Delineation of a Human Mendelian Disorder of the DNA Demethylation Machinery: TET3 Deficiency.

Germline pathogenic variants in chromatin-modifying enzymes are a common cause of pediatric developmental disorders. These enzymes catalyze reactions that regulate epigenetic inheritance via histone post-translational modifications and DNA methylation. Cytosine methylation (5-methylcytosine [5mC]) of DNA is the quintessential epigenetic mark, yet no human Mendelian disorder of DNA demethylation has yet been delineated. Here, we describe in detail a Mendelian disorder caused by the disruption of DNA demethylation. TET3 is a methylcytosine dioxygenase that initiates DNA demethylation during early zygote formation, embryogenesis, and neuronal differentiation and is intolerant to haploinsufficiency in mice and humans. We identify and characterize 11 cases of human TET3 deficiency in eight families with the common phenotypic features of intellectual disability and/or global developmental delay; hypotonia; autistic traits; movement disorders; growth abnormalities; and facial dysmorphism. Mono-allelic frameshift and nonsense variants in TET3 occur throughout the coding region. Mono-allelic and bi-allelic missense variants localize to conserved residues; all but one such variant occur within the catalytic domain, and most display hypomorphic function in an assay of catalytic activity. TET3 deficiency and other Mendelian disorders of the epigenetic machinery show substantial phenotypic overlap, including features of intellectual disability and abnormal growth, underscoring shared disease mechanisms.

Causal variants in Maturity Onset Diabetes of the Young (MODY) - A systematic review.

BACKGROUND: Maturity Onset Diabetes of the Young (MODY) is an autosomal dominant type of diabetes. Pathogenic variants in fourteen genes are reported as causes of MODY. Its symptoms overlap with type 1 and type 2 diabetes. Reviews for clinical characteristics, diagnosis and treatments are available but a comprehensive list of genetic variants, is lacking. Therefore this study was designed to collect all the causal variants involved in MODY, reported to date. METHODS: We searched PubMed from its date of inception to December 2019. The search terms we used included disease names and name of all the known genes involved. The ClinVar database was also searched for causal variants in the known 14 MODY genes. RESULTS: The record revealed 1647 studies and among them, 326 studies were accessed for full-text. Finally, 239 studies were included, as per our inclusion criteria. A total of 1017 variants were identified through literature review and 74 unpublished variants from Clinvar database. The gene most commonly affected was GCK, followed by HNF1a. The traditional Sanger sequencing was used in 76 % of the cases and 65 % of the studies were conducted in last 10 years. Variants from countries like Jordan, Oman and Tunisia reported that the MODY types prevalent worldwide were not common in their countries. CONCLUSIONS: We expect that this paper will help clinicians interpret MODY genetics results with greater confidence. Discrepancies in certain middle-eastern countries need to be investigated as other genes or factors, like consanguinity may be involved in developing diabetes.

Community-Based Assessment of Knowledge, Attitude, Practices and Risk Factors Regarding COVID-19 Among Pakistanis Residents During a Recent Outbreak: A Cross-Sectional Survey.

Exceptional precautionary measures have been adopted to stop the transmission and control of COVID-19 through the world and Pakistan is facing lockdown in this scenario. Public loyalty to precautionary measures is affected by their knowledge, attitude, risk factors and practices (KAP) towards COVID-19. The present study was conducted among the Pakistani residents to observe the knowledge, attitude, practices and risk factors towards COVID-19 outbreak in Pakistan. A questionnaire was designed, and a cross-sectional survey was conducted among participants of the study area. Participants were asked the questions regarding knowledge, attitude, practices and risk factors towards COVID-19. Data were analyzed by SPSS and t/F test and correlation was applied among the knowledge, attitude, risk factors and practices. A total of 1060 questionnaires were received. 1004 were included while 56 were excluded. The highest representation was from Punjab province (65.6%), female (63%) and age group of 21-30 years (62.1%). Most participants were single (85%), Muslim (99.4%), Urdu speaking (45.6%) and were graduates (51.5%). Most of the participants were students (52.9%) and were from economically middle-class families (40.8%). The knowledge was positively correlated with attitude and practices whereas negatively correlated with risk factors (P < 0.05). The attitude was negatively correlated with risk factor and positively correlated with practices. The risk factors and practices were positively correlated with each other. Health education program to improve the COVID-19 knowledge, attitude, practices and risk factors should be initiated to combat current health challenge.

