An Ancient Harappan Genome Lacks Ancestry from Steppe Pastoralists or Iranian Farmers.

We report an ancient genome from the Indus Valley Civilization (IVC). The individual we sequenced fits as a mixture of people related to ancient Iranians (the largest component) and Southeast Asian hunter-gatherers, a unique profile that matches ancient DNA from 11 genetic outliers from sites in Iran and Turkmenistan in cultural communication with the IVC. These individuals had little if any Steppe pastoralist-derived ancestry, showing that it was not ubiquitous in northwest South Asia during the IVC as it is today. The Iranian-related ancestry in the IVC derives from a lineage leading to early Iranian farmers, herders, and hunter-gatherers before their ancestors separated, contradicting the hypothesis that the shared ancestry between early Iranians and South Asians reflects a large-scale spread of western Iranian farmers east. Instead, sampled ancient genomes from the Iranian plateau and IVC descend from different groups of hunter-gatherers who began farming without being connected by substantial movement of people.

Evolution of system connectivity to support food production in the Indus Basin in Pakistan.

Sustainability challenges related to food production arise from multiple nature-society interactions occurring over long time periods. Traditional methods of quantitative analysis do not represent long-term changes in the networks of system components, including institutions and knowledge that affect system behavior. Here, we develop an approach to study system structure and evolution by combining a qualitative framework that represents sustainability-relevant human, technological, and environmental components, and their interactions, mediated by knowledge and institutions, with network modeling that enables quantitative metrics. We use this approach to examine the water and food system in the Punjab province of the Indus River Basin in Pakistan, exploring how food production has been sustained, despite high population growth, periodic floods, and frequent political and economic disruptions. Using network models of five periods spanning 75 y (1947 to 2022), we examine how quantitative metrics of network structure relate to observed sustainability-relevant outcomes and how potential interventions in the system affect these quantitative metrics. We find that the persistent centrality of some and evolving centrality of other key nodes, coupled with the increasing number and length of pathways connecting them, are associated with sustaining food production in the system over time. Our assessment of potential interventions shows that regulating groundwater pumping and phasing out fossil fuels alters network pathways, and helps identify potential vulnerabilities for future food production.

Towards a World Wide Web without digital inequality.

The World Wide Web (WWW) empowers people in developing regions by eradicating illiteracy, supporting women, and generating economic opportunities. However, their reliance on limited bandwidth and low-end phones leaves them with a poorer browsing experience compared to privileged users across the digital divide. To evaluate the extent of this phenomenon, we sent participants to 56 cities to measure the cost of mobile data and the average page load time. We found the cost to be orders of magnitude greater, and the average page load time to be four times slower, in some locations compared to others. Analyzing how popular webpages have changed over the past years suggests that they are increasingly designed with high processing power in mind, effectively leaving the less fortunate users behind. Addressing this digital inequality through new infrastructure takes years to complete and billions of dollars to finance. A more practical solution is to make the webpages more accessible by reducing their size and optimizing their load time. To this end, we developed a solution called Lite-Web and evaluated it in the Gilgit-Baltistan province of Pakistan, demonstrating that it transforms the browsing experience of a Pakistani villager using a low-end phone to almost that of a Dubai resident using a flagship phone. A user study in two high schools in Pakistan confirms that the performance gains come at no expense to the pages' look and functionality. These findings suggest that deploying Lite-Web at scale would constitute a major step toward a WWW without digital inequality.

