A mutation in the major autophagy gene, WIPI2, associated with global developmental abnormalities.

We describe a large consanguineous pedigree from a remote area of Northern Pakistan, with a complex developmental disorder associated with wide-ranging symptoms, including mental retardation, speech and language impairment and other neurological, psychiatric, skeletal and cardiac abnormalities. We initially carried out a genetic study using the HumanCytoSNP-12 v2.1 Illumina gene chip on nine family members and identified a single region of homozygosity shared amongst four affected individuals on chromosome 7p22 (positions 3059377-5478971). We performed whole-exome sequencing on two affected individuals from two separate branches of the extended pedigree and identified a novel nonsynonymous homozygous mutation in exon 9 of the WIPI2 (WD-repeat protein interacting with phosphoinositide 2) gene at position 5265458 (c.G745A;pV249M). WIPI2 plays a critical role in autophagy, an evolutionary conserved cellular pathway implicated in a growing number of medical conditions. The mutation is situated in a highly conserved and critically important region of WIPI2, responsible for binding PI(3)P and PI(3,5)P2, an essential requirement for autophagy to proceed. The mutation is absent in all public databases, is predicted to be damaging and segregates with the disease phenotype. We performed functional studies in vitro to determine the potential effects of the mutation on downstream pathways leading to autophagosome assembly. Binding of the V231M mutant of WIPI2b to ATG16L1 (as well as ATG5-12) is significantly reduced in GFP pull-down experiments, and fibroblasts derived from the patients show reduced WIPI2 puncta, reduced LC3 lipidation and reduced autophagic flux.

Diagnosis and phenotypic assessment of Pakistani Haemophilia B carriers.

OBJECTIVES: 1: To assess the diagnostic utility of three polymorphisms (DdeI, XmnI and TaqI) and direct sequencing in haemophilia B (HB) carrier detection in Pakistani families. 2: To compare phenotypes of HB carriers with those of healthy females. METHODS: The study was conducted from March 2014 till February 2016 at Khyber Medical University Peshawar and National Institute of Blood Diseases, Karachi. Individuals from HB families of Khyber Pakhtunkhwa (KP) and Federally Administered Tribal Areas (FATA) with known F9 mutation in the proband were enrolled into the study. FIX activity (FIX: C) levels were determined in all the participants. Bleeding scores (BS) and complete blood counts were performed in the female participants. Linkage analysis followed by targeted Sanger sequencing was carried out in all the study participants. Heterozygosity rate was determined for each polymorphism. Healthy females and the carrier groups were compared for bleeding phenotypes. RESULTS: A total of 30 males and 48 females from 13 HB families were studied. The polymorphisms had a low heterozygosity rate. Direct sequencing determined the carrier status in all cases. The mean FIX: C was reduced whereas BS was raised in the carriers when compared with healthy females. A significant raise in white blood cells (WBCs) count was observed in the carriers. CONCLUSION: The three polymorphisms have a low heterozygosity rate in HB families from KP and FATA. Sanger sequencing is conclusive in determining carrier status in all the cases. FIX: C is low and BS is raised in the HB carriers in comparison to that of normal females. The mean WBCs count is significantly higher in the HB carriers than the normal females.

Long-term clinical efficacy and safety of thalidomide in patients with transfusion-dependent ß-thalassemia: results from Thal-Thalido study.

