Identification of a Single Nucleotide Polymorphism of Vitamin D Receptor (VDR) and Vitamin D Binding Protein (VDBP) Gene and Its Dysregulated Pathway Through VDR-VDBP Interaction Network Analysis in Vitamin D-Deficient Infertile Females.

INTRODUCTION: The prevalence of female infertility in Pakistan is currently estimated at 22%, and emerging research suggests that vitamin D (VD) deficiency (VDD) may play a significant role in influencing female fertility. The focus of this study was to investigate the single nucleotide polymorphism (SNP) patterns within the VD binding protein (VDBP). The study aimed to explore dysregulated pathways and gene enrichment through an interaction network analysis, specifically focusing on the interplay between the VD receptor (VDR) and VDBP in females experiencing unexplained infertility (UI) coupled with VDD. METHODS: A cross-sectional study was conducted on VD-deficient, fertile, and UI female subjects. VDBP and VDR were assessed by enzyme-linked immunoassay and genotyping performed. FunRich (version 3.1.3; http://funrich.org/index.html) was employed for analysis of the identified proteins: VDR and VDBP and with their mapped gene datasets, gene enrichment, and protein-protein interaction (PPI) network. RESULTS: The mean VD and VDR values of infertile females were significantly lower than those of fertile females. VDBP in infertile females (median (IQR)): 296.05 (232.58-420.23)) was lower than that of fertile females (469.9 (269.57-875.55), (p=0.01)). On sequence analysis, a mutation rs 4588 SNP (Thr 436 Lys) was found in exon 11 of the VDBP gene of UI females, but no mutation in exons 8 and 9 of the VDR gene, with some insignificant intronic variants, was observed. The proteins such as plasma membrane estrogen receptor signaling pathway (p < 0.001), VDR, SMAD3, NCOR1, CREBBP, NCOA1, STAT1, GRB2, PPP2CA, TP53, and NCOA2 were enriched after biological pathway grouping when VDR was made the focused gene and directly interacting with VDBP. CONCLUSION: The females with UI exhibited significantly low VD, VDBP, and VDR. The plasma membrane estrogen receptor signaling pathway was enriched in VDD infertile females.

Selective screening for inherited metabolic disorders in a tertiary care hospital of Karachi - A retrospective chart review.

Background & Objective: Selective high-risk screening of children suspected of having inherited metabolic disorders was conducted jointly by Chemical Pathology section and the Pediatric Department of Indus Hospital and Health Network- (IHHN) from October 2020-March 2022. Tandem mass spectrometry (MS) for newborn screening was recently introduced in a local laboratory. We did a selective high screening of children for metabolic disorders by using MS for neonates and other relevant tests for older children in our hospital. The present study was undertaken to get an estimate of the number of metabolic cases screened and identified after inclusion of an extended workup. Methods: This is a retrospective chart review of children who were selectively screened for IMDs. Patients' records with ages ranging from birth to fourteen years of age were retrieved from the electronic records department of IHHN from October 2020 to March 2022. Records were searched for demographic data, history, signs, symptoms, and lab investigations. All relevant information was recorded on a pre-designed questionnaire. Results: A total of 178 children were screened for inherited metabolic disorders. Majority of the children screened were less than one month of age 96 (54%). Consanguinity was noted in 74 (41.5%) children. Most common symptoms observed were failure to thrive in 77 children (43%), hypoglycemia in 45 children (25%), and feeding difficulty in 36 children (20%). Inherited metabolic disorders were confirmed in 12 children out of which five had congenital adrenal hyperplasia, four had cystic fibrosis and three children had congenital hypothyroidism. Conclusion: In the present study, we were able to screen several children after inclusion of an extended metabolic workup. However, confirmation of many disorders like fatty acid oxidation defects, disorders of carbohydrate metabolism, and sphingolipidosis could not be done due to lack of confirmatory tests. We recommend that confirmatory tests of these disorders be included in local labs.

Kasabach-Merritt Syndrome: a case study of successful treatment with vincristine and propranolol.

Kasabach-Merritt syndrome is a rare condition, characterised by the presence of an enlarging vascular tumour associated with thrombocytopenia, microangiopathic haemolytic anaemia and consumptive coagulopathy. The syndrome manifests in infancy, with high morbidity and mortality rates. No standard guidelines have been established for the treatment of Kasabach-Merritt syndrome to date. To existing literature we add this report of a four-month-old female child with Kasabach-Merritt syndrome who was successfully treated with propranolol and vincristine. This drug combination helped reverse the severe thrombocytopenia as well as decrease in size of her haemangioma. Management of Kasabach-Merritt syndrome continues to be a challenge, with varying response to first line drugs. Early diagnosis and initiation of treatment in a closely monitored setting is essential to ensure good outcomes. Since this is a relatively rare condition and large studies are not feasible, documenting treatment experience for single cases or small series becomes even more important.

