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.

Recessive progressive symmetric erythrokeratoderma results from a homozygous loss-of-function mutation of KRT83 and is allelic with dominant monilethrix.

BACKGROUND: Progressive symmetric erythrokeratoderma (PSEK) is a rare skin disorder characterised by symmetrically distributed demarcated hyperkeratotic plaques, often with associated palmoplantar hyperkeratosis, with new plaques appearing over time. Most cases are inherited in an autosomal dominant manner, although a few cases exhibit apparent autosomal recessive inheritance. OBJECTIVE: To identify the gene underlying autosomal recessive PSEK in a large Pakistani kindred. METHODS: We first carried out autozygosity mapping using microsatellite markers in candidate regions of the genome. We then carried out exome sequencing of five family members, autozygosity mapping and mutation analysis using the exome data and verification by Sanger sequencing. RESULTS: Autozygosity mapping and exome sequencing identified a homozygous frameshift deletion (c.811delA; p.Ser271fs) in KRT83, which co-segregated with the PSEK phenotype in the family and which is expected to abolish keratin 83, a type II keratin of hair and skin. CONCLUSIONS: At least some cases of PSEK result from loss-of-function mutations in KRT83. Heterozygous missense substitutions in KRT83 have been implicated in autosomal dominant monilethrix, a rare hair disorder. Our findings indicate that at least some cases of autosomal recessive PSEK and autosomal dominant monilethrix are allelic, respectively resulting from loss-of-function and missense mutations in the KRT83 gene. Together, these findings indicate that different types of mutations in KRT83 can result in quite different skin and hair phenotypes.

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.

Mutation in NSUN2, which encodes an RNA methyltransferase, causes autosomal-recessive intellectual disability.

Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.

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.

Identification of Lead Compounds against Scm (fms10) in Enterococcus faecium Using Computer Aided Drug Designing.

(1) Background: Enterococcus faecium DO is an environmental microbe, which is a mesophilic, facultative, Gram-positive, and multiple habitat microorganism. Enterococcus faecium DO is responsible for many diseases in human. The fight against infectious diseases is confronted by the development of multiple drug resistance in E. faecium. The focus of this research work is to identify a novel compound against this pathogen by using bioinformatics tools and technology. (2) Methods: We screened the proteome (accession No. PRJNA55353) information from the genome database of the National Centre for Biotechnology Information (NCBI) and suggested a potential drug target. I-TASSER was used to predict the three-dimensional structure of the protein, and the structure was optimized and minimized by different tools. PubChem and ChEBI were used to retrieve the inhibitors. Pharmacophore modeling and virtual screening were performed to identify novel compounds. Binding interactions of compounds with target protein were checked using LigPlot. pkCSM, SwissADME, and ProTox-II were used for adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. (3) Results: Novel selected compounds have improved absorption and have better ADMET properties. Based on our results, the chemically identified inhibitor ZINC48942 targeted the receptor that can inhibit the activity of infection in E. faecium. This research work will be beneficial for the scientific community and could aid in the design of a new drug against E. faecium infections. (4) Conclusions: It was observed that novel compounds are potential inhibitors with more efficacy and fewer side effects. This research work will help researchers in testing and identification of these chemicals useful against E. faecium.

Determination of in vivo biological activities of Dodonaea viscosa flowers against CCL4 toxicity in albino mice with bioactive compound detection.

