MUTATION IN PHOSPHOLIPASE C, δ1 (PLCD1) GENE UNDERLIES HEREDITARY LEUKONYCHIA IN A PASHTUN FAMILY AND REVIEW OF THE LITERATURE
Khan AK, Khan SA, Muhammad Na, Muhammad No, Ahmad J, Nawaz H, Nasir A, Farman S, Khan S
*Corresponding Author: Saadullah Khan, Ph.D., Department of Biotechnology & Genetic Engineering, Kohat University of Science & Technology, Banu Road, Kohat 26000, Khyber Pakhtunkhwa, Pakistan. Tel: +92-333-506-8108. Fax: +92-0922- 554-556. E-mail: saadkhanwazir@gmail.com; saad@kust.edu.pk
page: 69

MATERIALS AND METHODS

Human Subjects and DNA Samples. In order to investigate at the molecular level, a three-generation family was collected from a remote region of Pakistan. Prior to commencement of the clinical and molecular investigations, written informed consent signed by the legal guardians, the parents, on behalf of the affected children, and they agreed to the publication of the study outcomes. Ethical approval of the study was obtained from the Institutional Review Board (IRB), Kohat University of Science and Technology (KUST), Khyber Pakhtunkhwa, Pakistan. In order to identify the causative gene defect, peripheral blood samples were collected from four affected (II-2, III-1, III-2, III-3) and two unaffected individuals (II-1, III-4) in EDTA-containing vacutainer sets (Becton Dickin-son & Company, Franklin Lakes, NJ, USA) (Figure 1; Figure 1A). Genomic DNA was extracted from the blood by using Nucleospin® Blood kit (Macherey-Nagel, Düren, Germany). Nanodrop-1000 spectrophotometer (Thermal Scientific, Wilmington, NC, USA) was used for DNA quantification, measuring optical density at 260 nm and diluted to 40.0-50.0 ng/μL-1 for amplification by polymerase chain reaction (PCR). Candidate Gene (PLCD1) Screening. Entire coding regions and splice junction sites of the gene were amplified by PCR and screened by DNA sequencing for potential sequence variants. The primer3 program (http://primer3. sourceforge.net/) was used to design intronic primer pairs for individual exons amplification, and basic local alignment search tool (BLAST; http://www.ncbi.nlm.nih. gov/ blast) was used to check for specificity. To obtain a DNA sequence for the gene, UCSC Human Genome browser (http://www.genome.ucsc.edu/cgi-bin/hgGateway) was used. Purification of the PCR-amplified DNA was performed with commercially available kits (Marligen Biosciences, Ijamsville, MD, USA). The amplification conditions were 5 min. at 95 °C., followed by 30 cycles of 12 seconds at 95 °C, 5 seconds at 50 °C, and 4 min. at 60 °C, with a final extension at 60 °C for 20 min. The ampli-fied PCR products were sequenced using an ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Bioedit sequence alignment tool (Bioedit editor version 6.0.7) was used to align the sequence of each amplicon. Protein Structure Prediction. The three-dimensional (3D) model of normal and mutant PLCD1 protein was predicted using the phosphoinositide-specific phospholipase c-delta1 structure (PDB ID 1DJZ) as a template. The structure for Cys209Arg mutant was developed by changing the selected residue into the desired residue and follow refinement of the structure by subjecting it to similar energy minimization protocol that was used for the wild structure. Comparative modeling was performed using molecular operating environment (MOE; https:// www. chemcomp.com/MOE-Molecular_Operating_ Environment. htm/) software and structure was visualized with the PyMOL Software (www.pymol.org) [7].



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