The effect of tranexamic acid on postpartum bleeding in women with moderate and severe anaemia (WOMAN-2): an international, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Tranexamic acid, given within 3 h of birth, reduces bleeding deaths in women with postpartum haemorrhage. We examined whether giving tranexamic acid shortly after birth can prevent postpartum haemorrhage in women with moderate or severe anaemia. METHODS: This international, randomised, double-blind, placebo-controlled trial was done in 34 hospitals across four countries (Nigeria, Pakistan, Tanzania, and Zambia). We recruited women of any age in active labour with moderate or severe anaemia (haemoglobin <100 g/L). We randomly assigned women (1:1) who had given birth vaginally to receive 1 g of tranexamic acid or matching placebo by slow intravenous injection (over 10 min) within 15 min of the umbilical cord being cut or clamped. Women were randomly assigned by selection of the lowest numbered treatment pack from a box containing 20 packs that were identical apart from the pack number. Participants, care givers, and those assessing outcomes were masked to group assignment. The primary outcome was a clinical diagnosis of primary postpartum haemorrhage, which might be an estimated blood loss of more than 500 mL or any blood loss sufficient to compromise haemodynamic stability within 24 h of randomisation, analysed on an intention-to-treat basis. Safety analyses were performed in all participants included in the intention-to-treat population. This trial was registered on ISRCTN (ISRCTN62396133), ClinicalTrials.gov (NCT03475342), and the Pan African Clinical Trial Registry (PACTR201909735842379) and is closed to recruitment. FINDINGS: From Aug 24, 2019, to Sept 19, 2023, 16 586 women aged 14-50 years were invited to take part and 1518 were excluded. 7580 women were randomly assigned to receive tranexamic acid and 7488 to receive placebo. Primary outcome data were unavailable for one woman in each group. The median time interval from the start of the administration of the trial treatment to the diagnosis of postpartum haemorrhage was 18·5 min (IQR 5-58); 20 min (8-64) in women with moderate anaemia and 13 min (7-44) in women with severe anaemia. 358 (35%) of 1024 with postpartum haemorrhage for whom time data were available were diagnosed before the trial treatment had been fully administered. Clinically diagnosed postpartum haemorrhage occurred in 530 (7·0%) of 7579 in the tranexamic acid group and in 497 (6·6%) of 7487 in the placebo group (risk ratio [RR] 1·05, 95% CI 0·94-1·19). There was no strong evidence against the null hypothesis of homogeneity of effects for any of the prespecified subgroup analyses: severity of anaemia (p=0·44), antepartum haemorrhage (p=0·044), birth canal trauma (p=0·37), use of pain control (p=0·37), and baseline risk of postpartum haemorrhage (p=0·31). There were no vascular occlusive events (pulmonary embolism, deep vein thrombosis, stroke, and myocardial infarction) reported in either group. There were no adverse events related to the treatment and no treatment-related deaths. INTERPRETATION: In women with moderate and severe anaemia, giving tranexamic acid within 15 min of the umbilical cord being clamped did not reduce the risk of clinically diagnosed postpartum haemorrhage. FUNDING: The Bill & Melinda Gates Foundation and the Wellcome Trust.

Congenital septal defects in Karachi, Pakistan: an update of mutational screening by high-resolution melting (HRM) analysis of MTHFR C677T.

BACKGROUND: Congenital heart defects (CHDs) are the heart structural malformations present at birth. Septal defects account for 40% of CHD, including atrial, ventricular and atrioventricular septal defects. In Pakistan, the prevalence of CHD is 3.4 in 1000, and a study estimated that 60,000 babies are born with CHD annually. Methylenetetrahydrofolate reductase (MTHFR), a chief enzyme, involved in the folate metabolism. The missense mutation, C677T (rs1801133), exists in MTHFR gene, results in a MTHFR thermolabile variant having low enzymatic activity. The study is aim to identify the MTHFR C677T variant association with septal defects. METHODS: Samples of 194 CHD patients (age [Formula: see text]= 5.8 ± 5.1) and 50 normal echo controls (age [Formula: see text]= 6.0 ± 4.9), confirmed by pediatric consultant, were collected. Extracted DNA, quantified by agarose gel electrophoresis and nanodrop, was screened for SNP by high-resolution melting (HRM). Further, HRM results were confirmed using restriction analysis and sequencing. HRM was simply and precisely genotyped the samples within 3 h at low cost. RESULTS: Genotypic data suggested that heterozygous mutant (CT) was frequent in congenital septal defect patients (0.26) which was higher than controls (0.143), p > 0.05. Mutant (TT) genotype was not found in this study. CONCLUSIONS: rs1801133 has lack of significant association with congenital septal defects. The absence of TT genotype in this study suggesting the role of natural selection in targeted population. HRM is an easy, fast and next generation of PCR, which may be used for applied genomics.

Public human microbiome data are dominated by highly developed countries.

