Whole-Exome sequencing identifies GYS2 biallelic variants in individuals with suspected epilepsy.

BACKGROUND: Adequate glucose supply is essential for brain function, therefore hypoglycemic states may lead to seizures. Since blood glucose supply for brain is buffered by liver glycogen, an impairment of liver glycogen synthesis by mutations in the liver glycogen synthase gene (GYS2) might result in a substantial neurological involvement. Here, we describe the phenotypes of affected siblings of two families harboring biallelic mutations in GYS2. METHODS: Two suspected families - a multiplex Pakistani family (family A) with three affected siblings and a family of Moroccan origin (family B) with a single affected child who presented with seizures and reduced fasting blood glucose levels were genetically characterized. Whole exome sequencing (WES) was performed on the index patients, followed by Sanger sequencing-based segregation analyses on all available members of both families. RESULTS: The variant prioritization of WES and later Sanger sequencing confirmed three mutations in the GYS2 gene (12p12.1) consistent with an autosomal recessive pattern of inheritance. A homozygous splice acceptor site variant (NM_021957.3, c. 1646 -2A>G) segregated in family A. Two novel compound heterozygous variants (NM_021957.3: c.343G>A; p.Val115Met and NM_021957.3: c.875A>T; p.Glu292Val) were detected in family B, suggesting glycogen storage disorder. A special diet designed to avoid hypoglycemia, in addition to change of the anti-seizure medication led to reduction in seizure frequency. CONCLUSIONS: This study suggests that the seizures in patients initially diagnosed with epilepsy might be directly caused, or influenced by hypoglycemia due to pathogenic variants in the GYS2 gene.

Plasmodium sporozoite excystation involves local breakdown of the oocyst capsule.

Plasmodium oocysts develop on the abluminal side of the mosquito midgut in relatively small numbers. Oocysts possess an extracellular cell wall-the capsule-to protect them from the insect's haemolymph environment. To further maximise transmission, each oocyst generates hundreds of sporozoites through an asexual multiplication step called sporogony. Completion of transmission requires sporozoite egress from the capsule (excystation), but this process remains poorly understood. In this study, we fused the parasite-encoded capsule protein Cap380 with green fluorescent protein in a transgenic P. berghei line, allowing live fluorescence imaging of capsules throughout sporogony and sporozoite excystation. The results show that capsules progressively weaken during sporulation ultimately resulting in sporozoite exit through small holes. Prior to formation of the holes, local thinning of the capsule was observed. Our findings support an excystation model based on local, rather than global, weakening of the capsule likely facilitated by local re-orientation of sporozoites and apical secretion.

High morbidity and mortality in children with untreated congenital deficiency of leptin or its receptor.

The long-term clinical outcomes of severe obesity due to leptin signaling deficiency are unknown. We carry out a retrospective cross-sectional investigation of a large cohort of children with leptin (LEP), LEP receptor (LEPR), or melanocortin 4 receptor (MC4R) deficiency (n = 145) to evaluate the progression of the disease. The affected individuals undergo physical, clinical, and metabolic evaluations. We report a very high mortality in children with LEP (26%) or LEPR deficiency (9%), mainly due to severe pulmonary and gastrointestinal infections. In addition, 40% of surviving children with LEP or LEPR deficiency experience life-threatening episodes of lung or gastrointestinal infections. Although precision drugs are currently available for LEP and LEPR deficiencies, as yet, they are not accessible in Pakistan. An appreciation of the severe impact of LEP or LEPR deficiency on morbidity and early mortality, educational attainment, and the attendant stigmatization should spur efforts to deliver the available life-saving drugs to these children as a matter of urgency.

Plasmodium berghei oocysts possess fatty acid synthesis and scavenging routes.

