Basis for high-affinity ethylene binding by the ethylene receptor ETR1 of Arabidopsis.

The gaseous hormone ethylene is perceived in plants by membrane-bound receptors, the best studied of these being ETR1 from Arabidopsis. Ethylene receptors can mediate a response to ethylene concentrations at less than one part per billion; however, the mechanistic basis for such high-affinity ligand binding has remained elusive. Here we identify an Asp residue within the ETR1 transmembrane domain that plays a critical role in ethylene binding. Site-directed mutation of the Asp to Asn results in a functional receptor that has a reduced affinity for ethylene, but still mediates ethylene responses in planta. The Asp residue is highly conserved among ethylene receptor-like proteins in plants and bacteria, but Asn variants exist, pointing to the physiological relevance of modulating ethylene-binding kinetics. Our results also support a bifunctional role for the Asp residue in forming a polar bridge to a conserved Lys residue in the receptor to mediate changes in signaling output. We propose a new structural model for the mechanism of ethylene binding and signal transduction, one with similarities to that found in a mammalian olfactory receptor.

Overlapping neurological phenotypes in two extended consanguineous families with novel variants in the CNTNAP1 and ADGRG1 genes.

BACKGROUND: Population diversity is important and rare disease isolates can frequently reveal novel homozygous or biallelic mutations that lead to expanded clinical heterogeneity, with diverse clinical presentations. METHODS: The present study describes two consanguineous families with a total of seven affected individuals suffering from a clinically similar severe syndromic neurological disorder, with abnormal development and central nervous system (CNS) and peripheral nervous system (PNS) abnormalities. Whole exome sequencing (WES) and Sanger sequencing followed by 3D protein modeling was performed to identify the disease-causing gene. RNA was extracted from the fresh blood of both families affected and healthy individuals. RESULTS: The families were clinically assessed in the field in different regions of Khyber Pakhtunkhwa. Magnetic resonance imagining was obtained in the probands and blood was collected for DNA extraction and WES was performed. Sanger sequencing confirmed a homozygous, likely pathogenic mutation (GRCh38: chr17:42684199G>C; (NM_003632.3): c.333G>C);(NP_003623.1): p.(Trp111Cys) in the CNTNAP1 gene in family A, previously associated with Congenital Hypo myelinating Neuropathy 3 (CHN3; OMIM # 618186) and a novel nonsense variant in family B, (GRCh38: chr16: 57654086C>T; NC_000016.10 (NM_001370440.1): c.721C>T); (NP_001357369.1): p.(Gln241Ter) in the ADGRG1 gene previously associated with bilateral frontoparietal polymicrogyria (OMIM # 606854); both families have extended CNS and PNS clinical manifestations. In addition, 3D protein modeling was performed for the missense variant, p.(Trp111Cys), identified in the CNTNAP1, suggesting extensive secondary structure changes that might lead to improper function or downstream signaling. No RNA expression was observed in both families affected and healthy individuals hence showing that these genes are not expressed in blood. CONCLUSIONS: In the present study, two novel biallelic variants in the CNTNAP1 and ADGRG1 genes in two different consanguineous families with a clinical overlap in the phenotype were identified. Thus, the clinical and mutation spectrum is expanded to provide further evidence that CNTNAP1 and ADGRG1 are very important for widespread neurological development.

A novel de novo variant in the PHF21A causes craniofacial abnormalities, intellectual disability and skeletal manifestations.

IDDBCS is a heterogeneous genetic syndrome with diverse clinical features including Intellectual disability and epilepsy. Using WES, Sanger sequencing, we identified a novel nonsense variant in the PHF21A gene responsible for IDDBCS syndrome. The patient has diverse and overlapping clinical phenotypes. The identified variant leads to abnormal secondary and tertiary structure of the protein and, consequently, affects its function.

Analysis of a non-lethal biallelic frameshift mutation in ZMPSTE24 reveals utilization of alternative translation initiation codons.

