Exome sequencing in four families with neurodevelopmental disorders: genotype-phenotype correlation and identification of novel disease-causing variants in VPS13B and RELN.

Neurodevelopmental disorders (NDDs) are a clinically and genetically heterogeneous group of early-onset pediatric disorders that affect the structure and/or function of the central or peripheral nervous system. Achieving a precise molecular diagnosis for NDDs may be challenging due to the diverse genetic underpinnings and clinical variability. In the current study, we investigated the underlying genetic cause(s) of NDDs in four unrelated Pakistani families. Using exome sequencing (ES) as a diagnostic approach, we identified disease-causing variants in established NDD-associated genes in all families, including one hitherto unreported variant in RELN and three recurrent variants in VPS13B, DEGS1, and SPG11. Overall, our study highlights the potential of ES as a tool for clinical diagnosis.

A recurrent rare intronic variant in CAPN3 alters mRNA splicing and causes autosomal recessive limb-girdle muscular dystrophy-1 in three Pakistani pedigrees.

Autosomal recessive limb-girdle muscular dystrophy-1 (LGMDR1) is an autosomal recessive disorder characterized by progressive weakness of the proximal limb and girdle muscles. Biallelic mutations in CAPN3 are reported frequently to cause LGMDR1. Here, we describe 11 individuals from three unrelated consanguineous families that present with typical features of LGMDR1 that include proximal muscle wasting, weakness of the upper and lower limbs, and elevated serum creatine kinase. Whole-exome sequencing identified a rare homozygous CAPN3 variant near the exon 2 splice donor site that segregates with disease in all three families. mRNA splicing studies showed partial retention of intronic sequence and subsequent introduction of a premature stop codon (NM_000070.3: c.379 + 3A>G; p.Asp128Glyfs*15). Furthermore, we observe reduced CAPN3 expression in primary dermal fibroblasts derived from an affected individual, suggesting instability and/or nonsense-mediated decay of mutation-bearing mRNA. Genome-wide homozygosity mapping and single-nucleotide polymorphism analysis identified a shared haplotype and supports a possible founder effect for the CAPN3 variant. Together, our data extend the mutational spectrum of LGMDR1 and have implications for improved diagnostics for individuals of Pakistani origin.

Epidemiological, clinical and genetic characterization of aplastic anemia patients in Pakistan.

Aplastic anemia (AA) is the most serious non-malignant blood disorder in Pakistan, ranked second in prevalence, after thalassemia. We investigated various epidemiological, clinical, and genetic factors of AA in a Pakistani cohort of 214 patients reporting at our hospital between June 2014 and December 2015. A control group of 214 healthy subjects was included for comparison of epidemiological and clinical features. Epidemiological data revealed 2.75-fold higher frequency of AA among males. A single peak of disease onset was observed between ages 10 and 29 years followed by a steady decline. AA was strongly associated with lower socioeconomic profile, rural residence, and high rate of consanguineous marriages. Serum granulocyte colony-stimulating factor and thrombopoietin levels were significantly elevated in AA patients, compared to healthy controls (P < 0.0001), while there was no statistical significance in other nine cytokine levels screened. Allele frequencies of DRB1*15 (56.8%) and DQB1*06 (70.3%) were predominantly high in AA patients. Ten mutations were found in TERT and TERC genes, including two novel mutations (Val526Ala and Val777Met) in exons 3 and 7 of TERT gene. Despite specific features of the AA cohort, this study suggests that epidemiologic and etiologic factors as well as host genetic predisposition exclusively or cooperatively trigger AA in Pakistan.

Autologous mesenchymal stromal cell transplantation for spinal cord injury: A Phase I pilot study.

BACKGROUND AIMS: Mesenchymal stromal cell (MSC) transplantation has immerged as promising therapeutic approach to treat spinal cord injury (SCI). In this pilot study, we investigated the safety of intrathecal injection of autologous bone marrow-derived MSCs in nine patients with SCI. METHODS: Patients with complete SCI at the thoracic level were divided into two groups: chronic (>6 months, group 1) and sub-acute SCI (<6 months, group 2), according to time elapsed since injury. MSCs were isolated by density gradient separation of autologous bone marrow harvested from the iliac crest. Cells were cultured in a Good Manufacturing Practice-compliant facility to produce clinical scale dose. After quality control testing, MSCs were injected back to patients by intrathecal injection. Safety was defined as absence of adverse event and side effects after 1 month after receiving the injection. RESULTS: Six patients had chronic SCI with a median duration of 33 months since date of injury (range: 10-55 months), and three patients were in sub-acute phase of disease. Each patient received two or three injections with a median of 1.2 × 10(6) MSCs/kg body weight. No treatment-related adverse event was observed during median follow-up of 720 days (range: 630-826 days) in group 1 and 366 days (range: 269-367 days) in group 2, respectively. DISCUSSION: This pilot study demonstrated that autologous MSCs can be safely administered through intrathecal injection in spinal cord injury patients. Further investigation through randomized, placebo-controlled trials is needed.