Malaria parasites carry out fatty acid synthesis (FAS) in their apicoplast organelle via a bacterially related (type II) enzymatic pathway. In the vertebrate host, exoerythrocytic Plasmodium stages rely on FAS, whereas intraerythrocytic stages depend on scavenging FA from their environment. In the mosquito, P. falciparum oocysts express and rely on FAS enzymes for sporozoite formation, but P. yoelii oocysts do not express, nor depend on, FAS enzymes and thus rely on FA scavenging to support sporogony. In P. berghei, FAS enzymes are similarly expendable for sporogony, indicating it conforms to the P. yoelii scenario. We show here that P. berghei, unexpectedly, expresses FAS enzymes throughout oocyst development. These findings indicate that P. berghei can employ FAS alongside FA scavenging to maximise sporogony and transmission, and is more similar to P. falciparum than previously assumed with respect to FA acquisition by the oocyst. The ability of oocysts to switch between FAS and scavenging could be an important factor in the non-competitive relationship of resource exploitation between Plasmodium parasites and their mosquito vectors, which shapes parasite virulence both in the insect and vertebrate.

NAD(P) transhydrogenase isoform distribution provides insight into apicomplexan evolution.

Membrane-located NAD(P) transhydrogenase (NTH) catalyses reversible hydride ion transfer between NAD(H) and NADP(H), simultaneously translocating a proton across the membrane. The enzyme is structurally conserved across prokaryotes and eukaryotes. In heterotrophic bacteria NTH proteins reside in the cytoplasmic membrane, whereas in animals they localise in the mitochondrial inner membrane. Eukaryotic NTH proteins exists in two distinct configurations (isoforms) and have non-mitochondrial functions in unicellular eukaryotes like Plasmodium, the causative agent of malaria. In this study, we carried out a systematic analysis of nth genes across eukaryotic life to determine its prevalence and distribution of isoforms. The results reveal that NTH is found across all major lineages, but that some organisms, notably plants, lack nth genes altogether. Isoform distribution and phylogenetic analysis reveals different nth gene loss scenarios in apicomplexan lineages, which sheds new light on the evolution of the Piroplasmida and Eimeriidae.

An operator splitting scheme for numerical simulation of spinodal decomposition and microstructure evolution of binary alloys.

This article compares the operator splitting scheme to linearly stabilized splitting and semi-implicit Euler's schemes for the numerical solution of the Cahn-Hilliard equation. For the purpose of validation, the spinodal decomposition phenomena have been simulated. The efficacy of the three schemes has been demonstrated through numerical experiments. The computed results show that the schemes are conditionally stable. It has been observed that the operator splitting scheme is computationally more efficient.

Whole Exome Sequencing Reveals Clustering of Variants of Known Vitiligo Genes in Multiplex Consanguineous Pakistani Families.

Vitiligo is an autoimmune complex pigmentation disease characterized by non-pigmented patches on the surface of the skin that affect approximately 0.5-2% population worldwide. The exact etiology is still unknown; however, vitiligo is hypothesized to be a multifactorial and genetically heterogeneous condition. Therefore, the current study is designed to investigate the anthropometric presentation and genetic spectrum of vitiligo in fifteen consanguineous Pakistani families. The clinical evaluation of participating individuals revealed varying degrees of disease severity, with 23 years as the average age of disease onset. The majority of the affected individuals had non-segmental vitiligo (NSV). Whole exome sequencing analysis revealed clustering of rare variants of known vitiligo-associated genes. For instance, in the affected individuals of family VF-12, we identified three novel rare variants of PTPN22 (c.1108C>A), NRROS (c.197C>T) and HERC2 (c.10969G>A) genes. All three variants replaced evolutionarily conserved amino acid residues in encoded proteins, which are predicted to impact the ionic interactions in the secondary structure. Although various in silico algorithms predicted low effect sizes for these variants individually, the clustering of them in affected individuals increases the polygenic burden of risk alleles. To our knowledge, this is the first study that highlights the complex etiology of vitiligo and genetic heterogeneity in multiplex consanguineous Pakistani families.

Biallelic Mutations in P4HTM Cause Syndromic Obesity.

