Haemochromatosis Detection

HAEMOCHROMATOSIS DETECTION USING PCR-RFLP

INTRODUCTION

Hereditary Haemochromatosis (HH), first described in 1865, is a genetic disorder of metabolism, characterized by progressive iron overload resulting from abnormalities in intestinal iron absorption and or release of iron from reticuloendothelial cells . It is an autosomal recessive disorder, where the body accumulates excessive iron, which is deposited in a variety of organs. Iron cannot be excreted, thus, the excess builds to toxic levels in tissues of major organs such as the liver, heart, pituitary, thyroid, pancreas, lungs, and synovium (joints). These organs cease to function adequately and eventually become diseased. Serious illnesses such as diabetes, cirrhosis, hepatoma, hypogonadotrophic hypogonadism, cardiomyopathy and arthritis may be a consequence of this disease . It affects one in every three hundred Caucasians, and one in nine is a carrier , hence, making its early detection vital. The gene responsible for HH (HLA-H) was recently identified on the short arm of chromosome 6 and is thought to be mainly caused by a mutation of a gene called HFE, which allows excess iron to be absorbed from the diet . This mutation is known as C282Y. A single point mutation occurs, in which the amino acid cysteine at position 282 changes to a tyrosine . To develop haemochromatosis two genes, one from each parent, are required to be C282Y. However, not everyone with the mutation may develop the disease and it may occur if only one C282Y gene is present (4). 77.5% of affected individuals have two copies of the C282Y mutation, one inherited from each parent, while about 4% have a single copy of the mutation and one normal HFE gene .

First proposed in early 1970’s, Polymerase Chain Reaction (PCR) has been identified as a simple, robust, speedy, and most of all, flexible method that can be used to detect haemochromatosis . In this technique, specific DNA sequences are amplified for the detection of mutations that may be present, allowing early diagnosis of hereditary heamochromatosis (see figure 1). It is a major development in the analysis of DNA and RNA. The requirements of the reaction are simple, consisting of deoxynucleotides to provide both the energy and nucleosides for the synthesis of DNA, template, primer, DNA polymerase, and buffer containing magnesium . The crux of the PCR procedure involves three s...

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...’Med J Aust. 2001 Oct 15;175(8):418-21(PubMed): http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11700835&dopt=Abstract

11 ‘DNA Testing for Hereditary Hemochromatosis’: Molecular Genetic Testing in Mainstream Medicine (Vol. 14: Spring, 1997) http://www.mostgene.org/gd/gdvol14c.htm

12 http://www.aiddiagnostika.com/english/kits/GenID/rdb_2045e_haemochromatosis.htm

13 http://www.mja.com.au/public/issues/179_10_171103/ger10460_fm-2.html

14 Marion K. Stott, Andrew P. Fellowes, Jeff D. Upton, Michael J. Burt and Peter M. Georgea : ‘Simple Multiplex PCR for the Simultaneous Detection of the C282Y and H63D Hemochromatosis (HFE) Gene Mutations’ http://www.clinchem.org/cgi/content/full/45/3/426

15 http://bip.weizmann.ac.il/mb/bioguide/pcr/PCRwhat.html

16 http://allserv.rug.ac.be/~avierstr/principles/pcr.html

17 Alka Dwivedi, B.C. Sarin, Dev Mittar, P.K. Sehajpal: (2003) ‘Optimization of 38kDA based PCR assay for detection of mycobacterium tuberculosis from clinical samples’

18 McPherson, M.J.(Author). ‘PCR’. - Oxford, , GBR: BIOS Scientific Publishers Ltd, 2000. p 70.

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