Seroprevalence of Transplacentally acquired Measles antibodies in unvaccinated infants at nine months of age and its relation to the feeding practices.

BACKGROUND: In recent years Pakistan has faced frequent measles outbreaks killing hundreds of children despite the availability of vaccine for decades. This study was undertaken to determine the persistence of maternal transferred measles antibody levels in infants before measles vaccination with relation to their feeding practices. METHODS: A cross sectional study was conducted at district Islamabad over 1 year between 1st October 2013 to 30th Sept. 2016. Any infant less than 9 months of age, not suffering from an acute or debilitating illness and not vaccinated was enrolled in the study. After taking written informed consent from parents / guardians, information was collected on a pretested questionnaire. About 3 cc venous blood was taken to quantify any measles IgG antibodies. Data was analyzed by using Epi Info 7.2 version. RESULTS: Three hundred eighty-four infants were enrolled and were divided into three age groups, 1-90, 91-180 and 181-270 days age groups. Mean age of infants was 4.4 months ±3.2 SD. Male to female ratio was 1.2:1. A level of maternal measles IgG antibodies ≥12 U/ml was taken as protective. Of total 384 infants, 91(24%) had protective measles antibody titters (> 12 U/ml). and 65 (73%) of them were on breast milk. Highest antibody levels were found in 1-90 days age group. Analysis showed that 181-270 days aged infants had 3.1875 more odds of having unprotected/ low levels of antibodies against measles than children aged less than 180 days. Age group < 180 days found to be statistically significant with protective IgG levels (OR: 3.1875, P value: < 0.000063). CONCLUSION: Measles protective antibodies were found in infants < 180 days age group. Breast feeding provides early protection against measles. Levels drop down to low levels immediately after birth and then after 06 months. It is, therefore, recommended that measles vaccination should be considered for administration at 6 months or even earlier if measles immunity is desired.

Novel candidate genes and variants underlying autosomal recessive neurodevelopmental disorders with intellectual disability.

Identification of Mendelian genes for neurodevelopmental disorders using exome sequencing to study autosomal recessive (AR) consanguineous pedigrees has been highly successful. To identify causal variants for syndromic and non-syndromic intellectual disability (ID), exome sequencing was performed using DNA samples from 22 consanguineous Pakistani families with ARID, of which 21 have additional phenotypes including microcephaly. To aid in variant identification, homozygosity mapping and linkage analysis were performed. DNA samples from affected family member(s) from every pedigree underwent exome sequencing. Identified rare damaging exome variants were tested for co-segregation with ID using Sanger sequencing. For seven ARID families, variants were identified in genes not previously associated with ID, including: EI24, FXR1 and TET3 for which knockout mouse models have brain defects; and CACNG7 and TRAPPC10 where cell studies suggest roles in important neural pathways. For two families, the novel ARID genes CARNMT1 and GARNL3 lie within previously reported ID microdeletion regions. We also observed homozygous variants in two ID candidate genes, GRAMD1B and TBRG1, for which each has been previously reported in a single family. An additional 14 families have homozygous variants in established ID genes, of which 11 variants are novel. All ARID genes have increased expression in specific structures of the developing and adult human brain and 91% of the genes are differentially expressed in utero or during early childhood. The identification of novel ARID candidate genes and variants adds to the knowledge base that is required to further understand human brain function and development.

Burden of Tobacco in Pakistan: Findings From Global Adult Tobacco Survey 2014.

Introduction: The Global Adult Tobacco Survey (GATS) is the global standard for systematically monitoring adult tobacco use and tracking key tobacco control indicators. Methods: Using a multistage stratified cluster design, 9856 households were sampled, and one individual was randomly selected from each household. Standard GATS questionnaire was used to collect information on tobacco use, cessation, second-hand smoke, knowledge, attitudes, and perceptions. Data were analyzed per standard GATS protocol. Results: Of 9856 individuals, 7831 individuals completed the interview. The response rate was 81%. Overall, 19.1% adults were currently using tobacco products and among them, 12.4% smoked tobacco, and 7.7% smokeless tobacco. Exposure to second-hand smoke was seen in 86% in a restaurant while it was 76% on public transportation. A total of 24.7% smokers made a quit attempt in the past 12 months. Anticigarette smoking information was observed by 37.7% adults, while 29.7% current smokers thought about quitting after reading health warning labels on cigarette packages. Most (85%) adults favored no smoking in public places, and 74.8% favored increasing taxes on tobacco products. Current cigarette smokers spent Pakistani Rupees 767.3 per month (7.78 USD) on manufactured cigarettes and consumed 4500 cigarette sticks (225 packs) annually. Conclusions: Besides 19.1% tobacco users, the majority (86%) were exposed to second-hand smoke at public places indicating that ban on tobacco use in public places is not being followed. A quarter of current smokers wants to quit smoking who may be provided assistance to reduce tobacco burden. Implications: This study provides national-level data about tobacco use and its burden and also indicates weak implantation of tobacco control laws. There is need to devise a strategy for proper implementation of these laws to reduce the tobacco burden in the country.