Restrictive dermopathy (RD) is a lethal condition caused by biallelic loss-of-function mutations in ZMPSTE24, whereas mutations preserving residual enzymatic activity of the ZMPSTE24 protein lead to the milder mandibuloacral dysplasia with type B lipodystrophy (MADB) phenotype. Remarkably, we identified a homozygous, presumably loss-of-function mutation in ZMPSTE24 [c.28_29insA, p.(Leu10Tyrfs*37)] in two consanguineous Pakistani families segregating MADB. To clarify how lethal consequences are prevented in affected individuals, functional analysis was performed. Expression experiments supported utilization of two alternative translation initiation sites, preventing complete loss of protein function consistent with the relatively mild phenotypic outcome in affected patients. One of these alternative start codons is newly formed at the insertion site. Our findings indicate that the creation of new potential start codons through N-terminal mutations in other disease-associated genes should generally be taken into consideration in the variant interpretation process.

AMFR dysfunction causes autosomal recessive spastic paraplegia in human that is amenable to statin treatment in a preclinical model.

Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins.

Determinants of women's empowerment in Pakistan: evidence from Demographic and Health Surveys, 2012-13 and 2017-18.

BACKGROUND: Women's empowerment has always remained a contested issue in the complex socio-demographic and cultural milieu of Pakistani society. Women are ranked lower than men on all vital human development indicators. Therefore, studying various determinants of women's empowerment is urgently needed in the Pakistani context. METHODS: The study empirically operationalized the concept of women's empowerment and investigated its determinants through representative secondary data taken from the Pakistan Demographic and Health Surveys among women at reproductive age (15-49 years) in 2012-13 (n = 13,558) and 2017-18 (n = 15,068). The study used simple binary logistic and multivariable regression analyses. RESULTS: The results of the binary logistic regression highlighted that almost all of the selected demographic, economic, social, and access to information variables were significantly associated with women's empowerment (p < 0.05) in both PDHS datasets. In the multivariable regression analysis, the adjusted odds ratios highlighted that reproductive-age women in higher age groups having children, with a higher level of education and wealth index, involved in skilled work, who were the head of household, and had access to information were reported to be more empowered. Results of the multivariable regression analysis conducted separately for two empowerment indicators (decision-making and ownership) corroborated the findings of the one indicator of women empowerment, except where ownership did not appear to be significantly associated with number of children and sex of household head in both data sets (2012-13 and 2017-18). CONCLUSIONS: A number of social, economic, demographic, familial, and information-exposure factors determine women's empowerment. The study proposes some evidence-based policy options to improve the status of women in Pakistan.

Identification of a novel biallelic missense variant in the KIAA0825 underlies postaxial polydactyly type A.

Postaxial polydactyly (PAP) is characterized by development of extra digits, which mostly segregates in autosomal recessive pattern. The underlying genetic cause of recessive non-syndromic PAP type A has been associated with sequence variants in five different genes (ZNF141, IQCE, GLI1, FAM92A, KIAA0825). The present study was aimed to investigate clinical and genetic causes of PAPA in a consanguineous family of Pakistani origin. Microsatellite-based linkage analysis was used to search for the disease-causing gene. Linkage in the family was established at chromosome 5q15 harbouring a candidate gene KIAA0825. Subsequently, Sanger sequencing revealed a novel homozygous missense variant [c.50T>C; p. (Leu17Ser)] in the gene, which co-segregated with the disease within the family. Protein structural analysis predicted a substantial change in the secondary structure of the mutant protein affecting its function. This is the third disease causing variant identified in the KIAA0825. This has not only expanded spectrum of the mutations in the gene but also further substantiated its role in the limb development in human.

Correction to: Homozygous missense variant in the TTN gene causing autosomal recessive limb-girdle muscular dystrophy type 10.

Please be advised that following publication of the original article [1], the authors have identified the following errors with the scientific content.

Homozygous missense variant in the TTN gene causing autosomal recessive limb-girdle muscular dystrophy type 10.

