Muhammad Farooq Sabar

Five most common prognostically important fusion oncogenes are detected in the majority of Pakistani pediatric acute lymphoblastic leukemia patients and are strongly associated with disease biology and treatment outcome.

BACKGROUND AND OBJECTIVES Acute lymphoblastic leukemia (ALL) is a complex genetic disease involving many fusion oncogenes (FO) having prognostic significance. The frequency of various FO can vary in different ethnic groups, with important implications for prognosis, drug selection and treatment outcome. METHOD We studied fusion oncogenes in 101 pediatric ALL patients using interphase FISH and RT-PCR, and their associations with clinical features and treatment outcome. RESULTS Five most common fusion genes i.e. BCR-ABL t (22; 9), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (del 1p32) were found in 89/101 (88.1%) patients. Frequency of BCR-ABL was 44.5% (45/101). BCR-ABL positive patients had a significantly lower survival (43.7±4.24 weeks) and higher white cell count as compared to others, except patients with MLL-AF4. The highest relapse-free survival was documented with ETV6-RUNX1 (14.2 months) followed closely by those cases in which no gene was detected (13.100). RFS with BCR-ABL, MLL-AF4, TCF3-PBX1 and SIL-TAL1 was less than 10 months (8.0, 3.6, 5.5 and 8.1 months, respectively). CONCLUSIONS This is the first study from Pakistan correlating molecular markers with disease biology and treatment outcome in pediatric ALL. It revealed the highest reported frequency of BCR-ABL FO in pediatric ALL, associated with poor overall survival. Our data indicate an immediate need for incorporation of tyrosine kinase inhibitors in the treatment of BCR-ABL+ pediatric ALL in this population and the development of facilities for stem cell transplantation.

Prognostically Significant Fusion Oncogenes in Pakistani Patients with Adult Acute Lymphoblastic Leukemia and their Association with Disease Biology and Outcome

BACKGROUND AND OBJECTIVES Chromosomal abnormalities play an important role in genesis of acute lymphoblastic leukemia (ALL) and have prognostic implications. Five major risk stratifying fusion genes in ALL are BCR-ABL, MLL-AF4, ETV6-RUNX11, E2A-PBX1 and SIL-TAL1. This work aimed to detect common chromosomal translocations and associated fusion oncogenes in adult ALL patients and study their relationship with clinical features and treatment outcome. METHODS We studied fusion oncogenes in 104 adult ALL patients using RT-PCR and interphase-FISH at diagnosis and their association with clinical characteristics and treatment outcome. RESULTS Five most common fusion genes i.e. BCR-ABL (t 9; 22), TCF3-PBX1 (t 1; 19), ETV6-RUNX1 (t 12; 21), MLL-AF4 (t 4; 11) and SIL-TAL1 (Del 1p32) were found in 82/104 (79%) patients. TCF3-PBX1 fusion gene was associated with lymphadenopathy, SIL-TAL positive patients had frequent organomegaly and usually presented with a platelets count of less than 50 x10(9)/l. Survival of patients with fusion gene ETV6-RUNX1 was better when compared to patients harboring other genes. MLL-AF4 and BCR-ABL positivity characterized a subset of adult ALL patients with aggressive clinical behaviour and a poor outcome. CONCLUSIONS This is the first study from Pakistan which investigated the frequency of 5 fusion oncogenes in adult ALL patients, and their association with clinical features, treatment response and outcome. Frequencies of some of the oncogenes were different from those reported elsewhere and they appear to be associated with distinct clinical characteristics and treatment outcome. This information will help in the prognostic stratification and risk adapted management of adult ALL patients.

Isolation and characterization of two new hepatitis E virus genotype 1 strains from two mini-outbreaks in Lahore, Pakistan.

BackgroundPakistan is a highly endemic area for hepatitis E virus (HEV) infection. The aim of the current study was to isolate and characterize strains of HEV in two mini outbreaks.ResultsRNA was extracted and reverse transcribed to cDNA. Nested PCR was done for the detection of HEV RNA. The positive bands were eluted, cloned in TA vector and sequenced in both directions using genetic Analyzer (Applied Biosystem Inc, USA). Phylogenetic analysis was done using MEGA4 software. We isolated two new HEV genotype-1 strains from Lahore, Pakistan, based on cloning and sequencing of ORF2 region.ConclusionsOur study suggest that both the human HEV strains were closely related to the Sar-55 but different from the Abb-2B and 87-Pakistan-B HEV isolates sharing 88-91% sequence identity to Pakistani isolate Sar-55. These results indicated that Sar-55 is the main endemic HEV strain in various areas of the country.