Mutation of ATF6 causes autosomal recessive achromatopsia.

Achromatopsia (ACHM) is an early-onset retinal dystrophy characterized by photophobia, nystagmus, color blindness and severely reduced visual acuity. Currently mutations in five genes CNGA3, CNGB3, GNAT2, PDE6C and PDE6H have been implicated in ACHM. We performed homozygosity mapping and linkage analysis in a consanguineous Pakistani ACHM family and mapped the locus to a 15.12-Mb region on chromosome 1q23.1-q24.3 with a maximum LOD score of 3.6. A DNA sample from an affected family member underwent exome sequencing. Within the ATF6 gene, a single-base insertion variant c.355_356dupG (p.Glu119Glyfs*8) was identified, which completely segregates with the ACHM phenotype within the family. The frameshift variant was absent in public variant databases, in 130 exomes from unrelated Pakistani individuals, and in 235 ethnically matched controls. The variant is predicted to result in a truncated protein that lacks the DNA binding and transmembrane domains and therefore affects the function of ATF6 as a transcription factor that initiates the unfolded protein response during endoplasmic reticulum (ER) stress. Immunolabeling with anti-ATF6 antibodies showed localization throughout the mouse neuronal retina, including retinal pigment epithelium, photoreceptor cells, inner nuclear layer, inner and outer plexiform layers, with a more prominent signal in retinal ganglion cells. In contrast to cytoplasmic expression of wild-type protein, in heterologous cells ATF6 protein with the p.Glu119Glyfs*8 variant is mainly confined to the nucleus. Our results imply that response to ER stress as mediated by the ATF6 pathway is essential for color vision in humans.

Novel VPS13B Mutations in Three Large Pakistani Cohen Syndrome Families Suggests a Baloch Variant with Autistic-Like Features.

BACKGROUND: Cohen Syndrome (COH1) is a rare autosomal recessive disorder, principally identified by ocular, neural and muscular deficits. We identified three large consanguineous Pakistani families with intellectual disability and in some cases with autistic traits. METHODS: Clinical assessments were performed in order to allow comparison of clinical features with other VPS13B mutations. Homozygosity mapping followed by whole exome sequencing and Sanger sequencing strategies were used to identify disease-related mutations. RESULTS: We identified two novel homozygous deletion mutations in VPS13B, firstly a 1 bp deletion, NM_017890.4:c.6879delT; p.Phe2293Leufs*24, and secondly a deletion of exons 37-40, which co-segregate with affected status. In addition to COH1-related traits, autistic features were reported in a number of family members, contrasting with the "friendly" demeanour often associated with COH1. The c.6879delT mutation is present in two families from different regions of the country, but both from the Baloch sub-ethnic group, and with a shared haplotype, indicating a founder effect among the Baloch population. CONCLUSION: We suspect that the c.6879delT mutation may be a common cause of COH1 and similar phenotypes among the Baloch population. Additionally, most of the individuals with the c.6879delT mutation in these two families also present with autistic like traits, and suggests that this variant may lead to a distinct autistic-like COH1 subgroup.

Dengue knowledge and its management practices among physicians of major cities of Pakistan.

