Expanding OBSL1 Mutation Phenotype: Disproportionate Short Stature, Barrel Chest, Thoracic Kyphoscoliosis, Hypogonadism, and Hypospadias.

We present a Pakistani kinship afflicted with a syndrome with features including short stature, reduced sitting height, orofacial symptoms including prominent forehead and thick eyebrows, short and broad thorax, and variable features such as long philtrum, short broad neck, barrel chest, thoracic kyphoscoliosis, hypogonadism, and hypospadias. Phenotypic variation even within different sibships was considerable. The unique combination of the phenotypic characteristics prompted us to determine the shared homozygosity regions in patient genomes and the pathogenic variants by next generation technologies like single nucleotide polymorphism (SNP) genotyping and whole exome sequencing (WES). Through these analyses, we detected homozygous OBSL1 c.848delG (p.Gly283AlafsTer54) as the causal variant. Biallelic variants in OBSL1 are known to cause Three M Syndrome 2 (3M2), a rare disorder of growth retardation with characteristic facial dysmorphism and musculoskeletal abnormalities. Affected members of the family do not have the 3M2 hallmark features of dolichocephaly, hypoplastic midface, anteverted nares, low nasal bridge, pectus excavatum, sacral hyperlordosis, spina bifida occulta, anterior wedging of thoracic vertebrae, prominent heels, and prominent talus. Moreover, they have some variable features not typical for the syndrome such as round face, disproportionate short stature, barrel chest, thoracic kyphoscoliosis, hypogonadism, and hypospadias. Our study facilitated genetic diagnosis in the family, expanded the clinical phenotype for 3M2, and unraveled the considerable clinical variation within the same kinship. We conclude that unbiased molecular analyses such as WES should be more integrated into healthcare, particularly in populations with high parental consanguinity, given the potential of such analyses to facilitate diagnosis.

A Recurrent Mutation in Growth Hormone Receptor (GHR) Gene Underlying Laron-type Dwarfism in a Pakistani Family.

Laron syndrome (LS) is a rare autosomal recessively segregating disorder of severe short stature. The condition is characterized by short limbs, delayed puberty, hypoglycemia in infancy, and obesity. Mutations in growth hormone receptor (GHR) have been implicated in LS; hence, it is also known as growth hormone insensitivity syndrome (MIM-262500). Here we represent a consanguineous Pakistani family in which three siblings were afflicted with LS. Patients had rather similar phenotypic presentations marked with short stature, delayed bone age, limited extension of elbows, truncal obesity, delayed puberty, childish appearance, and frontal bossing. They also had additional features such as hypo-muscularity, early fatigue, large ears, widely-spaced breasts, and attention deficit behavior, which are rarely reported in LS. The unusual combination of the features hindered a straightforward diagnosis and prompted us to first detect the regions of shared homozygosity and subsequently the disease-causing variant by next generation technologies, like SNP genotyping and exome sequencing. A homozygous pathogenic variant c.508G>C (p.(Asp170His)) in GHR was detected. The variant is known to be implicated in LS, supporting the molecular diagnosis of LS. Also, we present detailed clinical, hematological, and hormonal profiling of the siblings.

Homozygous deletion of MYADML2 in cranial asymmetry, reduced bone maturation, multiple dislocations, lumbar lordosis, and prominent clavicles.

A null mutation in a patient can facilitate phenotype assignment and uncovers the function of that specific gene. We present five sibs of a consanguineous Pakistani family afflicted with a new syndrome with an unusual combination of skeletal anomalies including cranial asymmetry, fused sagittal sutures deviating from the medial axis, mandibular prognathia, maxillary hypoplasia, misaligned and crowded teeth, delayed bone age, multiple dislocations, hypoplastic and malpositioned patellae, humeral intracondylar fissures, scapular dyskinesis, long limbs, lumbar lordosis, protruding chest, prominent clavicles, short 5th digital rays, and ventral transverse digital creases plus features of cutis laxa. We mapped the disease gene locus to a 3.62-Mb region at 17q25.3 and identified a homozygous deletion of maximal 7.3 kb deduced to totally inactivate MYADML2 and downstream gene PYCR1, biallelic variants in which cause autosomal recessive cutis laxa (ARCL). All five affected sibs had the most common features of ARCL but not many of the less common ones. We attributed the anomalies not typical for ARCL to MYADML2 deficit, because no other genetic defect possibly a candidate to underlie the skeletal phenotype was found. MYADML2 is a gene of unknown function, has not been studied, and has not been associated with disease. Our findings present a possible phenotype for MYADML2 deficit that includes impaired bone patterning and maturation, definitely show that the gene is not essential for survival, and provide a start point for future studies on the function of MYADML2 protein. Detection of new patients is needed to confirm and delineate MYADML2-deficiency phenotype.

