers of susceptibility to ASD. For example, studies show that DNA methylation differences can occur in many loci across the genome of ASD subjects. Among biomarkers that have been examined in ASD are metabolic, oxidative stress, mitochondrial dysfunction, methylation, immune dysregulation, amino acids and neuropeptides, peripheral blood gene expression, digit ratio and dysbiosis (gut inflammation) among others. In my talk today after giving an overview (see above) of biomarkers in ASD, I will discuss my own group’s approach that includes both gene markers (serotonin transporter, OXTR and AVPR1a) as well as an innovative endophenotype biomarker approach to better understanding ASD. In a recent study we found that 2D:4D digit ratio (also a proposed marker for ASD) relationship to cognitive empathy (a core deficit in ASD) is dependent in normal subjects on an OXTR SNP linked to ASD in some studies. We have also shown that OXTR and AVPR1a gene polymorphisms are associated in normal subjects with cognitive and emotional empathy respectively. Notably, common polymorphisms in both genes are also associated with ASD. Similarly, the DRD4 gene shows a gender-sensitive association with cognitive empathy. In a parallel talk at this meeting I discuss in some detail how gene expression studies in lymphoblastoid cells may be a marker for ASD as well as personality traits. I will also discuss a finding with a colleague identifying a functional rare variant in autism using genome-wide screen for monoallelic expression. Altogether, our studies have revealed genetic and other biomarkers that jointly contribute to the social cognitive deficits that represent core diagnostic features of ASD. The biomarkers are not specific to ASD but nevertheless shed considerable light on the underlying common polymorphisms and multiple etiologies of this highly heritable disorder characterized by intellectual and social dysfunctions. One promising area of future studies of biomarkers in ASD is prenatal diagnosis using ultrasound.

Genetic testing forms an integral part of clinical management of heritable genetic disorders as it provides options for molecular diagnosis, carrier identification, prenatal diagnosis, etc. The impetus for the development of diagnostic assays for genetic testing of a disease is often influenced by how much is known about its molecular basis, its incidence among the local population and the clinical demand for testing. Subsequent laboratory implementation of a research test for clinical diagnostic testing requires demonstration of clinical utility and validity. Next-generation sequencing (NGS) approaches are increasingly being adopted for clinical diagnostics as the costs becomes increasingly affordable and its utility to resolve diagnostic odysseys has brought resolutions to many families. Validation of such NGS assays is complex but follows similar established guidelines as for traditional genetic tests. There is high analytical sensitivity for most of these assays although the true clinical sensitivity may remain unknown for some disorders. Targeted analysis using NGS is now available for many gene panels, e.g., involving myopathies, cilipathies, cardiomyopathies, etc. as well as for single gene disorders such as Wilson disease, retinoblastoma, RYR1-related diseases, etc. As the costs for sequencing whole exomes and genomes progressively drop further, it is anticipated that these technologies will also begin to transit into the clinical realm. This talk discusses the impact and challenges of NGS in clinical testing and the diagnostic dilemmas in test interpretations for these lab tests.

We have developed a fast and comprehensive method to scan for point mutations in a gene on X chromosome. A target region of the gene is first amplified. Then, using the amplified product as a template, PCR is carried out with multiple short-length forward primers arrayed in tandem in the scanned region, and a common reverse primer. The absence of amplified product defines the site of a mutation within a narrow region of the primer recognition site. To evaluate our method, point mutations in exon 12 of the human glucose-6-phosphate dehydrogenase (G6PD) gene were used as a model system. Out of 12 Singaporean G6PD-deficient patients, 6 cases were shown by the method to have a nucleotide change in this exon. Sequence analysis confirmed the presence of a nucleotide change in the region identified by our scanning. Thus, our method is accurate in localizing mutations within a narrow region, and allows large numbers of samples to be handled simultaneously.

The allele frequency of a L1 retroposon insertion into intron 30 of the neurofibromatosis type 1 (NF1) gene was determined by analyzing amplified fragment lengths in seven Asian or Pacific population; namely, Japanese, Chinese. Indian, Malay, Filipino, Indonesian and New Guinean. Nearly 100 chromosomes from each group were analyzed. The presence of the L1 insertion was identified by the appearance of an abnormally large PCR-amplified product. The insertion frequency varied from 0.45 to 0.75, depending on the population group. Malay and Indonesian populations were found to have the highest insertion frequencies (0.75 and 0.72, respectively), while the wild-type genotype was more prevalent in Indians. The lowest insertion frequency (0.45), observed in Indians, was nearest to that reported in Westerners (0.35). The different L1 insertion frequencies found in Asian and Pacific groups reflect a major divergence in these human populations. Japanese and Chinese populations showed the highest heterozygosity (0.50), suggesting the usefulness of this polymorphism in linkage analysis in these populations.

