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Background: Mucopolysaccharidoses type II (MPS II) is an X-linked lysosomal storage disease (LSD). It is due to mutation in IDS gene encoding iduronate-2-sulphatase (IDS) involved in the catabolism of dermatan sulphate and heparan sulphate. Currently, the treatments for MPS II patients are enzyme replacement therapy (ERT) and bone marrow transplantation (BMT). However, ERT is not effectively reducing the central nervous system manifestation and finding the suitable donor maybe quite challenging in BMT. Over the past decades, pharmacological chaperone has been an alternative approach for management of MPS II patient. Here, we described the in vitro profiling of small molecules in group of chondroitin/dermatan (CD) sulphate disaccharide, heparin oligosaccharides, unsaturated heparin disaccharides and 6-O-desulphated heparin oligosaccharide, using recombinant human iduronate-2-sulphatase (rhIDS). Twenty-one small molecule compounds with several concentrations were each screened by inhibition and thermal stability assays. Results: Our study revealed that condroitin dermatan trisulphate (CD3S), heparin tetrasaccharide (H4Sac), heparin octasaccharide (H8Sac) and heparin octadecasaccharide (H18Sac) showed high inhibition constant, Ki and low inhibition concentration, IC50 in comparison to others. In the thermal stability study, only rhIDS incubated with CD3S was found to preserve enzyme activity (20%) after incubated at 67oC. Conclusion: Overall, our experiments discovered that CD3S was able to bind, inhibit and chaperone rhIDS. These features suggest a potential pharmacological chaperone for MPS II.

Kajian ini dijalankan untuk menilai kemampuan kaedah penentuan jumlah homosisteina dalam plasma dengan menggunakan kromatografi cecair berprestasi tinggi jenis penukar ion (HPLC-IEC) dan membandingkan kaedah ini dengan kit komersil yang menggunakan kromatografi cecair berprestasi tinggi jenis fasa berbalik (HPLC-RPC). Validasi kaedah bagi penentuan jumlah homosisteina menggunakan HPLC-IEC telah dilakukan mengikut panduan yang dikeluarkan oleh Jabatan Standard Malaysia. Sebanyak 57 sampel daripada pesakit yang dihantar untuk penentuan jumlah homosisteina telah diuji secara serentak untuk menentukan korelasi antara kedua kaedah tersebut. Hasil kajian ini mendapati bahawa validasi kaedah ini telah memenuhi keperluan ISO MS 15189 dan kelinearan sehingga 500 µM dengan pekali penentuan, r2 ialah 0.999. Kepersisan yang dikaji pada aras normal (10 µM) dan aras berpenyakit/abnormal (60 µM) bagi kebolehulangan adalah masing-masing 3.2% dan 3.8% manakala bagi kebolehasilan semula masing-masing adalah 8.0% dan 8.3%. Had pengesanan dan had pengiraan yang diperoleh adalah 0.9274 dan 3.0912 µM. Keputusan ketepatan bagi kembalian semula adalah 92% dan bias adalah -8.6%. Perbandingan kaedah yang telah divalidasi dengan kit komersil menunjukkan hubungan korelasi yang kuat antara kedua-dua kaedah ini (y = 0.721x + 4.034; r = 0.978; p

Pyruvate dehydrogenase (PDH) deficiency is caused by a number of pathogenic variants and the most common are found in the PDHA1 gene. The PDHA1 gene encodes one of the subunits of the PDH enzyme found in a carbohydrate metabolism pathway involved in energy production. Pathogenic variants of PDHA1 gene usually impact the α-subunit of PDH causing energy reduction. It potentially leads to increased mortality in sufferers. Potential treatments for this disease include dichloroacetate and phenylbutyrate, previously used for other diseases such as cancer and maple syrup urine disease. However, not much is known about their efficacy in treating PDH deficiency. Effective treatment for PDH deficiency is crucial as carbohydrate is needed in a healthy diet and rice is the staple food for a large portion of the Asian population. This review analysed the efficacy of dichloroacetate and phenylbutyrate as potential treatments for PDH deficiency caused by PDHA1 pathogenic variants. Based on the findings of this review, dichloroacetate will have an effect on most PDHA1 pathogenic variant and can act as a temporary treatment to reduce the lactic acidosis, a common symptom of PDH deficiency. Phenylbutyrate can only be used on patients with certain pathogenic variants (p.P221L, p.R234G, p.G249R, p.R349C, p.R349H) on the PDH protein. It is hoped that the review would provide an insight into these treatments and improve the quality of lives for patients with PDH deficiency.