Sequence variants on 17q21 are associated with the susceptibility of asthma in the population of Lahore, Pakistan

Abstract Objective: Single nucleotide polymorphisms (SNPs) on 17q21 are known to be associated with asthma disease in multiple populations. This study was designed to know whether this region is associated with asthma in Lahore region population or not. Methods: A total of 200 asthma patients and 100 healthy controls were enrolled from different hospitals of Lahore, Pakistan. Twelve SNPs from chromosomal region 17q21 were analyzed in cases and controls by single base extension method and capillary-based genetic analyzers. Associations with asthma were checked using basic allelic model, genotypic model, and results were adjusted by logistic regression analysis using PLINK v1.9. Pair-wise linkage disequilibrium among the SNPs was analyzed by using Haploview software. Results: SNP rs3816470 showed a strong association (p = 8.89 × 10−5, Odd Ratio = 3.082 [1.755-5.41]) with asthma, whereas rs3859192 and rs6503525 also showed a significant association with the development of asthma, especially in the case of positive family history. In LD block1 (93 kb) consisting of six SNPs (rs12936231, rs7216389, rs7216558, rs9894164, rs1007654 and rs7212938), none of the haplotypes show any significant association with asthma except the haplotype “CCTCAG”, which is a significant protective factor against asthma having frequency 0.051 in controls while 0.017 in cases (p = 3.56 × 10−2, χ2 = 4.415). Conclusion: The present study reports that the polymorphic genomic variant rs3816470 is significantly and independently associated with asthma in the studied population, while the variants, rs6503525 and rs3859192, also indicate a significant association with asthma in this population when family history of the disease is taken as a covariate.

Presence of novel compound BCR-ABL mutations in late chronic and advanced phase imatinib sensitive CML patients indicates their possible role in CML progression

ABSTRACT BCR-ABL kinase domain (KD) mutations are well known for causing resistance against tyrosine kinase inhibitors (TKIs) and disease progression in chronic myeloid leukemia (CML). In recent years, compound BCR-ABL mutations have emerged as a new threat to CML patients by causing higher degrees of resistance involving multiple TKIs, including ponatinib. However, there are limited reports about association of compound BCR-ABL mutations with disease progression in imatinib (IM) sensitive CML patients. Therefore, we investigated presence of ABL-KD mutations in chronic phase (n = 41), late chronic phase (n = 33) and accelerated phase (n = 16) imatinib responders. Direct sequencing analysis was used for this purpose. Eleven patients (12.22%) in late-CP CML were detected having total 24 types of point mutations, out of which 8 (72.72%) harbored compound mutated sites. SH2 contact site mutations were dominant in our study cohort, with E355G (3.33%) being the most prevalent. Five patients (45%) all having compound mutated sites, progressed to advanced phases of disease during follow up studies. Two novel silent mutations G208G and E292E/E were detected in combination with other mutants, indicating limited tolerance for BCR-ABL1 kinase domain for missense mutations. However, no patient in early CP of disease manifested mutated ABL-KD. Occurrence of mutations was found associated with elevated platelet count (p = 0.037) and patients of male sex (p = 0.049). The median overall survival and event free survival of CML patients (n = 90) was 6.98 and 5.8 y respectively. The compound missense mutations in BCR-ABL kinase domain responsible to elicit disease progression, drug resistance or disease relapse in CML, can be present in yet Imatinib sensitive patients. Disease progression observed here, emphasizes the need of ABL-KD mutation screening in late chronic phase CML patients for improved clinical management of disease.