Identification of mutations in G6PD gene is performed as an epidemiologic investigation of G6PD deficiency in many countries. In order to understand the hereditary background of G6PD deficiency in a population, screening of mutations is required not only in exonic regions but also for intron and promoter regions. One hundred male neonatal samples diagnosed as with G6PD deficiency by newborn screening in Singapore were used in this study. The multiplex PCR using the multiple tandem forward primers and common reverse primer (MPTP) method was carried out to detect the common 11 mutations in south-east Asia such as Gaohe 95AG, Orissa 131CG, Vanua-Lava 383TC, Mahidol 487GA, Mediterranean 563CT, Coimbra 592CT, Viangchan 871GA, Chatham 1003GA, Union 1360CT, Canton 1376GT and Kaiping 1388GA. Samples whose mutations were unidentified by MPTP method were scanned at cording region, intron and promoter region by direct sequencing.Out of 100 samples, 90 samples (90.0%) were identified with one of the above mentioned common mutations. Eight out of 10 samples whose mutations were unidentified by MPTP method carried exonic mutations which had been previously reported such as Murcia 209AG, Quing Yuan 392GT, Nankang 517TC, Chinese5 1024CT. Two novel mutations were identified in these samples: one had a novel mutation (25CT); the remaining sample carried a 49 bp deletion in intron 12.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is caused by loss of the survival motor neuron gene, SMN1. SMA treatment strategies have focused on production of the SMN protein from the almost identical gene, SMN2. Valproic acid (VPA) is a histone deacetylase inhibitor that can increase SMN levels in some SMA cells or SMA patients through activation of SMN2 transcription or splicing correction of SMN2 exon 7. It remains to be clarified what concentration of VPA is required and by what mechanisms the SMN production from SMN2 is elicited. We observed that in two fibroblast cell lines from Japanese SMA patients, more than 1mM of VPA increased SMN2 expression at both the transcript and protein levels. VPA increased not only full-length (FL) transcript level but also exon 7-excluding (Δ7) transcript level in the cell lines and did not change the ratio of FL/Δ7, suggesting that SMN2 transcription was mainly activated. We also found that VPA modulated splicing factor expression: VPA increased the expression of splicing factor 2/alternative splicing factor (SF2/ASF) and decreased the expression of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). In conclusion, more than 1mM of VPA activated SMN2 transcription and modulated the expression of splicing factors in our SMA fibroblast cell lines.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is common in malaria endemic regions and is estimated to affect more than 400 million people worldwide. Deficient subjects are mostly asymptomatic but clinical manifestations range from neonatal jaundice due to acute hemolytic anemia to chronic non-spherocytic hemolytic anemia. To date, biochemical parameters allowed more than 400 different G6PD variants to be distinguished thereby suggesting a vast genetic heterogeneity. So far, only a small portion of this heterogeneity has been confirmed at the DNA level with the identification of about 90 different point mutations in the G6PD coding sequence. To determine the molecular background of G6PD deficiency in Southeast Asian countries, we conducted molecular analyses of G6PD patients from the Philippines, Malaysia, Singapore, Vietnam and Indonesia. The most prevalent mutation identified differs from country to country, thus suggesting independent mutational events of the G6PD gene.

We report a Chinese patient with amyloidotic polyneuropathy associated with a novel transthyretin mutation (V32A). He presented with slowly progressive sensorimotor polyneuropathy accompanied by autonomic dysfunction and cardiomyopathy by echocardiography. This mutation is likely to be associated with late onset and low-penetrance phenotype.

Part 1 Emerging technologies: FISH for the obstetrician and gynaecologist - a rapid and reliable tool aiding clinical analysis, L.A. Gole et al silico biotech, P. Kanguene and M.K. Sakharkar bioinformatics integration simplified - the Kleisli way, L.S. Wong issues in secondary structure prediction quality, B. Cheng. Part 2 Genes and diseases: G6PD deficiency and application of the MPTP technique, P.S. Lai et al analysis of deletion breakpoints in dystrophin transcripts, H.H. Khng et al maple syrup urine disease - a report of 26 cases in the Philippines, C.D. Padilla et a the heterogeneity of thalassaemia in Southeast Asia, S. Fucharoen and P. Winichagoon genetics and susceptibility to tuberculosis - a review, E.M.C. Cutiongco identification of RB1 gene mutations with constitutional origin, R. Rong et al. Part 3 Gene therapy: molecular medicine - potential therapies for genetic diseases, S.D. Wilton development of HVJ-liposomes and cancer gene therapy, Y. Kaneda naked plasmids -muscling into gene transfer, O.L. Kon et al treatment of Duchenne muscular dystrophy at the mRNA level, M. Matsuo and Y. Takeshima strategy of gene therapy for liver cirrhosis and liver cancer, J. Fujimoto. (Part contents).