MOLECULAR GENETIC STUDIES OF PAKISTANI POPULATIONS
Introduction
Revolutionary discoveries in molecular biology have opened up new vistas for research. It has been known for years that genetic characters are transmitted from parents to their offspring. However, it has been few decades ago that it is possible to study this phenomen at the molecular level.
All this has become due to the abilities to cut DNA molecules into smaller fragments with Restriction Endonucleases:
to engineer them into microorganisms for producing unlimited number of a particular DNA piece,
to sequence DNA,
to identify genes and ultimately their protein products.
The genetic etiology of many diseases have been discovered due to the awesome advancement of modern Nucleic Acid technology. In order to determine the gene responsible for a particular disease two basic approaches have been employed:
If the Biochemical lesion responsible for the disease is known, a candidate gene is analysed, as in the extensively studied B-Thalassaemia(Diaz-Chico JC., 1988) and Sickle-cell Anaemia(Novitski E., 1977).
If the chemical pathway is unknown and the genetic etiology of the diseased gene is to be discovered, an indirect statistical approach called linkage mapping has been used.
The gene responsible for Cystic Fibrosis was discovered using this technique(Kerem B., et al, 1989). It depends on the availability of gene probes that provide polymorphic genetic markers which are interpersed throughout the human genome and of statistical analysis using highly sophisticated computer programmes(Ott J., 1974). Not only it is now possible to attempt to unravel causes of diseases but also to examine the extraordinary diversity between and within livings species including man. Research on all aspects of life whether it be agricultural, clinical or anthropological etc., has continuously progressed in the developed world. Unfortunately, such a tradition is in absentia in Pakistan, only few laboratories have conducted some primitive research in this field.
Pakistan has few advantages with respect to such investigations which deals with the transmission of genetic characters. For several years, cousin marriages have led to the segregation of disease gene(s) in large, multigenerational families that are still largely nuclear. in addition, the practice of marrying within ethnic groups for social, religious or logistic purposes have also created pockets of populations that inhabit various areas of Pakistan.
It was mentioned earlier that no work has been carried out on Pakistani populations whether they may be of clinical or anthropological interest. A few examples of work done on other populations of the world in lieu of conducting the similar studies to be carried out in Pakistani populations.
During the past years, the availability of genetic markers that segregate with diseases in families has allowed:
The localisation of diseased gene and
The isolation of diseased gene using positional cloning approaches(Collins, 1992).
These markers are originally detected by Southern hybridisation and were termed Restriction fragment length Polymorphism(RFLP),(Botstein et al., 1980). Frequently used technique in this domain is PCR(Polymerase Chain Reaction) which enables the rapid amplification and detection of specific genomic sequence, has rapidly replaced RFPL-based studies. For PCR-based analysis, simple sequence repeats(SSR), like di-, tri-, and tetra-nucleotide repeats are very useful markers becuase of their variable numbers throughout the genome(Weber and May, 1989; Litt and Luty, 1989; Edwards et al., 1991). They have proved to be of great value in construction of linkage maps of chromosomes(Bowcock A., 1993) and the localisation of the diseased gene e.g., Wilson disease(WD), (Stewart EA., 1993).
At the biochemical and genetic engineering division of Dr. A.K Research Laboratories, RFLP was used to search human genome for the disease susceptibilty locus(DSL) for Tubercle bacillus diseases, which has been shown to be present on chromosome 1 of the mouse(Skamene et al., 1982). These studies have shown the abscence of of such a gene in a homologus chromosome 2. Furthermore, these studies established the relative sequence of nine genes excluding the disease susceptibility locus(Lsh\Beg\Ity), on the long arm of chromosome 2(Khaliq S, 1994). On the other hand, in a large scale search using microsatellite markers, a new gene responsible for retinitis pigmentosa (arRP) was found to be proximal to the microsatellite marker D1S53 in large Pakistani family(Leutelt et al., 1998).
Migraine headaches frequently runs in families suggesting that hereditary factors are the culprits. Typical migraine is a complex neurological disorder comprises of two main subtypes: migraine with aura (MA) and without aura (MO). The Migraine is classified as a chronic, painful and debilitating disease which manifest recurrent and severe headache attacks accompany with nausea, vomiting and photophobia or sonophobia in certain cases(Rod A, 2003). The disease etiology is still unknown, but family studies provide strong evidence that defective genes play an important role. In recent years a gene for familial hemiplegic migraine(FMH), a rare autosomal dominant subtype of migraine with aura, was mapped to chromosome 19p13. The findings of mutations in the gene 19p13, coding for the pore-forming subunit (alpha1A) of neuronal voltage-dependent P/Q-type calcium channels (FHM1), and in the ATP1A2 gene (1q21-23), encoding the alpha2-subunit of the Na+, K+ ATPase ionic pump (FHM2). A dysfunction of these channels modifies neuronal function and resulting in migraine with aura(Fumal and Schoenen, 2004).
Plan of Work:
It is the objective of this work to conduct similar studies among Pakistani population, using modern and complementry gene-mapping technologies.
Materials:
Blood samples from the screened patients. Lymphocytes will be separated by overlaying blood samples on Ficoll hypaque, centrifuging and collection of cells from the interface(Boyum A., 1968). Immortalised B-Lymphoblastoid cell lines will be generated by transformation with EBV as a substantial amount of DNA will be required to analyse the samples. the cell lines will be cryopreserved in a freezing mixture containing 10% DMSO, 45% heat inactivated foetal calf serum and 45% RPMI-1640(Walls EV., et al., 1987).
DNA Preparation:
DNA will be prepared from periphral blood cells and EBV-transformed cells by the method described in Maiatis et al., 1982. The techniques described below will be used to search disease gene.
RFLP Analysis
PCR
SNP and dinucleotide Repeat Marker
Agarose gel Electrophoresis.
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