Rare and heterogeneous manifestations of leucocyte adhesion deficiency type 1: report of two cases with diagnostic dilemmas and novel ITGB2 mutation.

BACKGROUND: Primary immunodeficiency disorders (PID) are rare disorders with heterogeneous manifestations, overlapping with other diseases such as autoimmunity, malignancy, and infections. This makes the diagnosis very challenging and delays management. Leucocyte adhesion defects (LAD) are a group of PIDs in which patients lack adhesion molecules on leukocytes needed for their emigration through blood vessels to the site of infection. Patients with LAD can present with diverse clinical features including severe and life-threatening infections, early in life, and the absence of pus formation around infection or inflammation. There is often delayed umbilical cord separation, omphalitis, late wound healing, and a high white blood cell count. If not recognized and managed early, can lead to life-threatening complications and death. CASE PRESENTATION: LAD 1 is characterized by homozygous pathogenic variants in the integrin subunit beta 2 (ITGB2) gene. We report two cases of LAD1 with unusual presentations (post-circumcision excessive bleeding and chronic inflammation of the right eye) which were confirmed by flow cytometric analysis and genetic testing. We found two disease-causing ITGB2 pathogenic variants in both cases. CONCLUSIONS: These cases highlight the importance of a multidisciplinary approach to recognizing clues in patients with uncommon manifestations of a rare disease. This approach initiates a proper diagnostic workup of primary immunodeficiency disorder leading to a better understanding of the disease, and appropriate patient counseling, and helps clinicians to be better equipped to deal with complications.

Lemierre's syndrome in a child.

Lemierre's Syndrome (LS) is a rare syndrome most frequently due to an anaerobic organism, Fusobacterium Necrophorum. It is commonly a complication of an acute oropharyngeal infection, but there are exceptions to its presentations. In our case the cause of LS was otitis media caused by Streptococcus species. This is a rather unusual presentation of LS. LS is caused due to septic complications of oropharyngeal infections, which lead to thrombophlebitis of internal jugular vein leading to thrombosis formation. In this case report, we present a case of Lemierre's syndrome in a seven-year-old male child. The patient presented with high grade fever spikes and earache, which were unresponsive to oral antibiotics. LS was diagnosed in this patient on the basis of clinical, microbiological and radiological findings. After the diagnosis, treatment involved using broad spectrum antibiotics and anticoagulants, followed by surgery. Though role of anticoagulants is controversial in LS, but there is no specific guideline contraindicating the use of anti-coagulants. In our case, timely diagnosis and management enabled us to discharge the patient without any symptoms.

Comparison of Clinical and Diagnostic Features of Pediatric Oncology Patients With or Without COVID-19 Infection: A Retrospective Chart Review.

There is a scarcity of data summarizing the clinical picture, laboratory, and imaging findings and outcome in children with malignancy and coronavirus disease 2019 (COVID-19) infection. This study characterizes a detailed comparison of pediatric oncology patients with and without COVID infection. A retrospective study was conducted at The Indus Hospital, Karachi, from March 2020 to June 2020. Clinical presentation, laboratory and imaging findings, disease severity, and outcome were compared between cohorts. The mean age of children with and without COVID was 8.0±4.9 and 7.4±4.1 years, respectively. Hematologic malignancy comprised the largest number of patients, followed by solid tumors. Lymphocytosis and low neutrophil-lymphocyte ratio was observed in the COVID positive group. Cardiac dysfunction (1.4% vs. 0%), acute respiratory distress syndrome (8% vs. 0%) and lower peripheral capillary oxygen saturation/fraction of inspired oxygen ratio (473 vs. 486) found to be associated with severe disease in COVID positive group (P<0.05). Overall mortality in children with COVID was 6.8% versus 2.7% in children without COVID. Pediatric patients with malignancy have different clinical features and laboratory parameters as compared with children without malignancy. Acute respiratory distress syndrome, absolute lymphocytosis and low neutrophil-lymphocyte ratio is associated with severe disease in children with malignancy and COVID infection. In contrast to adults, biochemical markers and complete blood count parameters do not help recognize COVID infection in pediatric patients with malignancy.

The Dawn of next generation DNA sequencing in myelodysplastic syndromes- experience from Pakistan.