We previously demonstrated that 50% of children with obesity from consanguineous families from Pakistan carry pathogenic variants in known monogenic obesity genes. Here, we have discovered a novel monogenetic recessive form of severe childhood obesity using an in-house computational staged approach. The analysis included whole-exome sequencing data of 366 children with severe obesity, 1,000 individuals of the Pakistan Risk of Myocardial Infarction Study (PROMIS) study, and 200,000 participants of the UK Biobank to prioritize genes harboring rare homozygous variants with putative effect on human obesity. We identified five rare or novel homozygous missense mutations predicted deleterious in five consanguineous families in P4HTM encoding prolyl 4-hydroxylase transmembrane (P4H-TM). We further found two additional homozygous missense mutations in children with severe obesity of Indian and Moroccan origin. Molecular dynamics simulation suggested that these mutations destabilized the active conformation of the substrate binding domain. Most carriers also presented with hypotonia, cognitive impairment, and/or developmental delay. Three of the five probands died of pneumonia during the first 2 years of the follow-up. P4HTM deficiency is a novel form of syndromic obesity, affecting 1.5% of our children with obesity associated with high mortality. P4H-TM is a hypoxia-inducible factor that is necessary for survival and adaptation under oxygen deprivation, but the role of this pathway in energy homeostasis and obesity pathophysiology remains to be elucidated.

A rare human variant that disrupts GPR10 signalling causes weight gain in mice.

Disruption of brain-expressed G protein-coupled receptor-10 (GPR10) causes obesity in animals. Here, we identify multiple rare variants in GPR10 in people with severe obesity and in normal weight controls. These variants impair ligand binding and G protein-dependent signalling in cells. Transgenic mice harbouring a loss of function GPR10 variant found in an individual with obesity, gain excessive weight due to decreased energy expenditure rather than increased food intake. This evidence supports a role for GPR10 in human energy homeostasis. Therapeutic targeting of GPR10 may represent an effective weight-loss strategy.

Demographic and Clinical Determinants of Tuberculosis and TB Recurrence: A Double-Edged Retrospective Study from Pakistan.

Objective: TB recurrence is the second episode of TB after initial treatment bringing about an additional 7% load in TB burden intensified by 17.7% of multidrug-resistant recurrent cases. It is necessary to curb recurrence so that attempts to deal with active disease can be made more effective. This study aimed to characterize sociodemographic and clinical factors associated with recurrent TB in a high-burden setting. Methodology. A retrospective case-control study was carried out at two hospitals in Rawalpindi, Pakistan. TB patients and controls were included in the study. Sociodemographic and clinical data were collected by questionnaire from all subjects. Multivariate regression analysis was performed to determine factors associated with TB and TB recurrence respectively. Results: In our study cohort, factors significantly associated with TB were low BMI (OR: 0.961 (CI 0.954-0.968), p < 0.001), female gender (OR: 2.065 (CI 1.922-2.219), p < 0.001), being single/unmarried (OR: 1.214 (CI 1.109-1.328), p=0.003), middle-income status (OR: 1.935 (CI 1.616-2.323), p < 0.001), smoking (OR: 1.567 (CI 1.435-1.710), p < 0.001), and diabetes mellitus (OR: 1.142 (CI 1.017-1.278), p=0.023). TB recurrence constituted 11.2% of patients presenting to the hospital. Compared with the first episode of TB, cases with recurrence were more likely to be older (OR: 1.011 (CI 1.004-1.017), p < 0.001), have disease awareness (OR: 1.906 (CI 1.486-2.437), p < 0.001), smear positive (OR: 2.384 (CI 1.650-3.536), p < 0.001), and be drug-resistant (OR: 5.615 (CI 4.265-7.386), p < 0.001). Conclusion: In the present study cohort, low BMI, female gender, being single, middle-income status, being unemployed, smoking, and being diabetic came out to be the sociodemographic and clinical risk factors for TB. Further exploring the TB cases increasing age, drug resistance and smear positivity stood out to be the major sociodemographic and clinical factors of TB recurrence despite high disease awareness.

A Novel Missense Mutation in ERCC8 Co-Segregates with Cerebellar Ataxia in a Consanguineous Pakistani Family.