Association of leukocyte telomere attrition in coronary artery disease in Pakistani population: A case-control study with meta-analysis.

BACKGROUND: Coronary arteries disease (CAD) is one of the primary causes of mortality worldwide. Genetic, epigenetic and environmental factors have been hypothesized in the pathogenesis of CAD. Leukocyte telomere length (LTL) has been proposed as a potential biomarker for early detection of atherosclerosis. Telomere is the DNA-Protein structure that maintains stability and integrity of chromosomes and is associated with the aging-related cellular mechanisms. This study is designed to investigate the association of LTL with CAD pathogenesis. MATERIAL AND METHOD: This prospective case-control study included 100 patients and 100 control individuals. DNA was extracted from the peripheral blood samples, and LTL was measured using real-time PCR. Data were normalized with single copy gene and presented as relative telomere length T/S ratio. Comprehensive meta-analysis was conducted to ascertain the pivotal role of telomere length in CAD pathology across multiple populations. RESULTS: Our results showed shorter telomere length in CAD patients as compared to control. The correlation analysis revealed a significant (P-value <0.01) negative correlation between telomere length with basal metabolic index (BMI), total cholesterol, and low-density lipoprotein cholesterol (LDL-C) and a positive correlation with high-density lipoprotein cholesterol (HDL-C). Meta-analysis results indicated a significantly shorter telomere length in the Asian population and a non-significant shorter telomere length in other populations. Receiver operator curve (ROC) analysis demonstrated an area under the curve (AUC) of 0.814 with cut-off value of 0.691 exhibited sensitivity of 72.2%, and specificity of 79.1%, for the diagnosis of CAD. CONCLUSION: In conclusion, LTL is associated with the onset of CAD and could be used as a diagnostic predictor to screen individuals with CAD.

To determine the frequency of aldehyde dehydrogenase type 2 (aldh2) deficiency in aplastic anaemia: A single center experience from pakistan.

BACKGROUND: Aplastic anaemia is a rare bone marrow failure syndrome and is defined by pancytopenia associated with a hypo-cellular bone marrow with no increase in reticulin and the absence of any abnormal infiltrate. The objective of the study was to determine the frequency of Aldehyde Dehydrogenase type 2 (ALDH2) deficiency in patients with Aplastic Anaemia and investigate its correlation with patient and disease characteristics. It was a descriptive cross-sectional study conducted at Armed Forces Bone Marrow Transplant Centre Rawalpindi from 01-08-2022-01-02-2023, over 6 months. METHODS: A total of 56 patients who were diagnosed with aplastic anaemia during this period, fulfilling inclusion criteria were enrolled. Patients were genotyped as GG (homozygous) and GA (heterozygous). GG had normal ALDH2, while GA were patients with ALDH2 deficiency. Data was collected on the patient's demographics, type and severity of anaemia, type of hematopoietic stem cell transplant (HSCT) and frequency of ALDH2 deficiency. Results were analyzed for ALDH2 deficiency and its correlation with patient and disease characteristics was investigated. RESULTS: A total of 56 patients were included in the study. The median age of the patients was 28 years (20-39). According to the type of aplastic anaemia, 2 (3.6%) had Fanconi anaemia and 54 (96.4%) had acquired aplastic anaemia. In our study, 18 (32.1%) patients had undergone HSCT while the remaining 38 (67.9%) could not undergo HSCT. The frequency of the presence of ALDH2 deficiency was 2 (3.6%). There was no statistically significant correlation between the frequency of ALDH2 deficiency with variables like gender, age distribution, type of aplastic anaemia, the severity of aplastic anaemia and hematopoietic stem cell transplant. CONCLUSIONS: We concluded from our study the frequency of ALDH2 was rare in patients with aplastic anaemia. There was no statistically significant correlation between the frequency of ALDH2 deficiency with variables like gender, age distribution, type of aplastic anaemia, the severity of aplastic anaemia and hematopoietic stem cell transplant.

Primary Hypertrophic Osteoarthropathy With Myelofibrosis.

Primary hypertrophic osteoarthropathy (PHO) is a rare autosomal recessive inherited multi-system disorder characterized by a triad of pachydermia, periostosis, and clubbing. PHO was revealed to be caused by the HPGD gene producing 15-prostaglandin dehydrogenase and the SLCO2A1 gene expressing one kind of prostaglandin transporter. It is primarily a benign disorder, but coexisting myelofibrosis can lead to clinically significant cytopenias. In this case report, we present the case of a 21-year-old boy with a history of transfusion-dependent anemia and a progressive increase in transfusion requirements over the course of seven years. On basis of the patient's medical history, family history, and clinical examination genetic testing was done. The patient was found to have homozygous c.664G>A (p. Gly222Arg) mutation in the SLCO2A1 gene; confirming the diagnosis of PHO.