BACKGROUND: Limb-girdle muscular dystrophies (LGMDs) are large group of heterogeneous genetic diseases, having a hallmark feature of muscle weakness. Pathogenic mutations in the gene encoding the giant skeletal muscle protein titin (TTN) are associated with several muscle disorders, including cardiomyopathy, recessive congenital myopathies and limb-girdle muscular dystrophy (LGMD) type10. The phenotypic spectrum of titinopathies is expanding, as next generation sequencing (NGS) technology makes screening of this large gene possible. AIM: This study aimed to identify the pathogenic variant in a consanguineous Pakistani family with autosomal recessive LGMD type 10. METHODS: DNA from peripheral blood samples were obtained, whole exome sequencing (WES) was performed and several molecular and bioinformatics analysis were conducted to identify the pathogenic variant. TTN coding and near coding regions were further amplified using PCR and sequenced via Sanger sequencing. RESULTS: Whole exome sequencing analysis revealed a novel homozygous missense variant (c.98807G > A; p.Arg32936His) in the TTN gene in the index patients. No heterozygous individuals in the family presented LGMD features. The variant p.Arg32936His leads to a substitution of the arginine amino acid at position 32,936 into histidine possibly causing LGMD type 10. CONCLUSION: We identified a homozygous missense variant in TTN, which likely explains LGMD type 10 in this family in line with similar previously reported data. Our study concludes that WES is a successful molecular diagnostic tool to identify pathogenic variants in large genes such as TTN in highly inbred population.

Genetic study of Khyber-Pukhtunkhwa resident Pakistani families presenting primary microcephaly with intellectual disability.

OBJECTIVE: To investigate the genetic factor responsible for causing microcephaly and determine allelic heterogeneity of Abnormal spindle microtubule gene. METHODS: The genetic study was conducted at the Kohat University of Science and Technology, Kohat, and Gomal University, D.I.Khan, Pakistan, during 2017-18, and comprised 5 consanguineous families from South Waziristan, Kurram Agency, Karak, Bannu and Dera Ismail Khan regions of the country's Khyber Pakhtukhwa province. Blood samples from all available and cooperative family members (including normal and affected) were obtained, and molecular analysis was carried out through whole genome single nucleotide polymorphisms genotyping, exome sequencing and Sanger sequencing. RESULTS: Of the 15 patients, 9(60%) were males and 6(40%) were females. Genetic mapping revealed linkage to the MCPH5 locus which harbours the microcephaly-associated abnormal spindle-like microcephaly gene. Mutation analysis of the gene identified missense mutation c.3978G>A (p.Trp1326*) in families A, B and C, a deletion mutation c.7782_7783delGA (p.(Lys2595Serfs*6)) in family D, and a splice site defect c.2936+5G>A in family E. CONCLUSIONS: There was suggestion of strong founder effect of mutation c.3978G>A (p.Trp1326*).

Role of secondary metabolites in plant defense against pathogens.

Pathogens get entry into host cell, reproduce there and use biological machinery of host plants which is threat to global crop production. Integrated management strategies based upon minimizing population and use of resistant cultivars can address this potential problem. In developing world farmers are less likely to adopt these approaches instead they prefer the use of chemical pesticides. Reckless use of chemical pesticides is destroying our ecosystem. That's why it is required to explore ecofriendly alternatives, like plant based metabolites to control pathogens. Studies conducted on different plant-metabolites reported that these metabolite can potentially combat plant pathogens. In this study we have also discussed some of plant secondary metabolites including alkaloids, flavonoids and phenolics. In this review we tried to highlight the new trends in utilizing secondary metabolites for controlling bacterial, viral and fungal pathogens with the hope that upcoming drugs will be human and ecosystem friendly.

Long non-coding RNAs as molecular players in plant defense against pathogens.