The importance of sampling from globally representative populations has been well established in human genomics. In human microbiome research, however, we lack a full understanding of the global distribution of sampling in research studies. This information is crucial to better understand global patterns of microbiome-associated diseases and to extend the health benefits of this research to all populations. Here, we analyze the country of origin of all 444,829 human microbiome samples that are available from the world's 3 largest genomic data repositories, including the Sequence Read Archive (SRA). The samples are from 2,592 studies of 19 body sites, including 220,017 samples of the gut microbiome. We show that more than 71% of samples with a known origin come from Europe, the United States, and Canada, including 46.8% from the US alone, despite the country representing only 4.3% of the global population. We also find that central and southern Asia is the most underrepresented region: Countries such as India, Pakistan, and Bangladesh account for more than a quarter of the world population but make up only 1.8% of human microbiome samples. These results demonstrate a critical need to ensure more global representation of participants in microbiome studies.

Phylodynamic signatures in the emergence of community-associated MRSA.

Community-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 y. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole-genome sequencing data for the Australian sequence type (ST) ST93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australian populations, dissemination of the ST93-MRSA-IV genotype into population centers on the Australian East Coast, and subsequent importation into the highlands of Papua New Guinea and Far North Queensland. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia, and Europe. Surges in Re were observed at the divergence of antibiotic-resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed among drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA in the late 20th century was driven by a combination of antibiotic-resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers.

Two-Year Duration of Immunity of Inactivated Poliovirus Vaccine: A Follow-up Study in Pakistan in 2020.

This was a follow-up study conducted in 2020 assessing changes in levels of type 2 poliovirus-neutralizing antibodies 2 years postimmunization in children who received inactivated poliovirus vaccine (IPV) in Karachi, Pakistan. Unexpectedly, the findings revealed an increase in seroprevalence of type 2 antibodies from 73.1% to 81.6% 1 year and 2 years after IPV, respectively. The increase in type 2 immunity could result from the intensive transmission of circulating vaccine-derived poliovirus type 2 (cVDPV2) in Karachi during the second year of IPV administration. This study suggests that the cVDPV2 outbreak detected in Pakistan infected large proportions of children in Karachi. Clinical Trials Registration . NCT03286803.

Mucosal Immunity to Poliovirus in Children 0-15 Years of Age: A Community-Based Study in Karachi, Pakistan in 2019.

This study assesses poliovirus type 1 (PV1) immunity in children to inform the contribution of mucosal immunity in and prevention of poliovirus circulation. A community-based study was conducted in periurban Karachi, Pakistan. Randomly selected children (0-15 years of age) received oral poliovirus vaccine (OPV) challenge dose. Blood and stool samples were collected at several time points and evaluated for polio-neutralizing antibodies and serotype-specific poliovirus, respectively. Eighty-one of 589 (14%) children excreted PV1 7 days post-OPV challenge; 70 of 81 (86%) were seropositive at baseline. Twelve of 610 (2%) were asymptomatic wild poliovirus type 1 (WPV1) excretors. Most poliovirus excretors had humoral immunity, suggesting mucosal immunity in these children likely waned or never developed. Without mucosal immunity, they are susceptible to poliovirus infection, shedding, and transmission. Asymptomatic WPV1 excretion suggests undetected poliovirus circulation within the community.

Autosomal recessive non-syndromic hearing loss genes in Pakistan during the previous three decades.

Hearing loss is a clinically and genetically heterogeneous disorder, with over 148 genes and 170 loci associated with its pathogenesis. The spectrum and frequency of causal variants vary across different genetic ancestries and are more prevalent in populations that practice consanguineous marriages. Pakistan has a rich history of autosomal recessive gene discovery related to non-syndromic hearing loss. Since the first linkage analysis with a Pakistani family that led to the mapping of the DFNB1 locus on chromosome 13, 51 genes associated with this disorder have been identified in this population. Among these, 13 of the most prevalent genes, namely CDH23, CIB2, CLDN14, GJB2, HGF, MARVELD2, MYO7A, MYO15A, MSRB3, OTOF, SLC26A4, TMC1 and TMPRSS3, account for more than half of all cases of profound hearing loss, while the prevalence of other genes is less than 2% individually. In this review, we discuss the most common autosomal recessive non-syndromic hearing loss genes in Pakistani individuals as well as the genetic mapping and sequencing approaches used to discover them. Furthermore, we identified enriched gene ontology terms and common pathways involved in these 51 autosomal recessive non-syndromic hearing loss genes to gain a better understanding of the underlying mechanisms. Establishing a molecular understanding of the disorder may aid in reducing its future prevalence by enabling timely diagnostics and genetic counselling, leading to more effective clinical management and treatments of hearing loss.