Genetic analysis of osteopetrosis in Pakistani families identifies novel and known sequence variants.

Osteopetrosis is a genetically heterogenous, fatal bone disorder characterized by increased bone density. Globally, various genetic causes are reported for osteopetrosis with all forms of inheritance patterns. A precise molecular diagnosis is necessary for prognosis and for prescribing treatment paradigms in osteopetrosis. Here we report on thirteen individuals diagnosed with infantile malignant osteopetrosis coming from ten unrelated Pakistani families; nine of whom are consanguineous. We performed whole exome sequencing and Sanger sequencing in all families and identified homozygous variants in genes previously reported for autosomal recessive inheritance of osteopetrosis. All the identified variants are expected to affect the stability or length of gene products except one nonsynonymous missense variant. TCIRG1 was found as a candidate causal gene in majority of the families. We report six novel variants; four in TCIRG1 and one each in CLCN7 and OSTM1. Our combined findings will be helpful in molecular diagnosis and genetic counselling of patients with osteopetrosis particularly in populations with high consanguinity.

Neutrophil-to-Lymphocyte Ratio, derived Neutrophil-to-Lymphocyte Ratio, Platelet-to-Lymphocyte Ratio and Lymphocyte-to-Monocyte Ratio as risk factors in critically ill COVID-19 patients, a single centered study.

BACKGROUND: A lot remains anonymous about the characteristics and laboratory findings that may evaluate poor outcomes in patients with Coronavirus disease 2019.The aim of this study was to determine the relationship of change in the peripheral blood factors of Neutrophil-to-Lymphocyte Ratio, derived-Neutrophil-to-Lymphocyte Ratio, Lymphocyte-to-Monocyte Ratio, and Platelet-to-Lymphocyte Ratio in hospitalized patients with COVID-19 and its severity. METHODS: Cross-sectional analytical study was performed at Department of Haematology in Pak Emirates Military Hospital affiliated with Army Medical College, Rawalpindi, Pakistan from March-July 2020. We included 735 patients confirmed by real-time reverse transcriptase polymerase-chain-reaction test for subacute respiratory syndrome corona virus-2 of all ages, irrespective of gender and were classified in groups of severe and non-severe groups. RESULTS: Data of blood and baseline characteristics were compared in between the two groups and found to be significant (p-value <0.001). The median age was 46.3 years, and 82 cases were only females. Receiver operator curve demonstrated larger area under the curve of NLR, d-NLR, and PLR and showed them as independent diagnostic biomarkers which were significantly associated with the severity of illness. Binary logistic regression performed in the form of forest plot also showed these factors were significantly linked with the severity (p-value <0.001). CONCLUSION: NLR, d-NLR, and PLR along with pre-existing co morbidities can be used as an independent biomarker for the poor clinical outcome of COVID-19 illness.

Epidemiology of aplastic anemia: a study of 1324 cases.

Objective: Prevalence of aplastic anemia (AA) is high in the Asian population. This study was done to explore the etiology and association of AA with various socio-economic and environmental factors.Study design and setting: Study included 1324 consecutive AA cases registered at Armed Forces Bone Marrow Transplant Centre Rawalpindi, Pakistan, from March 2001 to August 2016. The study questionnaire was completed through an interview. It included patients' socio-demographic details, personal and family medical history, environmental attributes and clinico-hematological features.Results: The median age of patients was 20 years, 997 were male and 327 female. Distribution of non-severe, severe and very severe AA was 230 (17.4%); 598 (45.2%) and 496 (37.4%), respectively. The majority of patients were from low (n = 761, 57.5%) or middle socioeconomic class (n = 543, 41%). Consanguinity among patients (n = 806, 61%) was slightly higher than the national statistics. History of chemical exposures included fertilizers (n = 116, 8.7%), pesticides (n = 56, 4.2%) and industrial chemicals (n = 37, 2.8%). PNH clone was found in 63 of AA patients. After excluding 298 patients undergoing HSCT and 660 deaths/lost to follow-up, disease evolution was observed in 38(10.4%) patients out of 366 evaluable patients. These included PNH = 18, MDS = 11 and AML = 9.Discussion: Due to lack of funding and adequate human resource at the center, age and sex-matched controls could not be included. Other limitations were a lack of molecular testing to exclude the possibility of inherited bone marrow failure syndromes on a genetic basis.Conclusion: Younger age, male predominance and higher consanguinity point toward genetic factors in AA etiology among the South Asian population.