An Efficient Trio-Based Mini-Haplotyping Method for Genetic Diagnosis of Phenylketonuria


Saeed Talebi, M.D., Ph.D, 1Mona Entezam, M.Sc, 1Neda Mohajer, M.Sc, 1Golnaz-Ensieh Kazemi-sefat, M.Sc, 1,2Masoumeh Razipour, M.Sc, 1Somayeh Ahmadloo, M.Sc, 1Aria Setoodeh, M.D, 3Mohammad Keramatipour, M.D. Ph.D., 1,*
Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
Department of Endocrinology, Children’s Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran
Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
Department of Endocrinology, Children’s Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran
*Corresponding Address: P.O.Box: 1417613151 Department of Medical Genetics School of Medicine Tehran University of Medical Sciences Poursina Ave. Keshavarz Blvd Tehran Iran Email:keramatipour@sina.tums.ac.ir
Any use, distribution, reproduction or abstract of this publication in any medium, with the exception of commercial purposes, is permitted provided the original work is properly cited This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Talebi Saeed, Entezam Mona, Mohajer Neda, Kazemi-sefat Golnaz-Ensieh, Razipour Masoumeh, Ahmadloo Somayeh, Setoodeh Aria, Keramatipour Mohammad. An Efficient Trio-Based Mini-Haplotyping Method for Genetic Diagnosis of Phenylketonuria . Cell J. 2016; 18(2): 229-236.

Abstract

Objective

The phenylalanine hydroxylase (PAH) locus has high linkage disequilibrium. Haplotypes related to this locus may thus be considered sufficiently informative for genetic diagnosis and carrier screening using multi-allelic markers. In this study, we present an efficient method for haplotype analysis of PAH locus using multiplexing dyes. In addition, we explain how to resolve the dye shift challenge in multiplex short tandem repeat (STR) genotyping.

Materials and Methods

One hundred family trios were included in this descriptive study. The forward primer of a tetra-nucleotide STR and the reverse primer of a variable number tandem repeat (VNTR) were labeled with three different non-overlapping dyes 5-carboxyfluorescein (FAM), 6-carboxy-N,N,N’,N’-tetramethylrhodamine (HEX) and 6-carboxy-N,N,N’,N’-tetramethylrhodamine (TAMRA). The polymerase chain reaction (PCR) products from each family trio were multiplexed for capillary electrophoresis and results were analyzed using Peak Scanner software.

Results

Multiplexing trio products decreased the cost significantly. The TAMRA labeled products had a significant predictable shift (migrated at a slower electrophoretic rate) relative to the HEX and FAM labeled products. Through our methodology we achieve, the less inter-dye shift than intra-dye shift variance. Correcting the dye shift in the labeled products, according to the reference allele size, significantly decreased the inter-dye variability (P<0.001).

Conclusion

Multiplexing trio products helps to detect and resolve the dye shift accurately in each family, which otherwise would result in diagnostic error. The dye system of FAM, HEX and TAMRA is more feasible and cheaper than other dye systems.