Genome sequencing reveals novel causative structural and single nucleotide variants in Pakistani families with congenital hypogonadotropic hypogonadism.

BACKGROUND/OBJECTIVES: This study aims to elucidate the genetic causes of congenital hypogonadotropic hypogonadism (CHH), a rare genetic disorder resulting in GnRH deficiency, in six families from Pakistan. METHODS: Eighteen DNA samples from six families underwent genome sequencing followed by standard evaluation for pathogenic single nucleotide variants (SNVs) and small indels. All families were subsequently analyzed for pathogenic copy number variants (CNVs) using CoverageMaster. RESULTS: Novel pathogenic homozygous SNVs in known CHH genes were identified in four families: two families with variants in GNRHR, and two others harboring KISS1R variants. Subsequent investigation of CNVs in the remaining two families identified novel unique large deletions in ANOS1. CONCLUSION: A combined, systematic analysis of single nucleotide and CNVs helps to improve the diagnostic yield for variants in patients with CHH.

Genomic analysis uncovers novel candidate genes related to adaptation to tropical climates and milk production traits in native goats.

BACKGROUND: Since domestication, both evolutionary forces and human selection have played crucial roles in producing adaptive and economic traits, resulting in animal breeds that have been selected for specific climates and different breeding goals. Pakistani goat breeds have acquired genomic adaptations to their native climate conditions, such as tropical and hot climates. In this study, using next-generation sequencing data, we aimed to assess the signatures of positive selection in three native Pakistani goats, known as milk production breeds, that have been well adapted to their local climate. RESULTS: To explore the genomic relationship between studied goat populations and their population structure, whole genome sequence data from native goat populations in Pakistan (n = 26) was merged with available worldwide goat genomic data (n = 184), resulting in a total dataset of 210 individuals. The results showed a high genetic correlation between Pakistani goats and samples from North-East Asia. Across all populations analyzed, a higher linkage disequilibrium (LD) level (- 0.59) was found in the Pakistani goat group at a genomic distance of 1 Kb. Our findings from admixture analysis (K = 5 and K = 6) showed no evidence of shared genomic ancestry between Pakistani goats and other goat populations from Asia. The results from genomic selection analysis revealed several candidate genes related to adaptation to tropical/hot climates (such as; KITLG, HSPB9, HSP70, HSPA12B, and HSPA12B) and milk production related-traits (such as IGFBP3, LPL, LEPR, TSHR, and ACACA) in Pakistani native goat breeds. CONCLUSIONS: The results from this study shed light on the structural variation in the DNA of the three native Pakistani goat breeds. Several candidate genes were discovered for adaptation to tropical/hot climates, immune responses, and milk production traits. The identified genes could be exploited in goat breeding programs to select efficient breeds for tropical/hot climate regions.

Clinical genomics expands the link between erroneous cell division, primary microcephaly and intellectual disability.

Primary microcephaly is a rare neurogenic and genetically heterogeneous disorder characterized by significant brain size reduction that results in numerous neurodevelopmental disorders (NDD) problems, including mild to severe intellectual disability (ID), global developmental delay (GDD), seizures and other congenital malformations. This disorder can arise from a mutation in genes involved in various biological pathways, including those within the brain. We characterized a recessive neurological disorder observed in nine young adults from five independent consanguineous Pakistani families. The disorder is characterized by microcephaly, ID, developmental delay (DD), early-onset epilepsy, recurrent infection, hearing loss, growth retardation, skeletal and limb defects. Through exome sequencing, we identified novel homozygous variants in five genes that were previously associated with brain diseases, namely CENPJ (NM_018451.5: c.1856A > G; p.Lys619Arg), STIL (NM_001048166.1: c.1235C > A; p.(Pro412Gln), CDK5RAP2 (NM_018249.6 c.3935 T > G; p.Leu1312Trp), RBBP8 (NM_203291.2 c.1843C > T; p.Gln615*) and CEP135 (NM_025009.5 c.1469A > G; p.Glu490Gly). These variants were validated by Sanger sequencing across all family members, and in silico structural analysis. Protein 3D homology modeling of wild-type and mutated proteins revealed substantial changes in the structure, suggesting a potential impact on function. Importantly, all identified genes play crucial roles in maintaining genomic integrity during cell division, with CENPJ, STIL, CDK5RAP2, and CEP135 being involved in centrosomal function. Collectively, our findings underscore the link between erroneous cell division, particularly centrosomal function, primary microcephaly and ID.