Regular blood transfusion is the mainstay of treatment in transfusion-dependent ß-thalassemia (TDT); however, transfusions culminate in an array of serious complications. Therefore, a single-arm, non-randomized clinical trial was conducted in hydroxyurea refractory TDT patients to explore the long-term safety and efficacy of thalidomide. The primary outcomes for efficacy were rise in hemoglobin (Hb) level and changes in transfusion frequency. Whereas, several clinical and laboratory parameters were assessed for safety of thalidomide. Secondary outcomes included changes in serum ferritin, serum lactate dehydrogenase (LDH), serum uric acid, red blood cell indices, and size of liver and spleen. A total of 532 patients were followed for a period of 30 months. Significant increase in mean Hb level was identified at 6 months (1.4 g/dL, p ≤ 0.001) and 30 months (2 g/dL, p ≤ 0.001) in comparison with baseline. A total of 408 (76.7%) patients responded to thalidomide therapy (excellent responders 25.8%, good responders 31%, and partial responders 19.9%) and attained transfusion independence within 6 months of therapy. A significant decline in mean ferritin, LDH level, liver size, and spleen size was observed. No unfavorable effects were observed on kidney and liver functions. Mild adverse events were reported in 48 (9%) patients and serious adverse events, including cerebral vascular accident and portal vein thrombosis were reported in two patients each. This study concludes that thalidomide is an effective and well-tolerated drug that can improve Hb levels and reduce transfusion burden in hydroxyurea refractory TDT patients.Trial registration: This trial is registered at http://www.clinicaltrial.gov as # NCT03651102.

Effects of transfusion of stored blood in patients with transfusion-dependent thalassemia.

OBJECTIVES: The aim of this study was to investigate the hematological and biochemical effects of stored blood transfusion on patients with transfusion-dependent thalassemia (TDT). METHODS: In this quasi-experimental study, 20-patients with TDT were enrolled. Each participant received on first visit, freshly collected red cell concentrate (RCC) (<2-days storage) and 15-days later on second visit, 7-days stored blood. Blood samples were obtained immediately before and 24-hours after each transfusion. Differences in the Complete blood counts, bilirubin, LDH, C-Reactive protein, ferritin, and iron levels in the pre- and post-transfusion samples were compared between the first and second transfusion. RESULTS: Fresh blood transfusion resulted in a higher (but non-significant) increase in hemoglobin and other red cell parameters. Notably, a significant increase in white cell counts (WCC) was seen in 7-days stored blood vs fresh blood (1.82×109/l vs 1.01×109/l, P=0.002). No statistically significant difference was found in LDH, direct and indirect bilirubin, creatinine, blood glucose, serum uric acid, serum ferritin, and serum Iron levels. There was a statistically significant rise in C-reactive protein levels in stored (6.43±7.46 mg/dl) versus fresh RCC (1.89±2.38 mg/dl), p-value =0.012. CONCLUSIONS: We show that in patients with chronic TDT, an increase in inflammation-associated markers (WCC and CRP) is observed. Further studies to assess the extent and duration of this increase are needed.

TET2 mutations in acute myeloid leukemia: a comprehensive study in patients of Sindh, Pakistan.

BACKGROUND: The tet oncogene family member 2 (TET2) gene has been reported to be involved in DNA methylation and epigenetic regulation in acute myeloid leukemia (AML). Various studies have proven functional role of TET2 mutations in AML. We herein studied the frequency and genotype-phenotype correlation of TET2 gene in AML patients in Sindh, Pakistan. PATIENTS AND METHODS: The current study was carried out at Liaquat University of Medical & Health Sciences, Jamshoro, Pakistan, in collaboration with National Institute of Blood Disease & Bone Marrow Transplant, Karachi, Pakistan, during the period from June 2019 to June 2020. A total of 130 patients diagnosed with AML were screened for TET2 mutations. Whole exome sequencing of 14 individuals was carried out to find the genetic variants in TET2 gene. The pathogenicity of the variants was predicted by SIFT, PolyPhen2, Mutation Taster and CADD Phred scores. The allele frequency of the variants was compared with global population using 1000 genomes project and Exome Aggregation Consortium (ExAC). Furthermore, exon 3 and exon 5 of the TET2 gene were sequenced by using Sanger sequencing. The findings were correlated with subtypes of AML and corresponding karyotypes. RESULTS: Through the exome sequencing, 17 genetic variants (13 SNPs and four indels) were identified in 14 individuals. Of these, four variants that is, one frameshift deletion, one frameshift insertion and two nonsense variants were novel and not present in dbSNP151 database. Three novel variants were found in exon 3 including two frameshift variants that is, p.T395fs and G494fs, predicted as deleterious by CADD Phred scores, and one stop-gain variant (p.G898X) predicted as deleterious by Mutation Taster and CADD Phred scores. One novel non sense variant (p.Q1191X) was found in the exon 5 predicted as deleterious by SIFT, Mutation Taster and CADD Phred scores. Sanger sequencing analysis revealed one novel deletion at g105233851: del.TAGATAGA, and one novel SNP g;105233861 T>G identified in the TET2 gene. Majority of the exon 3 mutations were seen in the patients diagnosed with AML with maturation, and had a normal karyotype. CONCLUSION: TET2 mutations were identified in around 16% of the total patients of our study indicating other mechanisms being involved in pathophysiology of AML in this cohort. The TET2 mutations provide a prognostic value in determining AML classification.