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.

Homozygous CHST11 mutation in chondrodysplasia, brachydactyly, overriding digits, clino-symphalangism and synpolydactyly.

BACKGROUND: Carbohydrate sulfotransferase 11 (CHST11) is a membrane protein of Golgi that catalyses the transfer of sulfate to position 4 of the N-acetylgalactosamine residues of chondroitin. Chondroitin sulfate is the predominant proteoglycan in cartilage, and its sulfation is important in the developing growth plate of cartilage. A homozygous deletion encompassing part of the gene and the embedded miRNA MIR3922 had been detected in a woman with hand/foot malformation and malignant lymphoproliferative disease. Chst11-deficient mouse has severe chondrodysplasia, congenital arthritis and neonatal lethality. We searched for the causative variant for the unusual combination of limb malformations with variable expressivity accompanied by skeletal defects in a consanguineous Pakistani kindred. METHODS: We performed detailed clinical investigations in family members. Homozygosity mapping using SNP genotype data was performed to map the disease locus and exome sequencing to identify the underlying molecular defect. RESULTS: The limb malformations include brachydactyly, overriding digits and clino-symphalangism in hands and feet and syndactyly and hexadactyly in feet. Skeletal defects include scoliosis, dislocated patellae and fibulae and pectus excavatum. The disease locus is mapped to a 1.6 Mb region at 12q23, harbouring a homozygous in-frame deletion of 15 nucleotides in CHST11. Novel variant c.467_481del (p.L156_N160del) is deduced to lead to the deletion of five evolutionarily highly conserved amino acids and predicted as damaging to protein by in silico analysis. Our findings confirm the crucial role of CHST11 in skeletal morphogenesis and show that CHST11 defects have variable manifestations that include a variety of limb malformations and skeletal defects.

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.

Assessment of risk factors associated with outbreak of hepatitis A in Shakrial, Rawalpindi, Pakistan.

INTRODUCTION: Two patients with jaundice reported to the National Institute of Health (NIH), Islamabad from Shakrial, Rawalpindi in April 2017. An outbreak investigation team was formulated to assess the disease magnitude, risk factors and control measures. METHODOLOGY: A case-control study was conducted in 360 houses in May 2017. The case definition was: onset of acute jaundice with any symptom including fever, right upper-quadrant pain, loss of appetite, dark urine, nausea and vomiting among Shakrial residents from March 10 - May 19, 2017. Four age and gender matched controls were selected for each case. Blood samples were sent to the NIH for laboratory confirmation. Frequencies, attack rates (AR), odd ratios, and logistic regression were computed at 95% confidence interval and p < 0.05. RESULTS: A total of 25 cases (23 new) were identified with mean age 8 years and male to female ratio 1.5:1. Overall AR was 1.39% and the most severely affected age-group was 5-10 years (AR of 3.92%). Multivariate analysis revealed that raw vegetable consumption, lack of awareness and poor handwashing practices had significant association with disease spread. All blood samples were positive for hepatitis A, and no resident was previously vaccinated. Lack of awareness of disease spread among the community was the most probable reason for the outbreak. There were no new cases during follow up until May 30, 2017. CONCLUSIONS: Healthcare departments should implement public policies towards the management of hepatitis A in Pakistan. Health awareness sessions and vaccination for children ≤ 16 years age is recommended.

A Two-Base Pair Deletion in IQ Repeats in ASPM Underlies Microcephaly in a Pakistani Family.

Aims: Autosomal recessive primary microcephaly (MCPH) is a clinically rare and genetically highly heterogeneous developmental disorder. Biallelic variants in the abnormal spindle-like microcephaly-associated (ASPM) gene account for 40% to 68% of all MCPH cases. This study was designed to elucidate the genetic basis of MCPH in an extended family. To highlight recurrent mutations useful in implementing genetic testing programs, we further aimed to carry out a descriptive review of the reported ASPM mutations. Materials and Methods: A large inbred kindred with seven affected members was investigated, and detailed clinical and behavioral assessments were carried out. Single nucleotide polymorphism (SNP)-based homozygosity mapping and exome sequencing were performed. Results: Affected individuals had characteristic features, including small head, receding forehead, mild to moderate intellectual disability, developmental delay, short stature, apraxia, and behavioral anomalies. We mapped the disease gene locus and detected a rare frameshift deletion c.6854_6855del (p.(Leu2285GlnfsTer32)) in exon 18 of ASPM. A total of 215 mutations in ASPM have been reported in at least 453 families, nearly 50% of which are of Pakistani origin. These mutations can be classified as recurrent, founder or private in Pakistani and other populations. Conclusion: SNP-based homozygosity mapping and exome sequencing are essential in delineating the genetically distinct microcephaly types. The highlighted recurrent mutations in ASPM could be useful in implementing genetic testing programs for MCPH.