Induced pluripotent stem cells (iPSCs) have been generated using different reprogramming strategies. Lentiviruses remain a strategic method for cell reprogramming as it is highly efficient in gene transfer. The latest fourth-generation lentiviral packaging systems claimed to be efficient and safe. However, modifications made to enhance safety of lentiviral vectors have been shown to affect vector performance. In this study, we established that the fourth-generation lentiviral packaging system can produce high-titre lentiviruses with high-transduction efficiencies. Subsequently, the robustness and reproducibility of generating iPSCs from adult human dermal fibroblasts were tested using these lentiviruses. The use of fourth-generation lentiviruses consistently generates iPSCs with similar efficiency and quality in different primary cell lines. This study demonstrated that the human-derived iPSCs can be maintained using mitomycin-C inactivated feeder cells. The iPSC clones highly expressed key pluripotency markers and can spontaneously differentiate into cells from the three embryonic germ layers. The iPSCs generated were able to differentiate into neural stem cell lineages, producing cells expressing Nestin and Sox2 as well as able to further differentiate into neurons with more than 70% efficiency. The data demonstrated that the use of the fourth-generation lentiviral packaging to produce lentiviruses for iPSCs generation is robust and reproducible as it can generate iPSCs from different adult dermal fibroblasts with the potential to differentiate into neural stem cells and neurons. The use of safer lentiviral packaging systems combined with established vector plasmids will help to expedite the generation of iPSCs for clinical applications.

Background: Lysosomal storage disorders (LSD) are storage disorders involving malfunction of degradation enzymes in lysosome. More than 50 types of LSD have been discovered, which includes the group of mucopolysaccharidoses (MPS), sphingolipidoses, oligosaccharidoses, mucolipidoses, lipoprotein storage disorders, lysosomal transport defects and neuronal ceroid lipofuscinoses and others. The aims of this study were to calculate the birth prevalence and carrier frequency of LSDs in the Malaysian population; to compare our results with reported epidemiologic data from other populations, and to describe the mutation spectrum in Malaysia. From 2008 to 2017, 2.1% (92/4338) suspected patients were diagnosed with LSD. Results: The prevalence of LSD in Malaysia was 1/231,904 live births. The combined prevalence of MPS was 1/292,401 with its subtype of MPS II presented the highest calculated birth prevalence of 1/221,425. Within the group of sphingolipidoses, the combine prevalence was 1/770,777 with Fabry as the most common disorder with calculated prevalence of 1/193,203 followed by metachromatic leukodystrophy (MLD) (1/494,514). MLD is more common among people of Iban ethnicity with the prevalence of 1/6,981. Pompe and mucolipidoses type II are the less common subtypes of LSD with a prevalence of 1/1,694,634 and 1/2,229,516, respectively.Conclusion: Overall, although the prevalence of LSD in Malaysia may be underestimated, the prevalence of MPS is consistent with other reported in East Asian countries.

IntroductionMucopolysaccharidoses Type II is caused by an X-linked mutation in IDS gene which produce an enzyme called iduronate 2-sulfatase (IDS) that is essential for the breakdown of glycosaminoglycans. Lack of IDS enzyme activity leads to the accumulation of heparan sulfate and dermatan sulfate within the lysosomes. Apart from enzyme replacement therapy, current treatments of MPS II have been a challenge especially in finding donor for bone marrow transplantation. Thus, this study aims to evaluate the various heparin oligosaccharides (HO) on IDS activities as potential pharmacological chaperone (PC). Materials & MethodsHO (H004, H008, H010, H012, H014, H016, H018 and H020) in several concentrations were used in this study. Enzyme inhibition assay is as illustrated in Figure 1. All HO candidates were also tested on ATCC cell for specificity testing. ResultsThe results of this study is summarized in Table 1. Three selected inhibition profiling of potential small molecules are depicted in Figure 2. No significant difference (p>0.05) was observed in the inhibition of IDS activity in ATCC cells which proves its specificity on IDS. Discussion and ConclusionPCs are small molecules that have an affinity to mutant enzymes and directly bind to them which improve their folding, stability, and enzymatic activity. Ideal PC of potent inhibitor exhibits low concentration of 50% inhibition of enzyme activity (IC50) with high inhibition constant (Ki) of the dissociation between the enzyme-inhibitor complex or the reciprocal of the binding affinity of the inhibitor to the enzyme. Out of eight species of HO tested, three demonstrate as PC with Ki and IC50 (µM) of 59.27 & 19.40; 34.40 & 95.51; and 11.55 & 7.19 for H004, H008 and H018, respectively.As conclusion, three potential small molecules (H004, H008 and H018) with high Ki with low IC50 have been identified as potential pharmacological chaperone. Future research will be done on thermal stability analysis and cell-based experiments.