BACKGROUND: Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells exhibiting ineffective hematopoiesis and tendency for transformation into acute myeloid leukemia (AML). The available karyotyping and fluorescent in situ hybridization provide limited information on molecular abnormalities for diagnosis/prognosis of MDS. Next generation DNA sequencing (NGS), providing deep insights into molecular mechanisms being involved in pathophysiology, was employed to study MDS in Pakistani cohort. PATIENTS AND METHODS: It was a descriptive cross-sectional study carried out at National institute of blood diseases and bone marrow transplant from 2016 to 2019. Total of 22 cases of MDS were included. Complete blood counts, bone marrow assessment and cytogenetic analysis was done. Patients were classified according to revised WHO classification 2016 and IPSS score was applied for risk stratification. Baseline blood samples were subjected to analysis by NGS using a panel of 54 genes associated with myeloid malignancies. RESULTS: The median age of patients was 48.5 ± 9.19 years. The most common presenting complaint was weakness 10(45.45%). Cytogenetics analysis revealed abnormal karyotype in 10 (45.45%) patients. On NGS, 54 non-silent rare frequency somatic mutational events in 29 genes were observed (average of 3.82 (SD ± 2.08) mutations per patient), including mutations previously not observed in MDS or AML. Notably, two genes of cohesin complex, RAD21 and STAG2, and two tumor suppressor genes, CDKN2A and TP53, contained highest number of recurrent non-silent somatic mutations in the MDS. Strikingly, a missense somatic mutation p.M272Rof Rad21 was observed in 13 cases. Overall, non-silent somatic mutations in these four genes were observed in 21 of the 22 cases. The filtration with PharmGKB database highlighted a non-synonymous genetic variant rs1042522 [G > C] located in the TP53. Genotype GG and GC of this variant are associated with decreased response to cisplatin and paclitaxel chemotherapy. These two genotypes were found in 13 cases. CONCLUSION: Sequencing studies suggest that numerous genetic variants are involved in the initiation of MDS and in the development of AML. In countries like Pakistan where financial reservation of patients makes the use of such analysis even more difficult when the availability of advanced techniques is already a prevailing issue, our study could be an initiating effort in adding important information to the local data. Further studies and large sample size are needed in future to enlighten molecular profiling and ultimately would be helpful to compare and contrast the molecular characteristics of Asian versus global population.

Clinical features and outcome of COVID-19 positive children from a tertiary healthcare hospital in Karachi.

: As the coronavirus disease 2019 (COVID-19) pandemic continues to evolve, differences in epidemiological and clinical features among pediatrics have been noticed across different countries. We describe the spectrum of COVID-19 in pediatric patients treated in tertiary health care. We conducted a retrospective chart review of pediatric patients admitted to Indus Hospital & Health care network, Korangi campus, Karachi; from April 1st, 2020 to July 31st, 2020. A total of 141 COVID-19 cases were reported, males were 81 (57%) and the median age was 8 (0.3-17) years. Moderate and severe infections were noted in 36(26%), and 17(12%) children respectively. Fever (50%) was the most common clinical feature. The SF ratio less than 264 was significantly associated with severe disease (p < .05). Lab investigations that differed significantly across disease severity groups included IL-6 levels (p < .01) and Prothrombin time (p < .05). Majority of children were advised home isolation 89 (63%), 29 (20.5%) were admitted while mortality was observed in 10 (7%) children. No significant difference was observed between children with and without malignancy. Pre-existing comorbidities are significantly associated with COVID-19 infections among children. Reduced SF ratio, elevated Prothrombin time, and interleukin-6 levels are associated with greater disease severity.

Novel Genetic Variations in Acute Myeloid Leukemia in Pakistani Population.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion of blast cells that exhibit great genetic heterogeneity. In this study, we describe the mutational landscape and its clinico-pathological significance in 26 myeloid neoplasm patients from a South Asian population (Pakistan) by using ultra-deep targeted next-generation DNA sequencing of 54 genes (∼5000×) and its subsequent bioinformatics analysis. The data analysis indicated novel non-silent somatic mutational events previously not reported in AML, including nine non-synonymous and one stop-gain mutations. Notably, two recurrent somatic non-synonymous mutations, i.e., STAG2 (causing p.L526F) and BCORL1 (p.A400V), were observed in three unrelated cases each. The BCOR was found to have three independent non-synonymous somatic mutations in three cases. Further, the SRSF2 with a protein truncating somatic mutation (p.Q88X) was observed for the first time in AML in this study. The prioritization of germline mutations with ClinVar, SIFT, Polyphen2, and Combined Annotation Dependent Depletion (CADD) highlighted 18 predicted deleterious/pathogenic mutations, including two recurrent deleterious mutations, i.e., a novel heterozygous non-synonymous SNV in GATA2 (p.T358P) and a frameshift insertion in NPM1 (p.L258fs), found in two unrelated cases each. The WT1 was observed with three independent potential detrimental germline mutations in three different cases. Collectively, non-silent somatic and/or germline mutations were observed in 23 (88.46%) of the cases (0.92 mutation per case). Furthermore, the pharmGKB database exploration showed a missense SNV rs1042522 in TP53, exhibiting decreased response to anti-cancer drugs, in 19 (73%) of the cases. This genomic profiling of AML provides deep insight into the disease pathophysiology. Identification of pharmacogenomics markers will help to adopt personalized approach for the management of AML patients in Pakistan.