Crimean-Congo Hemorrhagic Fever Virus Diversity and Reassortment, Pakistan, 2017-2020.

Sporadic cases and outbreaks of Crimean-Congo hemorrhagic fever (CCHF) have been documented across Pakistan since 1976; however, data regarding the diversity of CCHF virus (CCHFV) in Pakistan is sparse. We whole-genome sequenced 36 CCHFV samples collected from persons infected in Pakistan during 2017-2020. Most CCHF cases were from Rawalpindi (n = 10), followed by Peshawar (n = 7) and Islamabad (n = 4). Phylogenetic analysis revealed the Asia-1 genotype was dominant, but 4 reassorted strains were identified. Strains with reassorted medium gene segments clustered with Asia-2 (n = 2) and Africa-2 (n = 1) genotypes; small segment reassortments clustered with the Asia-2 genotype (n = 2). Reassorted viruses showed close identity with isolates from India, Iran, and Tajikistan, suggesting potential crossborder movement of CCHFV. Improved and continuous human, tick, and animal surveillance is needed to define the diversity of circulating CCHFV strains in Pakistan and prevent transmission.

Successful Treatment of Confirmed Naegleria fowleri Primary Amebic Meningoencephalitis.

Primary amebic meningoencephalitis caused by Naegleria fowleri is a rare but nearly always fatal parasitic infection of the brain. Globally, few survivors have been reported, and the disease has no specific treatment. We report a confirmed case in Pakistan in a 22-year-old man who survived after aggressive therapy.

Effect of a multicomponent quality improvement strategy on sustained achievement of diabetes care goals and macrovascular and microvascular complications in South Asia at 6.5 years follow-up: Post hoc analyses of the CARRS randomized clinical trial.

BACKGROUND: Diabetes control is poor globally and leads to burdensome microvascular and macrovascular complications. We aimed to assess post hoc between-group differences in sustained risk factor control and macrovascular and microvascular endpoints at 6.5 years in the Center for cArdiovascular Risk Reduction in South Asia (CARRS) randomized trial. METHODS AND FINDINGS: This parallel group individual randomized clinical trial was performed at 10 outpatient diabetes clinics in India and Pakistan from January 2011 through September 2019. A total of 1,146 patients with poorly controlled type 2 diabetes (HbA1c ≥8% and systolic BP ≥140 mm Hg and/or LDL-cholesterol ≥130 mg/dL) were randomized to a multicomponent quality improvement (QI) strategy (trained nonphysician care coordinator to facilitate care for patients and clinical decision support system for physicians) or usual care. At 2.5 years, compared to usual care, those receiving the QI strategy were significantly more likely to achieve multiple risk factor control. Six clinics continued, while 4 clinics discontinued implementing the QI strategy for an additional 4-year follow-up (overall median 6.5 years follow-up). In this post hoc analysis, using intention-to-treat, we examined between-group differences in multiple risk factor control (HbA1c <7% plus BP <130/80 mm Hg and/or LDL-cholesterol <100 mg/dL) and first macrovascular endpoints (nonfatal myocardial infarction, nonfatal stroke, death, revascularization [angioplasty or coronary artery bypass graft]), which were co-primary outcomes. We also examined secondary outcomes, namely, single risk factor control, first microvascular endpoints (retinopathy, nephropathy, neuropathy), and composite first macrovascular plus microvascular events (which also included amputation and all-cause mortality) by treatment group and whether QI strategy implementation was continued over 6.5 years. At 6.5 years, assessment data were available for 854 participants (74.5%; n = 417 [intervention]; n = 437 [usual care]). In terms of sociodemographic and clinical characteristics, participants in the intervention and usual care groups were similar and participants at sites that continued were no different to participants at sites that discontinued intervention implementation. Patients in the intervention arm were more likely to exhibit sustained multiple risk factor control than usual care (relative risk: 1.77; 95% confidence interval [CI], 1.45, 2.16), p < 0.001. Cumulatively, there were 233 (40.5%) first microvascular and macrovascular events in intervention and 274 (48.0%) in usual care patients (absolute risk reduction: 7.5% [95% CI: -13.2, -1.7], p = 0.01; hazard ratio [HR] = 0.72 [95% CI: 0.61, 0.86]), p < 0.001. Patients in the intervention arm experienced lower incidence of first microvascular endpoints (HR = 0.68 [95% CI: 0.56, 0.83), p < 0.001, but there was no evidence of between-group differences in first macrovascular events. Beneficial effects on microvascular and composite vascular outcomes were observed in sites that continued, but not sites that discontinued the intervention. CONCLUSIONS: In urban South Asian clinics, a multicomponent QI strategy led to sustained multiple risk factor control and between-group differences in microvascular, but not macrovascular, endpoints. Between-group reductions in vascular outcomes at 6.5 years were observed only at sites that continued the QI intervention, suggesting that practice change needs to be maintained for better population health of people with diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT01212328.

