Johari Mohd Ali

Mutations in the TSC1 gene account for a minority of patients with tuberous sclerosis.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by tumour-like malformations (hamartomas) of the brain, skin, and other organs, often associated with seizures and learning disability. There is genetic heterogeneity with loci for TSC on chromosomes 9q34 (TSC1) and 16p13.3 (TSC2). The recently cloned TSC1 gene has 23 exons spanning some 40 kb of genomic DNA with an 8.6 kb transcript. We now report the results of mutation screening by SSCP and heteroduplex analysis of genomic DNA for all 21 coding exons of TSC1 in 83 unrelated cases of tuberous sclerosis. TSC1 gene mutations were found in 16 of the 83 cases (19%). These comprised base substitutions, small insertions, or small deletions giving rise to six nonsense mutations, eight frameshifts, and two splice site mutations, all of which would be expected to result in a truncated or absent protein. In the 10 cases predicted to have TSC1 mutations by linkage analysis or loss of heterozygosity studies, the mutation was identified in eight (80%). In the remaining 73 unassigned cases, only eight mutations were found (11%). From these data we estimate that TSC1 mutations accounted for 24% of the cases in this sample (and an estimated 22% of all TSC cases). This contrasts with data from linkage studies suggesting that TSC1 and TSC2 mutations account for approximately equal numbers of families.

Permanent neonatal diabetes due to a novel insulin signal peptide mutation

We report a rare case of permanent neonatal diabetes (PND) due to insulin (INS) gene mutation in a 51‐month‐old girl who presented with hyperglycemia in the neonatal period. Mutational analysis of KCNJ11 and INS was performed and this detected a novel heterozygous c.38T>G (p.Leu13Arg) INS de novo mutation. The non‐conservative change substitutes the highly conserved L13 residue within the hydrophobic core region of the preproinsulin signal peptide. Given the frequent tendency of heterozygous INS mutations to exhibit dominant negative disease pathogenesis, it is likely that the mutant preproinsulin perturbed the non‐mutant counterpart progression and processing within the β‐cells, and this resulted to a permanent form of congenital diabetes.

Anti-proliferative, in vitro antioxidant, and cellular antioxidant activities of the leaf extracts from Polygonum minus Huds: Effects of solvent polarity

ABSTRACT The present study reports the antioxidant and anti-proliferative activities of Polygonum minus leaf extracts obtained through sequential extraction using four solvents of varying polarities (i.e. hexane (HX), ethyl acetate (EA), methanol, and water). The antioxidant potential was evaluated by measuring the total phenolic content (TPC), total flavonoid content (TFC), ferric reducing antioxidant power (FRAP), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical-scavenging, 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical-scavenging, superoxide anion and nitric oxide scavenging, ferrous ion-chelating (FIC), and cellular antioxidant activity (CAA) assays. The highest antioxidant potential was generally shown by the methanol extract (PM-MeOH). PM-MeOH exhibited the highest values for TPC (174.00 ± 0.18 mg GAE/g), TFC (53.19 ± 0.71 mg GAE/g), FRAP (1728.33 ± 0.96 µmol Fe2+/g), ABTS (226.25 ± 4.25 µmol TE/g), DPPH (1276.81 ± 7.08 µmol TE/g), and nitric oxide scavenging assays (IC50, 675 ± 32.33 µg/mL). In the CAA assay, PM-MeOH dose-dependently inhibited the peroxyl radical-induced oxidation of 2ʹ,7ʹ-dichlorodihydrofluorescein (DCFH2) to 2ʹ,7ʹ-dichlorofluorescein (DCF) in HCT116 cells, with an EC50 value of 263.92 ± 21.60 µg/mL. Liquid chromatography and mass spectrometry analyses of PM-MeOH suggested the presence of tannins and flavonoids including apigetrin, hyperoside, isoquercetin, astragalin, miquelianin, quercetin, and quercitrin. P. minus hexane (PM-HX) and ethyl acetate (PM-EA) extracts showed selective cytotoxicity towards HCT116 with IC50 values of 40.00 ± 0.83 µg/mL and 43.18 ± 0.67 µg/mL, respectively. Taken together, these results highlight the potential of P. minus as a source of bioactive phytochemicals that may be useful in cancer therapeutics and nutraceutical industry.