IntroductionMucopolysaccharidoses Type II (MPS II) is a rare inherited disease caused by mutation in IDS gene encoding iduronate-2-sulphatase (IDS), an important enzyme in the glycosaminoglycans degradation pathway of heparan sulphate and dermatan sulphate. Current treatments such as enzyme replacement therapy is not effective in reducing the central nervous system manifestation while finding the suitable donor quite challenging in bone marrow transplantation. The use of small molecules in assisting misfolding protein has been explored as an alternative approach for potential therapy in MPS II treatment. In this study, we performed the inhibition assay of desulphated heparin oligosaccharides (DSH) small molecules using recombinant human iduronate-2-sulphatase (rhIDS) to identify potential candidate as pharmacological chaperone for MPS II. Material and MethodsThe small molecules of DSH (DSH004, DSH006, DSH008, DSH010 & DSH012) were used in this study. All of the DSH candidates were tested in the inhibition assay as described in product insert of recombinant human iduronate 2-sulphatase (rhIDS) (2) (Figure 1). All DSH candidates were also tested on ATCC cell for specificity testing. ResultsThe results of inhibition assay for DSH candidates were shown in table 1. ▪ Figure 2 illustrated the inhibition profiling of each small molecules of DSH. There were no significant different in the inhibition of IDS activity in ATCC cells (p>0.05). Discussion and ConclusionInhibition concentration (IC50) referring to concentration required to produce 50% inhibition of enzyme activity. A lower IC50 generally means a more potent inhibitor (3) while inhibition constant (Ki ) indicates the binding affinity of the inhibitors. It was observed that DSH004 shows the highest value of IC50 and Ki compared to other DSH candidates. Although DSH004 demonstrate the highest Ki value, high IC50 value may cause toxicity to the cells. Overall, among all DSH candidates, DSH006 shows a promising outcome in inhibiting the IDS activity and therefore may have the potential to be a suitable candidate as the pharmacological chaperone for MPS II.

Background Serum protein electrophoresis (SPE) is a widely used laboratory technique to diagnose patients with multiple myeloma (MM) and other disorders related to serum protein. In patients with MM, abnormal monoclonal protein can be detected by SPE and further characterized using immunofixation electrophoresis (IFE). There are several semi‐automated agarose gel‐based systems available commercially for SPE and IFE. In this study, we sought to evaluate the analytical performance of fully automated EasyFix G26 (EFG26) and semi‐automated HYDRASYS 2 SCAN (H2SCAN) for both SPE and IFE. Methods Both instruments were operated according to manufacturer's instructions. Samples used include a commercially available normal control serum (NCS) and patients' specimens. The following were evaluated: precision and comparison studies for SPE, and reproducibility and comparison studies for IFE. Statistical analyses were performed using Microsoft Excel. Results For SPE repeatability study, our results showed that EFG26 has higher coefficient of variation (%CV) compared with H2SCAN for both samples except for monoclonal component with %CV of 0.97% and 1.18%, respectively. Similar results were obtained for SPE reproducibility study except for alpha‐1 (4.16%) and beta (3.13%) fractions for NCS, and beta fractions (5.36%) for monoclonal sample. Subsequently, reproducibility for IFE was 100% for both instruments. Values for correlation coefficients between both instruments ranged from 0.91 to 0.98 for the five classic bands. Conclusion Both instruments demonstrated good analytical performance characterized by high precision, reproducibility and correlation.