Long non-coding RNAs (lncRNAs) has significant role in of gene expression and silencing pathways for several biological processes in eukaryotes. lncRNAs has been reported as key player in remodeling chromatin and genome architecture, RNA stabilization and transcription regulation, including enhancer-associated activity. Host lncRNAs are reckoned as compulsory elements of plant defense. In response to pathogen attack, plants protect themselves with the help of lncRNAs -dependent immune systems in which lncRNAs regulate pathogen-associated molecular patterns (PAMPs) and other effectors. Role of lncRNAs in plant microbe interaction has been studied extensively but regulations of several lncRNAs still need extensive research. In this study we discussed and provide as overview the topical advancements and findings relevant to pathogen attack and plant defense mediated by lncRNAs. It is hoped that lncRNAs would be exploited as a mainstream player to achieve food security by tackling different plant diseases.

Manual Vacuum Aspiration (MVA) - A safe option for evacuation of first trimester miscarriage in cardiac patients.

This case series was done at Armed Forces Institute of Cardiology, National Institute of Heart Disease, Rawalpindi, to observe safety and efficacy of manual vacuum aspiration and frequency of complications in cardiac patients with missed abortion. All cardiac patients presenting in first trimester with diagnosed early foetal demise (missed miscarriage) or incomplete miscarriage were included. Manual vacuum aspiration was done as an outpatient procedure. Cardiac and procedure related complications including arrhythmias, thromboembolism, heart failure and ischaemia were noted. A total of 34 patients were enrolled. Mean age and parity was 25.9±2.25 years and 1.18±1.02. Mitral valve was the dominant valve involved in 20(58.8%) followed by double valve replacement in 5(14.7%), dilated cardiomyopathy 4(11.76%), aortic valve involvement in 3(8.8%) and supra ventricular tachycardia in 2(5.9%) patients. Complete evacuation was achieved in 100% patients and there were no major cardiac or gynaecological complications except arrhythmia in 1(2.9%) patient. MVA seems to be a safe and cost effective intervention compared to other modalities of miscarriage management even in high risk cardiac patients.

Circumstances leading to intimate partner violence against women married as children: a qualitative study in Urban Slums of Lahore, Pakistan.

BACKGROUND: Child marriage (<18 years) is prevalent in Pakistan which is associated with negative health outcomes including intimate partner violence (IPV). Our aim is to describe the types and circumstances of IPV against women who were married as children in urban slums of Lahore, Pakistan. METHODS: Women of reproductive age (15-49 years) who were married prior to 18 years, for at least 5 years were recruited from most populous slum areas of Lahore, Pakistan. Themes for the interview guide were developed using published literature and everyday observations of the researchers. Interviews were conducted by trained interviewers in Urdu language and were translated into English. The interviews were tape-recorded, transcribed, analyzed and categorized into themes. RESULTS: All 19 participants were married between 11 and 17 years. Most respondents were uneducated, poor and were working as housemaids. Majority of participants experienced verbal abuse, and threatened, attempted and completed physical violence by their husbands. A sizeable number of women reported unwanted sexual encounters by their husbands. Family affairs particularly issues with in-laws, poor house management, lack of proper care of children, bringing insufficient dowry, financial problems, an act against the will of husband, and inability to give birth to a male child were some of the reasons narrated by the participants which led to IPV against women. CONCLUSIONS: Women married as children are vulnerable to IPV. Concerted efforts are needed from all sectors of society including academia, public health experts, policy makers and civil society to end the child marriage practice in Pakistan.

Knowledge and attitude towards child marriage practice among women married as children-a qualitative study in urban slums of Lahore, Pakistan.