Autosomal-recessive cerebellar ataxias (ARCAs) are heterogeneous rare disorders mainly affecting the cerebellum and manifest as movement disorders in children and young adults. To date, ARCA causing mutations have been identified in nearly 100 genes; however, they account for less than 50% of all cases. We studied a multiplex, consanguineous Pakistani family presenting with a slowly progressive gait ataxia, body imbalance, and dysarthria. Cerebellar atrophy was identified by magnetic resonance imaging of brain. Using whole exome sequencing, a novel homozygous missense mutation ERCC8:c.176T>C (p.M59T) was identified that co-segregated with the disease. Previous studies have identified homozygous mutations in ERCC8 as causal for Cockayne Syndrome type A (CSA), a UV light-sensitive syndrome, and several ARCAs. ERCC8 plays critical roles in the nucleotide excision repair complex. The p.M59T, a substitution mutation, is located in a highly conserved WD1 beta-transducin repeat motif. In silico modeling showed that the structure of this protein is significantly affected by the p.M59T mutation, likely impairing complex formation and protein-protein interactions. In cultured cells, the p.M59T mutation significantly lowered protein stability compared to wildtype ERCC8 protein. These findings expand the role of ERCC8 mutations in ARCAs and indicate that ERCC8-related mutations should be considered in the differential diagnosis of ARCAs.

Risk variants of obesity associated genes demonstrate BMI raising effect in a large cohort.

Obesity is highly polygenic disease where several genetic variants have been reportedly associated with obesity in different ethnicities of the world. In the current study, we identified the obesity risk or protective association and BMI raising effect of the minor allele of adiponectin, C1Q and collagen domain containing (ADIPOQ), cholesteryl ester transfer protein (CEPT), FTO alpha-ketoglutarate dependent dioxygenase (FTO), leptin (LEP), and leptin receptor (LEPR) genes in a large cohort stratified into four BMI-based body weight categories i.e., normal weight, lean, over-weight, and obese. Based on selected candidate genetic markers, the genotyping of all study subjects was performed by PCR assays, and genotypes and allele frequencies were calculated. The minor allele frequencies (MAFs) of all genetic markers were computed for total and BMI-based body weight categories and compared with MAFs of global and South Asian (SAS) populations. Genetic associations of variants with obesity risk were calculated and BMI raising effect per copy of the minor allele were estimated. The genetic variants with higher MAFs in obese BMI group were; rs2241766 (G = 0.43), rs17817449 (G = 0.54), rs9939609 (A = 0.51), rs1421085 (C = 0.53), rs1558902 (A = 0.63), and rs1137101 (G = 0.64) respectively. All these variants were significantly associated with obesity (OR = 1.03-4.42) and showed a high BMI raising effect (ß = 0.239-0.31 Kg/m2) per copy of the risk allele. In contrast, the MAFs of three variants were higher in lean-normal BMI groups; rs3764261 A = 0.38, rs9941349 T = 0.43, and rs7799039 G = 0.40-0.43). These variants showed obesity protective associations (OR = 0.68-0.76), and a BMI lowering effect per copy of the protective allele (ß = -0.103-0.155 Kg/m2). The rs3764261 variant also showed significant and positive association with lean body mass (OR = 2.38, CI = 1.30-4.34). Overall, we report six genetic variants of ADIPOQ, FTO and LEPR genes as obesity-risk markers and a CETP gene variant as lean mass/obesity protective marker in studied Pakistani cohort.

A generalized class of estimators for sensitive variable in the presence of measurement error and non-response.

In this paper, a general class of estimators is proposed for estimating the finite population mean for sensitive variable, in the presence of measurement error and non-response in simple random sampling. Expressions for bias and mean square error up to first order of approximation, are derived. Impact of measurement errors is examined using real data sets, including the survey conducted at Quaid-i-Azam University, Islamabad. Simulated data sets are also used to observe the performance of the proposed estimators in comparison to some other estimators. We obtain the empirical bias and MSE values for the proposed and the competing estimators.

Rare Variant Analysis of Obesity-Associated Genes in Young Adults With Severe Obesity From a Consanguineous Population of Pakistan.

