Javed Anver Qureshi

Compound heterozygous ASPM mutations in Pakistani MCPH families

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference (≤4 SD) and mental retardation without any other neurological manifestation. Of the four identified MCPH genes, homozygous truncating mutations in ASPM (MCPH5) account for >50% of all reported families. In spite of the high frequency of MCPH in Pakistan only one case of compound heterozygosity for mutations in ASPM has been reported yet. In this large MCPH study we ascertained 37 families including 319 persons (140 patients). Haplotype analysis of eight STS markers suggested linkage by homozygosity in 20 families, and re‐analysis of single sib ships in the remaining families demonstrated possible compound heterozygosity in two families. Direct sequencing indeed confirmed compound heterozygosity in two and homozygous mutations in 20 families, respectively, showing that up to 10% of families with MCPH caused by ASPM are compound heterozygous. In total we identified 16 different nonsense or frameshift mutations of which 12 were novel thereby increasing the number of mutations in ASPM significantly from 35 to 47. We found no correlation between the severity of the condition and the site of truncation. We suggest that the high frequency of compound heterozygosity observed in this study is taken into consideration as part of future genetic testing and counseling in Pakistani MCPH families.

A subunit vaccine against hydropericardium syndrome using adenovirus penton capsid protein

Hydropericardium syndrome (HPS) is a disease of poultry that is caused by fowl adenovirus-4. Inactivated liver homogenate from diseased birds is still the choice of vaccine in some countries which disseminates numerous pathogens along with inactivated virus. Moreover incomplete attenuation or inactivation, reversion to virulence and the oncogenic potential/genetic instability of the adenoviruses have prevented their use in routine vaccines. To address this problem an effort is made to develop a subunit vaccine. For this purpose penton base protein of HPS virus was expressed in Escherichia coli and used as subunit vaccine in broilers. Immunogenicity of the recombinant penton base protein and challenge protection test against pathogenic virus demonstrated the ability of recombinant penton base protein to confer (90%) protection. Results suggest that the recombinant penton base protein is a candidate for subunit vaccine against HPS.

Investigating the concurrent presence of HCV in serum, oral fluid and urine samples from chronic HCV patients in Faisalabad, Pakistan

Hepatitis C virus (HCV) is normally present in the blood of infected patients; however, it can also be present in some other body fluids. Therefore, in this study, a concurrent presence of HCV-RNA was investigated in oral fluid and urine of 80 Pakistani chronic HCV patients. HCV-RNA was detected in 31 (38.8%) oral fluid and 10 (12.5%) urine samples using RT-PCR in all 80 of the patients whose sera tested positive for HCV-RNA. From this study, it is concluded that, in addition to the blood, HCV RNA can also be found in oral secretions as well as urine of chronic HCV patients.

Detection and screening of bovine leukocyte adhesion deficiency in Pakistan using molecular methods

Bovine leukocyte adhesion deficiency (BLAD) is an autosomal recessive disease. Affected animals die because of extreme susceptibility to infections caused by the lack of a membrane glycoprotein called the leukocyte integrin beta-2 subunit of CD18. The present study was planned to standardize a technique for the diagnosis of BLAD and to get an estimation of BLAD allele in the Pakistani cattle population. The study was performed on 700 animals including Holstein-Friesian (HF) (n=280), Friesian-Sahiwal (FS) (n=120) Sahiwal (n=100) cows and HF calves (n=59) from Government as well as private farms. Similarly 141 bulls of Sahiwal (n=100), HF (n=18) and FS (n=23) from the Semen Production Unit Qadirabad and Kherimorat were also sampled. The identification of normal, carrier and affected animals were made by the PCR-RFLP method. No animal was found homozygous for BLAD while 10 animals including HF calve (n=1), FS bull (n=1), HF (n=6) and FS (n=2) cows were BLAD carrier. The Hardy-Weinberg frequency of the mutant allele in HF and FS population in Pakistan was calculated to 0.01. Thus there is a need of regular screening of the bulls used for artificial insemination to avoid the risk of spreading BLAD in the cattle population of Pakistan.

Molecular characterization of mutations causing β -thalassemia in Faisalabad Pakistan using the amplification refractory mutation system (ARMS-PCR)

BACKGROUND : Faisalabad is the third biggest city of Pakistan. Majority of the population is Punjabi while other ethnic groups are in minority. AIMS : The present study was undertaken to find the mutations causing β -thalassemia in Faisalabad Pakistan. MATERIALS AND METHODS : A total of 285 β -globin alleles from 143 unrelated families having at least one transfusion-dependent child were analyzed by using amplification refractory mutation system (ARMS-PCR). RESULTS : FSC-8/9 (+G) and IVS-I-5 (G ®C) were the most common mutations. The allele frequency for FSC-8/9 (+G) was 38.59% while frequency for IVS-I-5 (G ®C) was 37.89%. The high frequency (76.48%) of IVS-I-5 (G ®C) and FSC-8/9 (+G) on various alleles provides a strong evidence of intermarriages. CONCLUSIONS : By using ARMS-PCR, the mutations were successfully characterized in 95.79% of subjects, while 4.21% remain to be characterized. This study will facilitate the implementations of genetic counseling and prenatal diagnosis in the population of Faisalabad.