BACKGROUND: Child marriage (<18 years) is prevalent in Pakistan which is associated with negative health outcomes. Our aim is to describe women's knowledge and attitude towards child marriage practice who themselves were married as children. METHODS: Women of reproductive age (15-49 years) who were married prior to 18 years, for at least 5 years and had at least one child birth were recruited from most populous slum areas of Lahore, Pakistan. Themes for the interview were developed using published literature and everyday observations of the researchers. Interviews were conducted by trained interviewers in Urdu language and were translated into English. The interviews were tape-recorded, transcribed, analyzed and categorized into themes. RESULTS: Nineteen of 20 participants who agreed to participate were married between 11-17 years. Most respondents were uneducated, poor and were working as housemaids. The majority participants were unaware of the negative health outcomes of child marriages. They appeared satisfied by the decision of their parents of marrying them before 18 years, and even condemned banning child marriages in Pakistan. Strong influence of culture and community perceptions, varying interpretation of religion, and protecting family honor are some of the reasons that were narrated by the participants, which seems playing a role in continuation of child marriage practice in Pakistan. CONCLUSION: Raising awareness of the negative health outcomes of child marriage, implementing and enforcing strict laws against child marriage practice, promoting civil, sexual and reproductive health rights for women, can help eliminate child marriages in Pakistan.

Stability Analysis of SARS-CoV-2 with Heart Attack Effected Patients and Bifurcation.

The aim of this study is to analyze and investigate the SARS-CoV-2 (SC-2) transmission with effect of heart attack in United Kingdom with advanced mathematical tools. Mathematical model is converted into fractional order with the help of fractal fractional operator (FFO). The proposed fractional order system is investigated qualitatively as well as quantitatively to identify its stable position. Local stability of the SC-2 system is verified and test the proposed system with flip bifurcation. Also system is investigated for global stability using Lyponove first and second derivative functions. The existence, boundedness, and positivity of the SC-2 is checked which are the key properties for such of type of epidemic problem to identify reliable findings. Effect of global derivative is demonstrated to verify its rate of effects of heart attack in united kingdom. Solutions for fractional order system are derived with the help of advanced tool FFO for different fractional values to verify the combine effect of COVID-19 and heart patients. Simulation are carried out to see symptomatic as well as a symptomatic effects of SC-2 in the United Kingdom as well as its global effects, also show the actual behavior of SC-2 which will be helpful to understand the outbreak of SC-2 for heart attack patients and to see its real behavior globally as well as helpful for future prediction and control strategies.

Truncated DNM1 variant underlines developmental delay and epileptic encephalopathy.

Background: Developmental and epileptic encephalopathies (DEEs) signify a group of heterogeneous neurodevelopmental disorder associated with early-onset seizures accompanied by developmental delay, hypotonia, mild to severe intellectual disability, and developmental regression. Variants in the DNM1 gene have been associated with autosomal dominant DEE type 31A and autosomal recessive DEE type 31B. Methods: In the current study, a consanguineous Pakistani family consisting of a proband (IV-2) was clinically evaluated and genetically analyzed manifesting in severe neurodevelopmental phenotypes. WES followed by Sanger sequencing was performed to identify the disease-causing variant. Furthermore, 3D protein modeling and dynamic simulation of wild-type and mutant proteins along with reverse transcriptase (RT)-based mRNA expression were checked using standard methods. Results: Data analysis of WES revealed a novel homozygous non-sense variant (c.1402G>T; p. Glu468*) in exon 11 of the DNM1 gene that was predicted as pathogenic class I. Variants in the DNM1 gene have been associated with DEE types 31A and B. Different bioinformatics prediction tools and American College of Medical Genetics guidelines were used to verify the identified variant. Sanger sequencing was used to validate the disease-causing variant. Our approach validated the pathogenesis of the variant as a cause of heterogeneous neurodevelopmental disorders. In addition, 3D protein modeling showed that the mutant protein would lose most of the amino acids and might not perform the proper function if the surveillance non-sense-mediated decay mechanism was skipped. Molecular dynamics analysis showed varied trajectories of wild-type and mutant DNM1 proteins in terms of root mean square deviation, root mean square fluctuation and radius of gyration. Similarly, RT-qPCR revealed a substantial reduction of the DNM1 gene in the index patient. Conclusion: Our finding further confirms the association of homozygous, loss-of-function variants in DNM1 associated with DEE type 31B. The study expands the genotypic and phenotypic spectrum of pathogenic DNM1 variants related to DNM1-associated pathogenesis.