Recent advances in genetic analysis have significantly helped in progressively attenuating the heritability gap of obesity and have brought into focus monogenic variants that disrupt the melanocortin signaling. In a previous study, next-generation sequencing revealed a monogenic etiology in ∼50% of the children with severe obesity from a consanguineous population in Pakistan. Here we assess rare variants in obesity-causing genes in young adults with severe obesity from the same region. Genomic DNA from 126 randomly selected young adult obese subjects (BMI 37.2 ± 0.3 kg/m2; age 18.4 ± 0.3 years) was screened by conventional or augmented whole-exome analysis for point mutations and copy number variants (CNVs). Leptin, insulin, and cortisol levels were measured by ELISA. We identified 13 subjects carrying 13 different pathogenic or likely pathogenic variants in LEPR, PCSK1, MC4R, NTRK2, POMC, SH2B1, and SIM1. We also identified for the first time in the human, two homozygous stop-gain mutations in ASNSD1 and IFI16 genes. Inactivation of these genes in mouse models has been shown to result in obesity. Additionally, we describe nine homozygous mutations (seven missense, one stop-gain, and one stop-loss) and four copy-loss CNVs in genes or genomic regions previously linked to obesity-associated traits by genome-wide association studies. Unexpectedly, in contrast to obese children, pathogenic mutations in LEP and LEPR were either absent or rare in this cohort of young adults. High morbidity and mortality risks and social disadvantage of children with LEP or LEPR deficiency may in part explain this difference between the two cohorts.

Comparative analysis of the serum microbiome of HIV infected individuals.

HIV infects the CD4 cells which marks the suppression of our immune system. DNA from serum of healthy, treated and untreated HIV infected individuals was extracted. The DNA was subjected to 16S metagenomic sequencing and analyzed using QIIME2 pipeline. 16S sequencing analysis showed serum microbiome was dominated by Firmicutes, Proteobacteria, Bacteroidota and Actinobacteria. Treated HIV infection showed highest abundance of Firmicutes (66.40%) significantly higher than untreated HIV infection (35.88%) and control (41.89%). Bacilli was most abundant class in treated (63.59%) and second most abundant in untreated (34.53%) while control group showed highest abundance of class Gamma-proteobacteria (45.86%). Untreated HIV infection group showed Enterococcus (10.72%) and Streptococcus (6.599%) as the most abundant species. Untreated HIV infection showed significantly higher (p = 0.0039) species richness than treated and control groups. An altered serum microbiome of treated HIV infection and higher microbial abundance in serum of untreated HIV infection was observed.

Reproductive Rights Knowledge, Health Care Utilization, and Contraceptive Use in Pakistan: A Reproductive Rights Perspective.

INTRODUCTION: This study investigates the effects of utilization of health care and the level of reproductive rights knowledge on contraceptive use. Current family planning programs in developing countries utilize a two-pronged strategy involving improvement of level of reproductive rights and, right to access health care systems. The effectiveness of this strategy in developing countries such as Pakistan remains poorly investigated. This study aimed at examining the effect of reproductive rights knowledge on contraceptive use. METHODS: The target population is educated, 18- to 45-year-old married Pakistani women. The sample is composed of 160 nonrandomly selected purposive group of women. We gathered data using a structured questionnaire and analyzed the data using several multivariate methods such as latent class analysis and multiple classification analysis. RESULTS: Our results suggest that reproductive rights knowledge has no direct effect on contraceptive use. Furthermore, the level of utilization of the health care system plays a significant intervening role between reproductive rights knowledge and contraceptive use. DISCUSSION: Our results call for improving access and availability of health care while strengthening the awareness and knowledge of reproductive rights among women to improve their capacity to utilize family planning methods. Public awareness and educational programs are indeed necessary to promote knowledge of reproductive rights among women in developing countries. As public health campaigns propagandize the crucial role reproductive rights play in improving women's reproductive health, it is important to maintain a structurally harmonious relationship between health care systems and family planning programs promoting contraceptive use. This approach is more likely to result in significant returns for public awareness campaigns promoting reproductive rights in developing countries.