Monoallelic and bi-allelic variants in NCDN cause neurodevelopmental delay, intellectual disability, and epilepsy.

Neurochondrin (NCDN) is a cytoplasmatic neural protein of importance for neural growth, glutamate receptor (mGluR) signaling, and synaptic plasticity. Conditional loss of Ncdn in mice neural tissue causes depressive-like behaviors, impaired spatial learning, and epileptic seizures. We report on NCDN missense variants in six affected individuals with variable degrees of developmental delay, intellectual disability (ID), and seizures. Three siblings were found homozygous for a NCDN missense variant, whereas another three unrelated individuals carried different de novo missense variants in NCDN. We assayed the missense variants for their capability to rescue impaired neurite formation in human neuroblastoma (SH-SY5Y) cells depleted of NCDN. Overexpression of wild-type NCDN rescued the neurite-phenotype in contrast to expression of NCDN containing the variants of affected individuals. Two missense variants, associated with severe neurodevelopmental features and epilepsy, were unable to restore mGluR5-induced ERK phosphorylation. Electrophysiological analysis of SH-SY5Y cells depleted of NCDN exhibited altered membrane potential and impaired action potentials at repolarization, suggesting NCDN to be required for normal biophysical properties. Using available transcriptome data from human fetal cortex, we show that NCDN is highly expressed in maturing excitatory neurons. In combination, our data provide evidence that bi-allelic and de novo variants in NCDN cause a clinically variable form of neurodevelopmental delay and epilepsy, highlighting a critical role for NCDN in human brain development.

Sequence variants underlying severe combined immunodeficiency and leukocyte adhesion deficiency type 1 in six consanguineous families.

Inborn errors of immunity (IEI) are defined as genetic disorders affecting the immune system and resulting in diverse clinical signs and symptoms. Despite the lack of diagnosis and unavailability of IEI estimation in the Pakistani population, consanguinity is exacerbating its prevalence. The current study focuses on severe combined immunodeficiency (SCID) and leukocyte adhesion deficiency type 1 (LAD1). SCID is associated with the life-threatening symptoms developing at post-birth. LAD1 is clinically characterized by recurrent bacterial infections related to the skin, mouth, and respiratory tract owing to impaired leukocytes. Herein, in six consanguineous families, flow cytometry was used to evaluate the patient's immune status. Whole-exome sequencing (WES) was then conducted to search for the causative variations in immunodeficiency genes. Sanger sequencing was used to assess the segregation of the variants with the disorder within the families. Sequence analysis revealed five homozygous variants in four different causative genes. This included four novel nonsense variants in CD70 p.(Thr126Profs*33), CD3e p.(Trp151*), IL7R p.(Val138Ilefs*10), and ITGB2 p.(Ser627Valfs*61), and one previously reported in ITGB2 p.(Cys62*). In one of the families, two variants in two different genes, including DNAH6 p.(Tyr2653His) and NIPAL4 p.(Gly121Ser), were detected in an unclassified patient. All the identified variants were found in a homozygous state in the patient but in a heterozygous state in the available parents. The study will facilitate the diagnosis and management of IEI patients.

Sequence variants in the BTD underlying biotinidase deficiency in families of Pakistani origin.