Permanent neonatal diabetes due to a heterozygous INS mutation

Permanent Neonatal Diabetes Mellitus (PNDM) is a rare disorder where patient presents with diabetes within the first few months of life without autoantibodies associated with type 1 diabetes. The majority of PNDM cases have INS, ABCC8 or KCNJ11 mutations. We present a PNDM case with INS mutation. The proband is a second child of three siblings without family history of diabetes. She was born at term via emergency lower segment caesarean section with good APGAR score. Her birth weight was 2.0kg (<3rd percentile), length 49cm (50th percentile), and head circumference 34cm (50th percentile). She was discharged well at day 3 of life, but readmitted at day 17 of life with hyperglycaemia, sepsis and severe metabolic acidosis, requiring insulin infusion. Despite clinical improvement and resolving sepsis, she remained hyperglycaemic and hence neonatal diabetes was suspected. Her GAD-65 and ICA-512 antibodies were negative. HbA1c and c-peptide at diagnosis were 7.9% and 38 pmol/L (normal range: 297.9–1324) respectively. She was still hyperglycaemic despite receiving total daily insulin (TDI) of 1.5U/kg/day, but not ketotic. She was discharged after 20 days of hospitalization with insulatard administered three times daily (TDI 1.2U/kg/day). During the first 8 months of diagnosis, her metabolic control was good (HbA1c 6.7 - 7.6%) despite low insulin requirement, as low as 0.4U/kg/day. Her metabolic control deteriorated since then (HbA1c 11.9 - 13.2%), with TDI doses ranging 0.7 - 0.9 U/kg/day. Rapid acting insulin was not used due to episodes of hypoglycaemia and unpredictable eating habit and activity levels during her toddler years. Direct DNA sequencing revealed she is heterozygous for p.A24D INS mutation. This mutation has been reported in the literature and known to disrupt preproinsulin processing. Recent evaluation at 51 month showed, negative anti-islet cell antibodies and c-peptide of <30pmol/L (normal range: 297.9–1324). Currently, her developmental milestone is appropriate for her age. However, her height and weight were both below the 3rd centiles. She was recently started on insulin pump to improve her metabolic control and growth. Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Late onset X-linked adrenal hypoplasia congenita with hypogonadotropic hypgonadism due to a novel 4-bp deletion in exon 2 of NR0B1

Abstract We report a novel NR0B1 mutation in a patient affected with X-linked adrenal hypoplasia congenita (X-AHC). The proband first presented with a generalized convulsion at 11 years, 4 months. His clinical and biochemical presentations were consistent with adrenal insufficiency. His basal 17-hydroxyprogesterone (17-OHP) level was not high, and the poor response in 17-OHP on ACTH stimulation test excluded congenital adrenal hyperplasia. At 14 years of age, he did not show any signs of puberty, with low levels of LH, FSH, and testosterone and unresponsiveness to lutenizing hormone releasing hormone stimulation test. Direct DNA sequencing revealed that the proband is hemizygous for a novel NR0B1 mutation (c.1177_1180delGGCC, p.Gly393Cysfs*4). The mother is the conductor of the mutation, which is likely pathogenic as the C-terminus truncated protein lacks the activation function-2 (AF2-TA) transactivation domain, which is highly conserved among members of the nuclear receptor superfamily.

Microarray gene expression profiling in colorectal (HCT116) and hepatocellular (HepG2) carcinoma cell lines treated with Melicope ptelefolia leaf extract reveals transcriptome profiles exhibiting anticancer activity

Background We have previously reported anticancer activities of Melicope ptelefolia (MP) leaf extracts on four different cancer cell lines. However, the underlying mechanisms of actions have yet to be deciphered. In the present study, the anticancer activity of MP hexane extract (MP-HX) on colorectal (HCT116) and hepatocellular carcinoma (HepG2) cell lines was characterized through microarray gene expression profiling. Methods HCT116 and HepG2 cells were treated with MP-HX for 24 hr. Total RNA was extracted from the cells and used for transcriptome profiling using Applied Biosystem GeneChip™ Human Gene 2.0 ST Array. Gene expression data was analysed using an Applied Biosystems Expression Console and Transcriptome Analysis Console software. Pathway enrichment analyses was performed using Ingenuity Pathway Analysis (IPA) software. The microarray data was validated by profiling the expression of 17 genes through quantitative reverse transcription PCR (RT-qPCR). Results MP-HX induced differential expression of 1,290 and 1,325 genes in HCT116 and HepG2 cells, respectively (microarray data fold change, MA_FC ≥ ±2.0). The direction of gene expression change for the 17 genes assayed through RT-qPCR agree with the microarray data. In both cell lines, MP-HX modulated the expression of many genes in directions that support antiproliferative activity. IPA software analyses revealed MP-HX modulated canonical pathways, networks and biological processes that are associated with cell cycle, DNA replication, cellular growth and cell proliferation. In both cell lines, upregulation of genes which promote apoptosis, cell cycle arrest and growth inhibition were observed, while genes that are typically overexpressed in diverse human cancers or those that promoted cell cycle progression, DNA replication and cellular proliferation were downregulated. Some of the genes upregulated by MP-HX include pro-apoptotic genes (DDIT3, BBC3, JUN), cell cycle arresting (CDKN1A, CDKN2B), growth arrest/repair (TP53, GADD45A) and metastasis suppression (NDRG1). MP-HX downregulated the expression of genes that could promote anti-apoptotic effect, cell cycle progression, tumor development and progression, which include BIRC5, CCNA2, CCNB1, CCNB2, CCNE2, CDK1/2/6, GINS2, HELLS, MCM2/10 PLK1, RRM2 and SKP2. It is interesting to note that all six top-ranked genes proposed to be cancer-associated (PLK1, MCM2, MCM3, MCM7, MCM10 and SKP2) were downregulated by MP-HX in both cell lines. Discussion The present study showed that the anticancer activities of MP-HX are exerted through its actions on genes regulating apoptosis, cell proliferation, DNA replication and cell cycle progression. These findings further project the potential use of MP as a nutraceutical agent for cancer therapeutics.