LINKAGE ANALYSIS OF FIRST SIX MODY GENES TO KNOWN LOCI AND SCREENING OF THREE MOST PREVALENT MODY GENES IN PAKISTANI POPULATION

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METHODOLOGY Ascertainment of families
Family affected by Maturity-onset diabetes was obtained through personal contacts.As an initial step, the family history was obtained from the representative members of the family and pedigree was draw.The extensive clinical examination of both patients and their unaffected normal relatives will carry out by expert clinician.Pedigree drawing was performed using the Cyrillic (v2.10) program for genetic implication by the standard method described by [20].The exact genealogic relationships for all the affected individuals were obtained through the extensive personal interviews of elders of the families.Figure 1 shows the pedigree of the family that is clinically diagnosed with MODY.

Collection of blood samples
Blood samples were drawn with informed consent from all members of the family by 5 c.c. syringes in 10ml (to be checked) Vaccutainer tubes (Becton Dickinson, Franklin Lakes, and NJ.) containing acid citrate dextrose (ACD) or heparin.The blood was stored at 4 o C until DNA extraction.

Extraction of Genomic DNA
Genomic DNA was extracted from blood samples using standard methods known as Phenol chloroform method or organic method.This method of DNA extraction from peripheral blood lymphocytes involves 3 days procedure.

Primer Designing
A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing.Specific primers for region of interest in genomic DNA were designed at the Institute of Biomedical and Genetic engineering (IBGE), Islamabad using Bioinformatics Primer Designing Tool Primer3 (http://primer3.sourceforge.net/).These primers were then synthesized commercially (Operon, Germany).

Genotyping
To determine the linkage or exclusion study of the family to six known loci for MODY, a minimum of two microsatellite markers each of the candidate regions of these loci for MODY were genotyped in all the available individuals of this family.Microsatellite Markers used for genotyping this family were selected from Marshfield Map of STRPs set Version 8 available at IBGE.Table 2 shows the Microsatellite Markers that are used to test linkage to known loci of MODY.

Polymerase chain Reaction
Standard PCR reactions will be performed using Bioline or Promega products as illustrated by Batzer et al., (1996) to amplify the DNA.PCR reactions were carried out on ~40ng/ul of DNA in different reaction volumes.A typical reaction mixture consisted of 1 ul 10X PCR buffer [100mM Trizma-HCL, pH 8.3; 500mM KCL; 15mM MgCl2 ; 0.01% (w/v) gelatin )(Bioline)], 2mM dNTPs (Promega), 20uM Forward and reverse primers (Operon, Germany), 5U/ul ul of Taq DNA polymerase (Institute of Biomedical and genetic engineering, Islamabad, Pakistan, Fermentas) and autoclaved deionized water.Each PCR reaction will be consistently performed using the GeneAmp PCR system 9700 thermocycler (Applied Biosystems, foster city, California, USA).The amplifications conditions of each marker along with initial and final concentrations for required number of reactions are shown in the table 3.

Polyacrylamide Gel electrophoresis
Denaturing polyacrylamide gel electrophoresis technique will be employed to distinguish the different alleles of the polymorphic microsatellite markers used in linkage/homozygosity analysis.The standard apparatus used (Bio-Rad Sequi-Gen Cell) measured 38 * 50 cm and utilized thick spacers. .Amplified products were detected by placing the gel on UV transilluminator (Syngene Using GeneScan, ver 3.1.2software, Cambridge, UK), (UV light of wavelength 254 nm) and gel photographs were analysed to determine alleles.