Two different point mutations in ABL gene ATP-binding domain conferring Primary Imatinib resistance in a Chronic Myeloid Leukemia (CML) patient: A case report

Imatinib (Gleevec) is the effective therapy for BCR-ABL positive CML patients. Point mutations have been detected in ATP-binding domain of ABL gene which disturbs the binding of Gleevec to this target leading to resistance. Detection of mutations is helpful in clinical management of imatinib resistance. We established a very sensitive (ASO) PCR to detect mutations in an imatinib-resistant CML patient. Mutations C944T and T1052C were detected which cause complete partial imatinib resistance, respectively. This is the first report of multiple point mutations conferring primary imatinib resistance in same patient at the same time. Understanding the biological reasons of primary imatinib resistance is one of the emerging issues of pharmacogenomics and will be helpful in understanding primary resistance of molecularly-targeted cancer therapies. It will also be of great utilization in clinical management of imatinib resistance. Moreover, this ASO-PCR assay is very effective in detecting mutations related to imatinib resistance.

Fowl adenovirus: history, emergence, biology and development of a vaccine against hydropericardium syndrome

The poultry industry has emerged as one of the largest and fastest growing public sectors in the developed and developing countries. Unfortunately, this industry is under a major threat from diseases that are viral (Newcastle disease, infectious bursal disease, influenza, hydropericardium syndrome), bacterial (colibacillosis, pasteurellosis, salmonellosis, mycoplasmosis), parasitic (coccidiosis, histoplasmosis) or nutritional (dyschondroplasia, osteoporosis). Among these diseases, hydropericardium syndrome (HPS) is one of the important emerging diseases occurring in the specific areas of the world where broilers (chickens) are reared under intensive conditions. HPS was first observed in 1987 at Angara Goth, an area near Karachi, Pakistan, where broilers are raised. Since then, HPS has been reported in many countries of the world. From these reported cases, an adenovirus that was either isolated from or visualized electron microscopically in the liver of affected broilers has been implicated in the syndrome. The syndrome has been reproduced by inoculation of isolated fowl adenovirus (FAdV) strains, and hence, the syndrome is also called infectious hydropericardium syndrome. To our knowledge, HPS has not been observed in humans, so it is not considered a zoonotic disease, but it is of economic importance and causes huge losses to the poultry industry. Efforts have been made to develop conventional vaccines against this disease, which were formulated from infected liver homogenate. Formalin-inactivated liver organ vaccines have failed to protect the poultry industry. Hence, there is a dire need to develop a suitable vaccine to combat this disease. Currently, recombinant vaccine candidates are being developed by using molecular biology and biotechnological approaches for the prevention and control of infectious diseases, including HPS. Therefore, it is suggested that the immunogenicity of these recombinant proteins should be evaluated for their use as subunit vaccines.

Evidence for a plasmid conferring salt-tolerance in the plant-root associated, diazotrophKlebsiella sp. NIAB-I

SummaryUsing analytical and preparative methods, we demonstrated the presence of an indigenous plasmid (pNIAB-I) in a diazotroph,Klebsiella sp. NIAB-I isolated, from the roots of Kallar grass, growing on saline lands in Pakistan. The plasmid is approximately 50 kilobase (kb) in size. Transformation experiments indicated that non-halophilic bacteria such asE. coli K12 strain (MV10) andK. pneumoniae M5AI on acquiring this plasmid become tolerant to high salt (NaCl) and alkaline pH.

Prevention of β-Thalassemia in a Large Pakistani Family through Cascade Testing

Background: We report cascade testing of a large Pakistani family for β-thalassemia alleles. The family was still practicing consanguineous marriages and was at risk of having more affected births. Objective: The objective of this study was to show that identification of disease carriers in families with index cases in order to create awareness about disease and provide genetic counseling would result in reduction of the frequency of β-thalassemia in Pakistan. Methods: In this large family with an index case, 27 available living members were tested for β-thalassemia. Carriers of the disease were detected by measuring hemoglobin indices, and amplification refractory mutation system polymerase chain reaction was used for mutation analysis. Genetic counseling was provided to members of this family. Results: There were already 3 marriages between the carrier members and 1 between a carrier and noncarrier in this large family; 12 (44.4%) members were found to carry the mutant gene, representing a very high carrier rate compared to the 5.4% carrier frequency of β-thalassemia in the general population of Pakistan. The family was counseled for prevention of affected births. The initially reluctant family gradually became cooperative and seriously attended the genetic counseling sessions. Conclusion: Cascade testing is more practical than general population screening in a country with limited health facilities where consanguineous marriages are practiced. This report emphasizes the need of extensive testing within families with index cases to identify the carriers of β-thalassemia in order to reduce disease occurrence through awareness and genetic counseling.

β-Thalassemia in Pakistan: a pilot program on prenatal diagnosis in Multan.

Prenatal diagnosis (PND) of &bgr;-thalassemia has been underutilized in Pakistan because of a number of social and economic factors. National Institute for Biotechnology and Genetic Engineering Faisalabad in collaboration with Multan Institute of Nuclear Medicine and Radiotherapy Multan introduced free PND service for carrier couples of Multan district. Multan has a population of about 4 million. More than 170 couples registered for retrospective PND and in 2 years 105 PND were carried out through first trimester chorionic villus sampling. Almost 90% of these couples were unable to afford the cost of PND and would not have undergone the test as free service was not available. Monoplex and Multiplex Amplification Refractory Mutation System-polymerase chain reaction and genomic DNA sequencing were used for detection of IVS (intervening sequence)-I-5 (G-C), FSC (frameshift codon)-8/9 (+G), FSC-41/42 (-TTCT), IVS-I-1 (G-T), 619 bp deletion, and CD-15 (G-A) &bgr;-globin mutations. Eighty-one percent (85/105) couples analyzed were in a consanguineous marriage. Twenty-three fetuses were found homozygous mutant and all couples opted for discontinuation of affected pregnancies. More families are registering for PND after establishment of this free and accessible PND service.