Prevalence of different tick species on livestock and associated equines and canine from different agro-ecological zones of Pakistan.

Ticks are ectoparasites that act as vectors for transmission of various pathogens to wild and domesticated animals and pose a serious threat to human health. Because of the hot and humid conditions in different agro-ecological zones of Pakistan, ticks are abundant and parasitize a variety of animals. The aim of this study was to identify different tick species and distribution on different hosts especially livestock, such as sheep, goat, cattle, buffalo, and camel, and livestock associated canines and equines, such as horse, donkey, and dog, across different agro-ecological zones of Pakistan. The ticks samples were collected and morphologically identified at genus and species level using morphological keys under stereomicroscope. A total of 2,846 animals were examined for the tick infestation, and 408 animals were tick-infested. Eleven tick species belonging to 4 genera were identified: Hyalomma anatolicum, Hyalomma scupense, Hyalomma dromedarii, Hyalomma isaaci, Rhipicephalus microplus, Rhipicephalus haemaphysaloides, Rhipicephalus turanicus, Haemaphysalis cornupunctata, Haemaphysalis montgomeryi, Haemaphysalis bispinosa, and Ixodes kashmiricus. The overall tick prevalence was 14.3%; host-wise infestation rate was 12.2% in sheep; 12.6%, goat; 11.7%, buffalo; 11.7%, cattle; 19.6%, camel; 27.4%, donkey; 23.5%, horse; and 24.3%, dog. Tick infestation of different animals differed on the basis of the zones. Camels showed the highest tick infestation rate in zones 1 and 2 (21.4 and 26.7%, respectively), whereas donkeys showed the highest infestation rate in zones 3, 4, 6, and 7 (25, 39.3, 3.3, and 21.4%, respectively). The infestation rates of Hyalomma and Rhipicephalus were the highest in zone 2 (71.4 and 52.9%, respectively). The infestation rate of Hyalomma was the highest (47.4%) in sheep; Haemaphysalis (46.9%), goat; Rhipicephalus (69.7%), buffalo; Rhipicephalus (62.3%), cattle; Hyalomma (70%), camel; Ixodes (60.9%), donkey; Ixodes (75%), horse; and Rhipicephalus (61.1%), dog. This study showed the diversity and infestation rate of different ticks with respect to their hosts and agro-ecological zones of Pakistan. High tick burdens and infestation rates are responsible for the spread of different tick-borne infections, resulting in loss of animal productivity and posing a threat to animal and human health. Understanding different tick species and their distribution across different zones will be helpful for developing efficient control strategies against different tick born infections.

CRADD and USP44 mutations in intellectual disability, mild lissencephaly, brain atrophy, developmental delay, strabismus, behavioural problems and skeletal anomalies.

In a consanguineous Pakistani kinship afflicted with mild to moderate intellectual disability (ID), mild lissencephaly, brain atrophy and skeletal anomalies, we detected homozygous CRADD c.2T > G (p.Met1?) and USP44 c.873_886delinsT (p.Leu291Phefs*8), two good candidates 1.85-Mb apart that segregated with the disorder. Biallelic damaging variants in CRADD cause recessive mental retardation-34 (MRT34; MIM 614499) with mild to moderate ID, "thin" lissencephaly, and variable megalencephaly and seizures. For USP44, only a single ID family has been reported with a homozygous deleterious variant, which is the same as the variant we detected. In affected individuals we present, at ages 29-32 years, clinical findings are similar yet not fully concordant with phenotypes for either gene considering the skeletal findings, and ID is not as severe as would be expected for defects in two genes with additive effect. Some variable CRADD-related features such as language impairment and seizures are not observed in the presented family. The presence of the two variants in the family is a very rare example of familial linked homozygous variants, and whether the damaging USP44 variant contributed to the disease in the family we present is not clear. As for the skeletal findings, facial dysmorphism and digestive problems, we did not find a candidate variant. This study is an example of both clinical variation and difficulty in variant detection and evaluation. Our findings highlight that even an extensive exome sequence analysis can fail to fully uncover the complex molecular basis of a syndrome even if potentially causative variants are identified.

Demographic Attributes of Knowledge, Attitude, Practices, and One Health Perspective Regarding Diarrhea in Pakistan.