Mutation Detection
To identify genes involved in susceptibility to MODY and to discover new genes and mutations contributing to MODY, following three genes for four different types of MODY will be screened for mutation detection: 1. HNF4α (MODY 1) Bioinfo Publications

GCK (MODY 2)
3. HNF1α (MODY 3) Mutation detection in any of the genes will be done through Single Strand Conformation Polymorphism technique.Prior to SSCP we must set the condition and annealing temperature of each exon.Optimization of exons was done by polymerase chain reaction.Amplification was done in a final volume of 15µl containing 40ng genomic DNA, 1X PCR buffer (Bioline), 0.45mM MgCl2, 200µM dNTPs (Promega) 1µM each forward and reverse primer, 1U Taq DNA polymerase.Amplification was done with initial denaturizing at 94 o C for 2 min, 35 cycles each consisting of denaturation at 94 o C for 45 s, annealing for 45 s, extension at 72 o C for 45 s and final extension for 10 min at 72 o C. Forward and reverse primers for each exon with specific sequence were used in amplification.Amplified products were run on 2% (w/v) agarose gel containing 0.5µg/ml ethidium bromide at constant power supply of 200 volts for 30 minutes.

AGROSE GEL CASTING
The 2 percent agarose gel was prepared by adding 6 gms of agarose in 300 ml 1X TBE buffer (Tris Borate EDTA).Five µl of Orange G dye was added to 5 µl Amplified DNA sample (PCR product) and this mixture was loaded in wells using a micropipette.The 100 bp ladder (Fermentas) was also loaded in a well for size reference.After the gel was loaded, it was placed in a gel tank i.e.Maxicell electrophretic gel system filled with 1X TBE buffer and the negative and positive indicator on the cover and apparatus chamber were properly oriented.In order to visualize the DNA, the gel was placed on a transilluminator (Syngene, Cambridge, UK), (UV light of wavelength 254 nm).

Single Strand conformation polymorphism
Single-strand conformation polymorphism (SSCP), or single-strand chain polymorphism, is defined as conformational difference of single-stranded nucleotide sequences of identical length as induced by differences in the sequences under certain experimental conditions.This property allows distinguishing the sequences by means of gel electrophoresis, which separates the different conformations.A single nucleotide change in a particular sequence, as seen in a double-stranded DNA, cannot be distinguished by electrophoresis, because the physical properties of the double strands are almost identical for both alleles.After denaturation by formamide, single-stranded DNA undergoes a 3dimensional folding and may assume a unique conformational state based on its DNA sequence.The difference in shape between two single-stranded DNA strands with different sequences can cause them to migrate differently on an electrophoresis gel, even though the number of nucleotides is the same, which is, however, a shortfall of SSCP.

RESULTS
The unidentified MODY genes that facilitate to cause Maturity-onset diabetes of the young in European population may play a central role for diabetes characterized by autosomal dominant transmission in Pakistani population.From this research study it is concluded that the examined Pakistani family which is clinically diagnosed with MODY may be due to mutation in other MODY genes except the genes that are screened in the present research study for this family.The results indicate that there is no linkage to the known loci of MODY which are used for linkage in this study.Similarly through genetic screening the following findings come to our acknowledge that no mutation is indicated in this examined family in MODY genes.There may be some environmental factors that are involved in causing this disease in this family or may be this disease is due to mutation in other reported MODY genes that are not screened in this study.

DISCUSSION
In the present research work we aim to first find out the linkage of the most common MODY genes, HNF4α (MODY 1), GCK (MODY 2), HNF1α (MODY 3) to the known loci of MODY by using Microsatellite Markers.Our results of Exclusion study shows that there is no linkage of these known loci to this family that is clinically diagnosed with MODY.Our second objective after doing exclusion study or linkage analysis was to discover new genes or mutation other than known eleven genes or mutations that are reported so far.For this purpose we use the Single Strand Conformation polymorphism.Our findings highlighted that those unidentified MODY genes that facilitate to cause this form of diabetes in European population may play a central role for diabetes characterized by autosomal dominant transmission in Pakistani population.

Table 1 -
Specific primers 5' to 3' sequences of above mentioned four genes for region of interest in genomic

Table 2 -
Microsatellite Markers used to test linkage to known loci of MODY