Detection of Exon 12 and 14 Mutations in Janus Kinase 2 Gene Including a Novel Mutant in V617F Negative Polycythemia Vera Patients from Pakistan

The most frequently reported genetic aberration among polycythemia vera (PV) patients is a gain of function mutation V617F in exon 14 of Janus kinase 2 (JAK2) gene. However in many investigations, V617F negative PV patients have been reported to harbor mutations in JAK 2 exon 12. We investigated 24 patients with PV (diagnosed following 2016 WHO guidelines) to detect V617F mutation through allele specific PCR. The frequency of which was found to be 19/24 (79.2 %). Later on JAK2 exon 12 and 14 was amplified by conventional PCR in V617F negative patients and subjected to sequence analysis. A total of 03 mutated sites in exon 12 were detected in only two V617F-negative patients 2/5 (40%). All three substitutions were heterozygous i.e. F537F/I found in both patients and R528R/T, which is a novel mutation. In addition, one patient 1/5 (10%) manifested amino acid substitution V617A in JAK2 exon 14. Hematological parameters of individuals harboring mutations do not vary significantly than rest of the PV patients. Previous history and 2.3 years of follow-up studies reveal 15-year survival of V617F positive patients (n=19) to be 76%, while it is 94% for wild type V617 patients (n=05). Mean TLC of the patient cohort was 17.6± 9.1 x 109/L, mean platelet count was 552± 253 x 109/L, mean hemoglobin was 16.9± 3.2 g/dl, mean corpuscular volume (MCV) was 77.2± 13.0 fl and mean corpuscular hemoglobin (MCH) was 25.6± 3.9 pg. This is the very first attempt from Pakistan to screen JAK2-exon 12 mutations in PV patients. We further aim to investigate Jak2 exon 12 mutations in larger number of PV patients to assess their clinical relevance and role in disease onset, progression and transformation.

rs12603332 is associated with male asthma patients specifically in urban areas of Lahore, Pakistan

ABSTRACT Objective: rs12603332, an important regulatory site variant, is known to alter the regulatory motif E2A that is involved in the maturation of B-lymphocytes. The study was designed to check whether different environmental exposures alter its risk allele association with asthma or not. Methods: 200 Physician-diagnosed asthma patients and 108 healthy individuals were enrolled from hospitals of Lahore. After quantitation of DNA extracted from peripheral blood, amplification of genomic region with rs12603332, followed by single base extension (SBE), was performed. Allele and genotype frequencies were calculated by SHEsis and Haploview software packages. Statistical analyses on PLINK were also performed, taking different factors as covariates. HaploReg analysis was done to predict the effect of risk allele on different regulatory motifs. Results: Risk allele for rs12603332 i.e., “C” allele was found to be significantly associated with male patients residing in urban localities. Conclusion: The finding suggests that on exposure with urban environment, risk allele carriers tend to develop asthma symptoms via epigenetic regulation of motif associated with maturation of B-lymphocytes.

Genetic variants of ADAM33 are associated with asthma susceptibility in the Punjabi population of Pakistan.

Abstract Objective: A disintegrin and metalloproteinase 33 (ADAM33) gene has been considered as an asthma susceptibility gene due to its possible role in airway remodeling, abnormal cell proliferation, and differentiation. Association of this gene with asthma has been reported in several genetic studies on various populations. The current study aims to evaluate the association of ADAM33 gene polymorphisms with the risk of asthma in the Punjabi population of Pakistan. Method: A total of 101 asthma patients and 102 age-matched healthy controls from Lahore, a city in Punjab, were recruited. ADAM33 single nucleotide polymorphisms (SNPs) T + 1[rs2280089], T2[rs2280090], T1[rs2280091], ST + 5[rs597980], ST + 4[rs44707], S2[rs528557], Q − 1[rs612709], and F + 1[rs511898] were genotyped in both patients and controls using single base extension and capillary electrophoresis-based genetic analyzer. The basic allelic and genotypic model was analyzed for association of the SNPs with asthma using SHEsis software. Haploview software was used to calculate pairwise linkage disequilibrium (LD) among six of the genotyped SNPs. Results: Of the 8 SNPs genotyped, only S2[rs528557] showed significant association with asthma (Allele p = 0.0189, Genotype p = 0.021). SNPs T + 1[rs2280089], T2[rs2280090], T1[rs2280091], ST + 4[rs44707], S2[rs528557], and Q − 1[rs612709] were found to be in moderate to strong LD. The significantly higher frequency of haplotype “AAGTCG” in healthy controls suggests a protective effect against asthma risk in the studied population (p = 0.0059). Conclusion: These findings suggest that genetic variants of ADAM33 gene may play important roles in asthma susceptibility in the Punjabi population of Pakistan.