Background: Loose bowels is a clinical sign of gastrointestinal transport channel proteins, channels, and physical and chemical boundaries being harmed, prompting issues of water and electrolyte transport in the intestinal system. It is still considered as a major reason for emergency visits to hospitals in low-middle income countries. Zinc is a suitable treatment along with ORS for diarrhea. KAP surveys are usually conducted to collect information about general or specific topics of a particular population. The objective of this study was to investigate the knowledge, attitude, practices (KAP), and one health perspective regarding diarrhea among the participants from urban and rural populations of Rawalpindi and Islamabad, Pakistan. Methods: Data was collected by conducting a survey among residents of twin cities over a period of 6 months (from July 2020 to December 2020). The questionnaire compromised socio-demographic features and the degree of KAP with respect to diarrhea management and control. One way ANOVA tests were applied to observe the demographic relationship and various factors influencing knowledge, attitude, practices, and one health perspective about diarrhea. Results: A total of 338 subjects participated in the study. Female subjects were in the majority with 63% while the rest were male. A majority of the participants were between 15-25 years of age and 79.6% participants were un-married. The leading ethnic group was Punjabi with 52.7%; the lowest ethnic group were of Sindhi ethnicity with 8.6%. Age has a significant association with respect to knowledge and attitude. Religion has a significant association with respect to knowledge, practices, and one health, while education/qualification has an association with knowledge. The rest of the variables found no association with each other. Conclusion: It is concluded from the recent study that most residents of the twin cities of Pakistan knew about diarrhea and had a good attitude and practices toward it. Age, religion, and education have different roles regarding different diseases in the population of Pakistan. The current study has its limitations as well. Parts of the study were conducted in the capital of Pakistan which is more developed as compared to other areas of Pakistan. It would be better to explore the remote areas of Pakistan where basic amenities of life such as education, wealth, and unemployment are not available. It is important to create more awareness among community members. They should be aware how dangerous these viruses and bacteria can be. Other parts of Pakistan should also be explored for better understanding that will help in making a nationwide health policy.

Community Based Assessment of Behavior and Awareness of Risk Factors of Cystic Echinococcosis in Major Cities of Pakistan: A One Health Perspective.

Background: The parasitic disease, cystic echinococcosis (CE), is a serious health problem in Pakistan. Risk of disease transmission is increased by economic and political instability, poor living conditions, and limited awareness of hygienic practices. The current study aimed to investigate the community perception and awareness regarding the risk factors of CE in Pakistan, from a One Health perspective. Methods: We conducted a community-based survey involving 454 participants in the major cities of Pakistan. Quantitative data based on knowledge, attitude, and practices (KAP), the One Health concept, risk factors, and community perception of CE among the general population of the major cities of Pakistan were collected. The questions included those related to knowledge, attitude, practices, One Health concept, risk factors, and community perception. The Chi-squared test was applied to determine the associations regarding KAPs across socio-demographic parameters. Results: KAPs had no significant associations with sociodemographic aspects such as age, sex, religion, ethnicity, education, marital status, occupation, or financial status of the participants. The findings indicated a lack of awareness about CE among the participants. Respondents were unaware of the risk factors and the One Health concept of CE. However, the community attitude and perception were positive toward the control of CE. Conclusion: Illiteracy, deficient sanitation systems and lack of awareness are the contributing factors to CE in Pakistan. It is necessary to make the community aware regarding CE and its importance. Increasing this awareness represents an important step toward the eradication and control of CE.

A founder RDH5 splice site mutation leads to retinitis punctata albescens in two inbred Pakistani kindreds.

Background: Retinitis punctate albescens (RPA) is a rare form of retinal dystrophy characterized by congenital stationary night blindness and a characteristic fundus appearance. Missense or nonsense mutations in RDH5 in homozygous or heterozygous state have been implicated in RPA.Material and methods: Two consanguineous Pakistani kindreds with the highly variable manifestation of RPA were studied. Whole-exome sequencing was applied to the index subjects in both families. Sanger sequencing of the candidate RDH5 variant was carried out. Pathogenicity of the detected variant was assessed through bioinformatics tools.Results: The ophthalmic examination through full-field electroretinogram of affected patients in both families was consistent with RPA. A novel splice donor variant at the first exon/intron boundary of RDH5 (NM_002905.3: c.-33 + 2dup) segregated in recessive fashion with the clinical phenotype in both families. One of the heterozygous variant carriers was also observed to have a milder expression of retinal flecks. Haplotype analysis surrounding the splice variant and pattern of runs of homozygosity were suggestive of common ancestry in these families.Conclusion: This is the first report of any pathogenic splice variant at first exon/intron boundary implicated in RPA and suggests another mechanism through which RDH5 variants could be associated with eye phenotype. This study also highlights the importance of a thorough phenotypic evaluation of heterozygous mutation carriers who may exhibit milder symptoms.