OBJECTIVE: To assess knowledge and practices related to dengue management among physicians. METHODS: The cross-sectional study was conducted at hospitals in Islamabad, Lahore, Faisalabad, Peshawar, Quetta and Karachi between June and December 2012Physicians from public and private sectors filled a self-administered questionnaire about dengue knowledge and its management practices. A maximum score of 100 was assigned to the knowledge portion. Data was analysed using SPSS 15. RESULTS: A total of 400 subjects participated in the study; 200(50%) each from public and private hospitals. Of them, 223(56%) were males; 268(67%) were in the 21-30 years age bracket. The highest score was recorded in Quetta 67 followed by 65 in Karachi, 62 in Lahore, Faisalabad, Peshawar and 59 in Islamabad. Of the total, 200 (50%) were not aware that leucopenia is a criterion for diagnosing probable dengue. Similarly 140 (35%) did not know the criteria for diagnosing dengue haemorrhagic fever and warning signs of severe dengue. Total of 204 (51%) were not aware of the criteria for discharging of the admitted cases. There was no significant difference between dengue knowledge of the physicians belonging to public and private sectors (p>0.05). CONCLUSIONS: Quite a large number of physicians lacked knowledge of probable diagnosis of dengue and appropriate time to discharge the patients.

Evaluation of diagnostic potential of Rv3803c and Rv2626c recombinant antigens in TB endemic country Pakistan.

To overcome and eliminate tuberculosis (TB), definitive, reliable, and rapid diagnosis is mandatory. Presently, the diagnostic potential of acute and latent stage TB specific antigens i.e., Rv3803c and Rv2626c was determined. Immunogenic recombinant genes of Rv3803c and Rv2626c antigens were cloned in bacterial expression vector pET23b and expressed product was purified. The homogeneity and structural integrity was confirmed by Western blot analysis. Diagnostic potential of Rv3803c and Rv2626c antigens was analyzed using the sera of 140 active TB patients (AFB smear positive) by indirect ELISA. Ten patients of leprosy and 94 healthy individuals were taken as disease and normal control respectively. The data was analyzed using R statistical package. The sensitivity and specificity of Rv3803c in active TB patients was of 69.3% and 76.4% respectively with an area under ROC curve of 0.77, whereas sensitivity and specificity of Rv2626c 77.1% and 85.1%, respectively. The area under ROC curve of Rv2626c was 0.89 which was significantly higher than Rv3803c (p < 0.0001). Recombinant antigens Rv3803c and Rv2626c have potential to be used as diagnostic markers for TB and need to evaluate with other antigens for differential diagnosis of TB.

Dengue fever in patients admitted in tertiary care hospitals in Pakistan.

OBJECTIVES: To assess the gaps in the diagnosis and management of dengue fever cases. METHODS: The retrospective descriptive analytical study was done with a case record analysis of patients with dengue fever admitted from January to December 2010 at five tertiary care hospitals in different Pakistani cities. Using a questionnaire, information was gathered on demography, haematological profile, management, use of blood and platelet transfusions and the outcome. For comparison, data of serologically-confirmed dengue patients from a private laboratory in Islamabad was collected to see the age, gender and month-wise distribution of cases tested over the same period. SPSS 16 was used for statistical analysis. RESULTS: Out of the 841 confirmed dengue cases, 514 (79%) were males and 139 (21%) females. The overall mean age was 31.3 +/- 14.0 years. Dengue fever was seen in 653 (78%) and dengue haemorrhagic fever (DHF) in 188 (22%) patients. Most cases were between 20 and 49 years of age. A gradual increase in dengue fever and dengue haemorrhagic fever was seen from August, with a peak in October/November. Tourniquet test was done only in 20 (2.3%) cases, out of which 11 (55%) were positive and 9 (45%) were negative. Serial haematocrit was not done in any case. Total deaths were 5 (0.6%). CONCLUSIONS: Most cases were seen in October/November with the majority being in the 20-39 age group. Tourniquet test and serial haematocrit were infrequently used. No standard national guidelines were employed.

In-silico analysis of potential anticancer drug for NEK7 and PPP1CA proteins overexpressed in pancreatic ductal adenocarcinoma.

NIMA-related kinase 7 (NEK7) and phosphoprotein phosphatase-1 catalytic subunit alpha (PPP1CA) are the most common proteins overexpressed in pancreatic ductal adenocarcinoma, which is the most common type of pancreatic cancer. The goal of the current study was to identify a possible NEK7 and PPP1CA therapeutic inhibitor. For this investigation, 5000 compounds were retrieved from the IMPPAT library of phytochemicals, which were docked with our respective target proteins. Also, a reference compound, gemcitabine, which is a Food and Drug Administration (FDA) approved drug, was docked with the target proteins. The binding energy of the reference compound for both the targeted proteins was -6.5 kcal/mol. The common ligand with the lowest binding energy for both targets is boeravinone B (PubChem ID: 14018348) with -9.2 kcal/mol of NEK7 and -7.6 kcal/mol for PPP1CA. The compound was further investigated through density function theory (DFT) and molecular dynamic simulation analysis. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and hydrogen bonding analysis indicated the stability of the boeravinone B with the target proteins (NEK7 and PPP1CA).Communicated by Ramaswamy H. Sarma.

