Balkan Journal of Medical Genetics

GENE MAPPING IN AN ANOPHTHALMIC PEDIGREE OF A CONSANGUINEOUS PAKISTANI FAMILY OPENED NEW HORIZONS FOR RESEARCH
Saleha S, Ajmal M, Zafar S, Hameed A
*Corresponding Author: Dr. Shamim Saleha, Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat 26000, Khyber Paktunkhwa, Pakistan. Tel: +92-922-5291-4659. Cell: +92-333-964-2532. Fax: +92-922-554-556. E-mail: shamimsaleha@yahoo.com
page: 77

MATERIALS AND METHODS

Ascertainment of Family. This study was approved by the Ethics Committee of the Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Khyber Pakhtunkhwa, Pakistan. A consanguineous Pakistani family of the Pashtoon ethnic group with bilateral clinical anophthalmia, in which disease was segregating as an autosomal recessive trait, was ascertained. This family resided in the southern region of Khyber Paktunkhwa, Pakistan, known as Kohat, which is inhabited by various Pashtoon tribes (Figure 1). It was observed that ocular tissue was absent in the orbit of the eyes in affected offspring (Figure 2). On the basis of clinical features assessed by slit lamp examination and radiological assessment by CT scanning, the ophthalmologist diagnosed the anophthalmia as an isolated entity and having no syndromic presentation in the affected daughters. Written informed consent was obtained from the elders of this family to participate in the study. A pedigree of the family was created from information provided by the family using the Cyrillic (version 2.10) program (http://www. cherwell.com). Blood Sample Collection and DNA Extraction. Blood samples were collected in 10 mL vacutainer tubes (Becton Dickinson, Mountain View, CA, USA) with written informed consent from six individuals including four clinically normal (2MOP001, 2MOP002, 2MOP004, and 2MOP005), and two affected (2MOP003, and 2MOP006) daughters of this family. Genomic DNA was extracted from peripheral blood samples following the standard phenolchloroform extraction procedure [21]. Genotyping and Linkage Analysis. Identification of the locus responsible for the isolated clinical anophthalmia phenotype in the selected family, genomic DNA from each individual was genotyped using a microsatellite short tandem repeat (STR) marker for the known clinical anophthalmia loci (Table 1). The microsatellite markers for each locus were amplified by polymerase chain reaction (PCR). Each PCR reaction was performed in a 10 μL volume, containing 1.5 mM MgCl2, 0.6 μM of each forward and reverse primer, 0.2 mM dNTPs, 1 U Taq DNA polymerase and PCR buffer [16 mM (NH4) 2SO4, 67 mM Tris-HCl (pH 8.8), and 0.01% of the nonionic detergent Tween-20] (Bioline Reagents Ltd., London, UK). Amplification was performed with an initial denaturation for 4 min. at 94 °C, followed by 35 cycles of denaturation at 94 °C for 35 seconds, annealing at 55 °C for 35 seconds, extension at 72 °C for 35 seconds, and a final extension at 72 °C for 7 min. The PCR products were separated on 10.0% nondenaturing polyacrylamide gels (Protogel; National Diagnostics, Edinburgh, Scotland, UK). The gel was stained with ethidium bromide and photographed under UV illumination. Alleles were assigned to individuals and genotypic data was used to find genotypes of all individuals of this family. The phenotype was analyzed as an autosomal recessive trait. Mutation Screening. All individuals of this family were screened for mutations in the candidate gene. Polymerase chain reaction amplification of DNA of both normal and affected individuals was performed with forward and reverse primers sets, spanning the whole exonic region and promoter region of the candidate gene (Tables 2 and 3) that were designed using online available Primer 3 (http:// primer3plus.comweb_3.0.0/primer3web_input. htm) software. The PCR amplification was performed in a 50 μL reaction volume containing 250 ng of genomic DNA, amplification buffer containing 600 nM of each primer, 1.5 mM MgCl2, 200 mM of dNTPs and 2.5 U of Taq DNA polymerase (Applied Biosystems Ltd., Warrington, Cheshire, UK) in an PxE thermal cycler (Hybaid, Basingstoke, Hampshire, UK). The amplification conditions were 95 °C for 5 min., followed by 35 cycles of 95 °C for 45 seconds, primer specific annealing temperature (55 to 65 °C) for 45 seconds, and 72 °C for 45 seconds. Aliquots (5 μL) of the PCR products were analyzed by 2 to 2.5% agarose gel. The PCR products were then purified using QIAquick PCR Purification Kit (Qiagen Ltd., Crawley, West Sussex, UK) and sequenced directly using Big Dye® Terminator v3.1 cycle sequencing kit in an ABI PRISM® 3130 genetic analyzer (Applied Biosystems, Foster City, CA, USA).

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