Dodonaea viscosa L.Jacq. is an evergreen shrub and native to Asia, Africa, and Australia. It has been used as traditional medicine in different countries. The foremost objective of the current study was to discover the protective potential of D. viscosa flowers Methanol (DVM) and Chloroform (DVC) extracts against CCL4 induced toxicity in mice. This study was intended to identify phytochemicals through HPLC, GCMS, and FT-IR, as well as in vitro antioxidant and in vitro anti-tuberculosis activity. Our comprehensive findings indicate that Dodonaea viscosa is valuable and widespread herbal medicine through therapeutic potentials for curing various ailments. Dodonaeaviscosa flowersare found to have a protective effect against oxidative stress produced by CCL4 in the liver, kidney, and spleen. The intake of DV extracts restored the level of hepatic enzymes (ALP, AST ALT, and Direct bilirubin), hematological parameters (RBCs, WBCs, and Platelets), total protein, and liver antioxidant enzymes (SOD, GPx, and CAT) after a decline in levels by CCL4. Histopathological results discovered the defensive effect of 300 mg/kg of DVM extract against CCL4 induced damage, thus having an improved protective effect compared to DVC and control. As a result of metabolite screening, the total flavonoids and total phenolics were present in abundance. A phytochemical investigation by HPLC identified gallic acid, epicatechin, cumeric acid, flavonoids, while GCMS estimated oleic acid (Octadecenoic acid) (C18H34O2), Stearic acid (C18H36O2), Ricinoleic acid (C18H34O3), and Cedrol (C15H26O). DVM extract exhibited resistance against in vitro Mycobacterium tuberculosis strains. So this study proposed that the protective effect of DV against oxidative damage induced in the liver, kidney, and spleen can be correlated to the antioxidant compounds.

Exploration and determination of algal role as Bioindicator to evaluate water quality - Probing fresh water algae.

OBJECTIVES: To explore the algal floral diversity and its role to determine water quality. METHODS: The regular monthly collection of algal and water samples was made during 2018. Unicellular algae were preserved in 2 to 3% formalin while macroalgae in 4% formalin. Microphotographs of algae were taken at the biotechnological Lab of PCSIR Lahore, Pakistan. Palmer pollution index was used to determine water quality. RESULTS: The study identified 201 algal species distributed among 57 genera, 42 families, 25 orders, 10 classes and 7 divisions. The total score of Algal Genus Pollution Index of Banjosa Lake, Ali Sojal Dam, Dothan Dam, Drake Dam and Rawalakot Nullah (city) were 14, 9, 10, 18 and 25 respectively. It was revealed that Banjosa Lake has probable organic pollution, Ali Sojal Dam and Dothan Dam showed lack of organic pollution, Drake Dam indicated moderate pollution while Rawalakot Nullah (City) indicated confirm high organic pollution. CONCLUSION: We strongly recommend the conservation and managed status of algal species for sustainable resource of algal- derived products in future. It was revealed that the water quality of Banjosa Lake, Drak Dam and Rawalakot Nullah was affected from anthropogenic activities and needs to be managed.

FAM92A Underlies Nonsyndromic Postaxial Polydactyly in Humans and an Abnormal Limb and Digit Skeletal Phenotype in Mice.

Polydactyly is a common congenital anomaly of the hand and foot. Postaxial polydactyly (PAP) is characterized by one or more posterior or postaxial digits. In a Pakistani family with autosomal recessive nonsyndromic postaxial polydactyly type A (PAPA), we performed genomewide genotyping, linkage analysis, and exome and Sanger sequencing. Exome sequencing revealed a homozygous nonsense variant (c.478C>T, p.[Arg160*]) in the FAM92A gene within the mapped region on 8q21.13-q24.12 that segregated with the PAPA phenotype. We found that FAM92A is expressed in the developing mouse limb and E11.5 limb bud including the progress zone and the apical ectodermal ridge, where it strongly localizes at the cilia level, suggesting an important role in limb patterning. The identified variant leads to a loss of the FAM92A/Chibby1 complex that is crucial for ciliogenesis and impairs the recruitment and the colocalization of FAM92A with Chibby1 at the base of the cilia. In addition, we show that Fam92a-/- homozygous mice also exhibit an abnormal digit morphology, including metatarsal osteomas and polysyndactyly, in addition to distinct abnormalities on the deltoid tuberosity of their humeri. In conclusion, we present a new nonsyndromic PAPA ciliopathy due to a loss-of-function variant in FAM92A. © 2018 American Society for Bone and Mineral Research.