In silico identification of potential drug-like molecules against G glycoprotein of Nipah virus by molecular docking, DFT studies, and molecular dynamic simulation.

Nipah virus (NiV) is a novel zoonotic pathogen that belongs to the Paramyxovirus family. The pathogen has infected a number of people in countries like Bangladesh, India, Singapore, and Malaysia with high mortality rates. Although the NiV has been classified as a biosafety level four pathogen (BSL-4), there is no drug approved for treatment against it. In this study, the G glycoprotein of the NiV was chosen as an antiviral target. Based on ADMET criteria, BBB- and BBB + group compounds were screened out of the Gold & platinum Asinex library containing 211620 compounds. After careful evaluation, the selected ligands were then virtually screened to identify the potential inhibitors against the G glycoprotein of the NiV through molecular docking, density functional theory (DFT), and molecular dynamic (MD) simulation studies. In our study we identified 5-(1,3-Benzodioxol-5-yl)-2-[(3-fluorobenzyl)sulfanyl]-5,8-dihydropyrido[2,3-d]pyrimidine-4,7(1H,6H)-dione (from BBB- group) and 7,7-Dimethyl-1-(4-methylphenyl)-3-(4-morpholinylcarbonyl)-7,8-dihydro-2,5(1H,6H)-quinolinedione) (from BBB + group) as potential compounds for the prevention and treatment of NiV related diseases.Communicated by Ramaswamy H. Sarma.

A loss of function variant in AGPAT3 underlies intellectual disability and retinitis pigmentosa (IDRP) syndrome.

Intellectual disability (ID) and retinal dystrophy (RD) are the frequently found features of multiple syndromes involving additional systemic manifestations. Here, we studied a family with four members presenting severe ID and retinitis pigmentosa (RP). Using genome wide genotyping and exome sequencing, we identified a nonsense variant c.747 C > A (p.Tyr249Ter) in exon 7 of AGPAT3 which co-segregates with the disease phenotype. Western blot analysis of overexpressed WT and mutant AGPAT3 in HEK293T cells showed the absence of AGPAT3, suggesting instability of the truncated protein. Knockdown of Agpat3 in the embryonic mouse brain caused marked deficits in neuronal migration, strongly suggesting that reduced expression of AGPAT3 affects neuronal function. Altogether, our data indicates that AGPAT3 activity is essential for neuronal functioning and loss of its activity probably causes intellectual disability and retinitis pigmentosa (IDRP) syndrome.

Neurological disorders and disability in Pakistan: A cross-sectional multicenter study.

BACKGROUND/OBJECTIVE: The aim of this study is to identify frequencies of various neurological disorders (NDs) and associated disability in patients attending neurologic clinics in rural and urban centers in Pakistan. METHODS: This is an observational study conducted in 39 neurological centers in both rural and urban areas, public and private health sectors all over Pakistan. This study was conducted between august 2017 to December 2019. RESULTS: A total of 28,845 adults were enrolled. Mean age of the study participants was 46.2 ± 17.2 years, 15,252 (52.9%) were men and 13,593 (47.1%) were women. Most common comorbid medical condition was hypertension 7622(26.4%) followed by Diabetes 3409(11.8%). Among neurological diagnoses, vascular diseases (20%) were the most common followed by Headache disorders (18.6%), Epilepsy (12.5%), nerve and root diseases (12.4%), Psychiatric diseases (10%), Dementias (8%) and movement disorders (7.9%). Half of the patients 15,503(53.7%) had no neurological disability, while minor disability was present in 10,442(36.2%) of cases. Moderate to severe disability was present in 2876(10%) cases. Headache disorders, psychiatric diseases, muscle pain/muscle related disorders and demyelinating diseases were more common in women. Vascular diseases, movement disorders and Dementias were more common in 46 years and above age group whereas headache disorders, Epilepsy and Psychiatric disorders were more prevalent in <46 years age groups. CONCLUSION: Vascular diseases are the most common presentation of patients in neurology clinics followed by headache disorders and epilepsies. Minor disability was present in 36% while moderate to severe disability was present in 10% cases.