Homozygous CALR Mutation in Primary Myelofibrosis and Its Effect on Disease Phenotype: A Case Report and Review of the Literature.

Somatic mutations in CALR gene have been reported in 60%-88% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) who are negative for JAK2 and MPL mutations. Most of the CALR mutations analyzed to date are heterozygous mutations in exon 9 of the gene. Homozygosity in CALR gene is rarely reported, and its association with clinical behavior of disease and impact on outcome of patients is not studied so far. We herein report a case of intermediate-2 risk PMF (according to IPSS) diagnosed with homozygous mutation (c.1139delA p.E380fs ∗ 50) in CALR gene having severe disease manifestations at presentation.

Genomic profile of a patient with triple negative essential thrombocythemia, unresponsive to therapy: A case report and literature review.

Clonal analysis of patients with triple negative myeloproliferative neoplasm (MPN) has provided evidence of additional aberrations, including epigenetic alterations. To discover such novel genetic aberrations, patients were screened through next-generation sequencing using a myeloid sequencing panel of 54 genes using a genetic analyser. Genetic variants in 28 genes, including TET2, BCOR, BCR, and ABL1 were identified in a triple negative essential thrombocythemia (ET) patient. The individual role of some of these variants in disease pathogenesis has yet to be studied. Somatic mutations in the same genes have been reported with variable frequencies in myeloid malignancies. However, no pathogenic impact of these variants could be found; therefore, long-term follow up of patients with genetic analysis of a large cohort and the use of whole genome sequencing is required to assess the effects of these variants.

Unique molecular alteration patterns in von Hippel-Lindau (VHL) gene in a cohort of sporadic renal cell carcinoma patients from Pakistan.

BACKGROUND: Renal cell carcinoma (RCC) is the most frequent form of kidney cancer in adults. Somatic mutations that inactivate the von Hippel-Lindau (VHL) gene are the most common cause of RCC. The frequencies of molecular changes in the VHL gene in RCCs vary among different populations. So far, a single chromosomal-based study has been reported from a South Asian population. This report presents, for the first time, the somatic changes and promoter hypermethylation in VHL in a cohort of 300 RCC patients from Pakistan. METHODS: To identify mutations in the VHL gene, direct DNA sequencing was carried out. Epigenetic silencing was investigated by using methylation-specific polymerase chain reaction. RESULTS: Our data showed molecular alterations in the VHL gene in 163 (54%) renal cell carcinoma patients. Somatic mutations were found in 87 (29%) patients and 35 novel mutations were identified. VHL promoter hyper-methylation analysis showed epigenetic changes in 106 (35%) out of 300 patients. Patients who had no evidence of molecular alterations in the VHL gene were significantly younger than patients who carried some molecular change. Molecular alterations in the VHL gene were not restricted to clear-cell RCCs (ccRCCs). CONCLUSIONS: This is the first report that identifies molecular aberrations in the VHL gene from a South Asian population. The frequency of somatic mutation is lower and that of promoter hypermethylation is higher when compared with data from other parts of the world. The data has important implications in the population-specific application of tailored preventive and therapeutic regimens in non-familial RCCs.

Synthesis, immunomodulation and cytotoxic effects of vanadium (IV) complexes.

Vanadium is known to exhibit several bioactivities and shows potential as a pharmaceutical drug. The current studies were conducted with the goal of synthesizing a new generation of oxovanadium(IV) complexes, investigating their effects on cancer cell proliferation and their immunomodulatory properties, and predicting possible structure activity relationships. The elucidation of the structures of the synthesized complexes was achieved using elemental analysis, conductivity measurements, magnetic property measurements, and IR and electronic spectroscopies. These studies suggest that the synthesized complexes have a binuclear structure. All of the complexes were evaluated on different cancer cell lines, including HeLa, PC-3, and C33A, and on the normal 3T3 fibroblast cells. Some of the compounds exhibited prominent inhibitory activities on the cervical cancer cell lines and the prostate cancer PC-3 cells. The immunomodulatory activity of the vanadium compounds was evaluated on human phagocytes for ROS (reactive oxygen species) production using luminol- and lucigenin-based chemiluminescence assays. No potent effect was exerted by the majority of the tested compounds on whole blood oxidative burst activity. A study of human T-cells proliferation in vitro on vanadium complexes was also conducted. The majority of the compounds were observed to exhibit potent inhibitory effects. The superoxide, nitric oxide and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging properties were also determined.