Biallelic inheritance in a single Pakistani family with intellectual disability implicates new candidate gene RDH14.

In a multi-branch family from Pakistan, individuals presenting with palmoplantar keratoderma segregate in autosomal dominant fashion, and individuals with intellectual disability (ID) segregate in apparent autosomal recessive fashion. Initial attempts to identify the ID locus using homozygosity-by-descent (HBD) mapping were unsuccessful. However, following an assumption of locus heterogeneity, a reiterative HBD approach in concert with whole exome sequencing (WES) was employed. We identified a known disease-linked mutation in the polymicrogyria gene, ADGRG1, in two affected members. In the remaining two (living) affected members, HBD mapping cross-referenced with WES data identified a single biallelic frameshifting variant in the gene encoding retinol dehydrogenase 14 (RDH14). Transcription data indicate that RDH14 is expressed in brain, but not in retina. Magnetic resonance imaging for the individuals with this RDH14 mutation show no signs of polymicrogyria, however cerebellar atrophy was a notable feature. RDH14 in HEK293 cells localized mainly in the nucleoplasm. Co-immunoprecipitation studies confirmed binding to the proton-activated chloride channel 1 (PACC1/TMEM206), which is greatly diminished by the mutation. Our studies suggest RDH14 as a candidate for autosomal recessive ID and cerebellar atrophy, implicating either disrupted retinoic acid signaling, or, through PACC1, disrupted chloride ion homeostasis in the brain as a putative disease mechanism.

Mutation Spectrum and Genotype-Phenotype Analyses in a Pakistani Cohort With Hemophilia B.

This study aimed to (1) identify F9 genetic alterations in patients with hemophilia B (HB) of Pakistani origin and (2) determine the genotype-phenotype relationships in these patients. Diagnosed cases of HB were identified through registries at designated tertiary health-care centers across the country. Consenting patients were enrolled into the study. The factor IX (FIX) coagulation activity (FIX:C) and key clinical features were recorded. Direct sequencing of F9 was carried out in all patients. All the variants identified were analyzed for functional consequences employing in silico analysis tools. Accession numbers from National Center of Biotechnology Information ClinVar database were retrieved for the novel variants. Genotype-FIX:C relationships were determined followed by FIX:C clinical phenotype assessment. A total of 52 patients with HB from 36 unrelated families were identified, which mainly comprised patients with moderate HB (n = 35; 67.3%). Among these, 35 patients from 22 unrelated families could be contacted and enrolled into the study. Missense variants were the most frequent (58.8%), followed by nonsense variants (17.6%). A missense, a short insertion, and a nonsense novel variants in exon 2, 6, and 7, respectively, were also identified. The disease manifested FIX:C heterogeneity in relation to the corresponding mutation in a significant number of cases. Clinical phenotype heterogeneity was also observed in relation to FIX:C-based severity assessment. We concluded that the registered FIX-deficient population of Pakistan mainly comprises moderate HB. F9 mutation spectrum in Pakistani patients with HB is heterogeneous. The HB population of Pakistan manifests a significant amount of genotype-FIX:C and FIX:C-clinical phenotype heterogeneities.