Loss-of-function variant in the LRR domain of SLITRK2 implicated in a neurodevelopmental disorder.

Background: Neurodevelopmental disorders are characterized by different combinations of intellectual disability (ID), communication and social skills deficits, and delays in achieving motor or language milestones. SLITRK2 is a postsynaptic cell-adhesion molecule that promotes neurite outgrowth and excitatory synapse development. Methods and Results: In the present study, we investigated a single patient segregating Neurodevelopmental disorder. SLITRK2 associated significant neuropsychological issues inherited in a rare X-linked fashion have recently been reported. Whole-exome sequencing and data analysis revealed a novel nonsense variant [c.789T>A; p.(Cys263*); NM_032539.5; NP_115928.1] in exon 5 of the SLITRK2 gene (MIM# 300561). Three-dimensional protein modeling revealed substantial changes in the mutated SLITRK2 protein, which might lead to nonsense-medicated decay. Conclusion: This study confirms the role of SLITRK2 in neuronal development and highlights the importance of including the SLITRK2 gene in the screening of individuals presenting neurodevelopmental disorders.

A novel homozygous truncating variant in PPFIBP1 further delineates PPFIBP1-associated neurodevelopmental disorder.

Neurodevelopmental disorders (NDDs) are classified as a group of disorders affecting function and development of the brain and having wide clinical variability. Herein, we describe two affected individuals segregating a recessive NDD. The affected individuals exhibited phenotypes such as global developmental delay (GDD), intellectual disability (ID), microcephaly and speech delay. Whole-exome sequencing (WES) followed by bidirectional Sanger sequencing techniques identified a homozygous nonsense variant (c.466C > T; p.Gln156*) in the PPFIBP1 gene (NM_003622.4) that segregated with the disease phenotype. Further, to elucidate the effect of the variant on protein structure, 3D protein modelling was performed for the mutant and normal protein that suggested substantial reduction of the mutant protein. Our data support the evidence that PPFIBP1 has a pivotal role in neurodevelopment in humans, and loss-of-function variants cause clinically variable neurodevelopmental phenotypes.

A GHRHR founder mutation causes isolated growth hormone deficiency type IV in a consanguineous Pakistani family.

Background: Isolated growth hormone deficiency (IGHD) is caused by a severe shortage or absence of growth hormone (GH), which results in aberrant growth and development. Patients with IGHD type IV (IGHD4) have a short stature, reduced serum GH levels, and delayed bone age. Objectives: To identify the causative mutation of IGHD in a consanguineous family comprising four affected patients with IGHD4 (MIM#618157) and explore its functional impact in silico. Methods: Clinical and radiological studies were performed to determine the phenotypic spectrum and hormonal profile of the disease, while whole-exome sequencing (WES) and Sanger sequencing were performed to identify the disease-causing mutation. In-silico studies involved protein structural modeling and docking, and molecular dynamic simulation analyses using computational tools. Finally, data from the Qatar Genome Program (QGP) were screened for the presence of the founder variant in the Qatari population. Results: All affected individuals presented with a short stature without gross skeletal anomalies and significantly reduced serum GH levels. Genetic mapping revealed a homozygous nonsense mutation [NM_000823:c.G214T:p.(Glu72*)] in the third exon of the growth-hormone-releasing hormone receptor gene GHRHR (MIM#139191) that was segregated in all patients. The substituted amber codon (UAG) seems to truncate the protein by deleting the C-terminus GPCR domain, thus markedly disturbing the GHRHR receptor and its interaction with the growth hormone-releasing hormone. Conclusion: These data support that a p.Glu72* founder mutation in GHRHR perturbs growth hormone signaling and causes IGHD type IV. In-silico and biochemical analyses support the pathogenic effect of this nonsense mutation, while our comprehensive phenotype and hormonal profiling has established the genotype-phenotype correlation. Based on the current study, early detection of GHRHR may help in better therapeutic intervention.