The Development of an Amplification Specific PCR Protocol for the Detection of the Presence of Two Gaucher Mutations in the Ashkenazi Jewish Population




Yael Helin

Introduction

Gaucher disease is the most common lysosomal disease and is highly prevalent in the Ashkenazi Jewish (AJ) population. It is an autosomal recessive disorder and it caused by a deficiency in glucocerebrosidase enzymatic activity due to various mutations in the gene encoding lysosomal glucocerebrosidase (GBA). Glucocerebrosidase is found in the lysosomes of nearly all cell types, and is responsible for the degradation by hydrolysis of metabolic intermediates derived from the cellular turnover of cell membrane (Zuckerman et al, 2007).
In Gaucher disease those intermediates accumulate in various organs, especially in cells of mononuclear phagocyte origin and cause severe pathological consequences (Jmoudiak & Futerman, 2005) . These macrophage cells containing these accumulations are also known as ‘Gaucher cells" (fig. 1).
The phenotypes of the disease are classified into three types. Type 1 of the Gaucher disease is the most frequent type occurring in approximately 90% of the patients and also highly prevalent in AJ.
Types 2 & 3 are rare and violent forms of Gauchers disease. They include neurological involvement resulting in death in the first years of life in type 2, and in the forth decades in type 3 (Cox, 2010 and Jmoudiak & Futerman, 2005).
In this experiment an amplification specific PCR protocol for the detection of the presence of two Gaucher mutations in the AJ population was developed.
PCR using two sets of primers for each mutation (Fig. 2) was performed to produce two Gaucher mutations – positive controls. For the amplification of the two positive controls, PCR was performed using different sets of primers.

Results

Mutation IVS2+
For IVS2+1 mutation only two primers, 2B and 2D specifically produced a band in the presence of the mutation but not in the absence of the mutation. Primers 2C and 2E produced a band in the presence of the mutation and a different size band in the absence of the mutation. Primers MF and 2A produced a band in the presence and in the absence of the mutation. Primers 2C and 2D did not produce any band in the presence or the absence of the mutation (Fig. 3).

Mutation 1226G
For mutation 1226G only one primer, 2C’ produced a band in the presence of the positive control but not in the absence of it. Primers MF’, 2A’, 2B’ and 2D’ produced a band in the presence and in the absence of the positive control (Fig. 4).

Discussion
In this experiment, two amplification PCR protocols were developed for two Gaucher mutations in the AJ population.
More experiments should be performed using new designed primers in order to develop more amplification PCR protocols for other Gaucher mutations in the JA population and in non Jewish population.
Detection of Gaucher disease mutations is a valuable tool for determining gene frequency for the disease and for carrier screening in order to prevent severe, untreatable Gaucher disease by identifying couples at risk before the birth of an affected child.

Literature cited

Marina Jmoudiak and Anthony H. Futerman (2005). Gaucher disease: pathological mechanisms and modern Management. British Journal of Haematology, 129, 178–188.

Shachar Zuckerman, MSc Amnon Lahad, MD, MPH Amir Shmueli, PhD Ari Zimran, MD Leah Peleg, PhD Avi Orr-Urtreger, MD, PhD Ephrat Levy-Lahad, MD (2007). Carrier Screening for Gaucher Disease - Lessons for Low-Penetrance, Treatable Diseases. JAMA, Vol 298: 1281-1290.

Timothy M Cox (2010). Gaucher disease: clinical profile and therapeutic developments Biologics: Targets & Therapy:4 299–313.









Figures


Figure 1-Gaucher cells.


Figure 2-The normal sequences and the mutated sequences that used as primers for generating the positive controls.


Figure 3-A. PCR products using six different primers in the absence of and in the presence of a positive control (the plasmid). B. Six mutated primers to amplify IVS2+1 mutation.


Figure 4-A. PCR products using five different primers in the absence of and in the presence of a positive control (the plasmid). B. Five mutated primers to amplify 1226G mutation.


Abstract
Gaucher disease is an autosomal, recessive, lysosomal storage disease most prevalent in the Ashkenazi Jewish population. Gaucher disease is caused by a deficiency in glucocerebrosidase enzymatic activity, with subsequent accumulation of glucosylceramide in various organs. In the Jewish Ashkenazi population there are four known mutations that cause the disease:1226G (N370S),84GG,IVS2+1 and 1448C (L444P). By using PCR mutagenesis, here we developed an amplification specific PCR protocol for the detection of the presence of two Gaucher mutations in the Ashkenazi Jewish population. Developing an amplification specific PCR protocol for the mutations of Gaucher disease is very important both for detecting carriers and homozygotes for the disease and for determine mutations frequencies.

Full Paper

Acknowledgments

I would like to thank Alex Bulanov and Xie Xie for their help, infinite patience and support, without which this project would not have been possible. In addition I would like to thank Dr. Berish Rubin for his guidance and providing the opportunity to take a part in this project and to achieve this amazing experience. I would like to thank also Dr. Sylvia Anderson for helping and answering any question I had.


This document was last modified 05/19/2011.
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