BACKGROUND: Biotinidase deficiency (BTD) is a rare autosomal recessive metabolic disease, which develops neurological symptoms because of the impaired biotin recycling. Pathogenic mutations on BTD gene cause BTD deficiency. The clinical features and mutation analysis of Pakistani children with BTD deficiency have rarely been described. Herein, for the first time, we report the clinical features, BTD gene mutations and biochemical analysis of seven symptomatic children with BTD deficiency from Pakistan. METHODS: Seven suspected BTD-deficient patients who presented abnormal organic acid profiles and clinical features were subjected to Sanger sequencing to identify pathogenic mutations in the BTD gene. The results were analyzed by Mutation Surveyor Software. RESULTS: All seven patients exhibited common biotinidase deficiency symptoms including hypotonia, developmental delay and seizures. Biochemical analysis shows marked excretion of 3-hydroxy isovalerate in all cases, followed by 3-hydroxy propionate and methyl citrate. Sanger sequencing revealed one frame-shift mutation, c.98_104delinsTCC (p.Cys33Phefs), and two missense mutations, c.1612C>A (p.Arg538Ser) and c.1330G>C (p.Asp444His). All mutations were in the homozygous state and classified as pathogenic in published studies and mutation databases. CONCLUSIONS: This study has validated the BTD variants as the underlying cause of biotinidase deficiency in which molecular testing of BTD is supported by urinary organic acid analysis and clinical diagnosis. Secondly, the strength of the local availability of this test in Pakistan will paved the way for the neonatal screening of biotinidase deficiency.

Influence of maize genotypes and harvest stages on in-silo fermentation quality and nutritional value of corn silage during hot summer condition of the tropics.

The aim of the experiment was to evaluate the potential of promising summer maize genotypes and optimal stage of harvesting these genotypes for ensiling in terms of dry matter (DM), starch, and crude protein (CP) yields, silage fermentation quality, nutrients profile, total digestible nutrients, metabolizable energy (ME) content, Cornell Net Carbohydrate and Protein System (CNCPS) carbohydrate (CHO) subfractions composition, in vitro DM digestibility (DMD) and in situ starch degradation characteristics. Six maize genotypes were chosen for the study: DK9108 from Monsanto, P30Y87, P3939 from Pioneer, QPM-300 (quality protein maize) and W94 from the International Maize and Wheat Improvement Center (CIMMYT), and a local cultivar, Afgoii, from the Cereal Research Institute (Persabaq, KP). A total of 72 plots (8 m × 10 m) were blocked in three replicate fields, and within each field, each genotype was sown in four replicate plots according to a randomized complete block design. For the data analysis, the Proc-Mixed procedure of Statistical Analysis System with repeated measure analysis of variance was used. The DM yield was strongly influenced (P < 0.001) by maize genotypes, varying from 12.6 to 17.0 tons/ha. Except for total CHO and ammonia nitrogen (NH3-N), the contents of all measured chemical components varied (P < 0.001) among the genotypes. Further comparison revealed that, genotype P3939 had a higher (P < 0.05) content of CP (7.27 vs. 6.92%), starch (36.7 vs. 27.9%), DMD (65.4 vs. 60.0%), ME (2.51 vs. 2.30 Mcal/kg) and lactic acid (5.32 vs. 4.83%) and lowest content of NDF (37.3 vs. 43.1%), pH (3.7 vs. 4.10) compared to the local cultivar (Afgoii). Advancement of post-flowering maturity from 25 to 35% DM (23 to 41 days after flowering (DAF)) increased (P < 0.05) the DM yield (10.4 to 17.8 tons/ha), starch content (29.1 to 35.0%), DMD (65.3 to 67.3%) and ME (2.34 to 2.47 Mcal/kg), and decreased (P < 0.001) the contents of CP (7.42-6.73%), NDF (48.8-38.5%), pH (4.10 to 3.60), NH3-N (8.93-7.80%N) and effective degradability of starch (95.4 to 89.4). Results showed that for higher yields and silage nutritional and fermentation quality, maize crops should be harvested at whole crop DM content of 30-35% (34 to 41 DAF). It was further concluded that genotype P3939 is the most suitable summer maize genotype for silage production in terms of yields and silage nutritional and fermentation quality under the hot environmental conditions of the tropics.