Short reports 275

5 Hubert R, Weber JL, Schmitt K, Zhang L, Arnheim N. A 8 Barrett MT, Reid BJ, Joslyn G. Genotypic analysis of multi- new source of polymorphic DNA markers for sperm ple loci in somatic cells by whole genome amplification. typing: analysis of microsatellite repeats in single cells. Am NucleicAcids Res 1995;23:3488-92. JHum Genet 1992;51:985-91. 9 Roth Emmert-Buck 6 Zhuang Z, MJ, MR, Lubensky IA, Kristjansson K, Chong SS, Van den Veyver IB, Subrama- Liotta Solomon D. Detection von Mol Path: first published as 10.1136/mp.50.5.275 on 1 October 1997. Downloaded from nian S, Snabes MC, Hughes MR. Preimplantation single LA, of the Hippel- cell analyses of deletions using whole Lindau gene deletion in cytologic specimens using genome amplification. Nat Genet 1994;6:19-23. microdissection and the polymerase chain reaction. Acta 7 Snabes MC, Chong SS, Subramanian SB, Kristjansson K, Cytol 1994;38:671-5. DiSepio D, Hughes MR. Preimplantation single-cell analy- 10 Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, sis of multiple genetic loci by whole-genome amplification. Zhuang Z, Goldstein SR, et al. Laser capture microdissec- Proc Nail Acad Sci USA 1994;91:6181-5. tion. Science 1996;274:998-1000.

A PCR-SSP method for detecting the Cys282Tyr mutation in the HFE gene associated with hereditary haemochromatosis

David Smillie

Abstract G is replaced by A at nucleotide position 845 in Hereditary haemochromatosis is a com- the cDNA sequence (845G-A), results in the mon genetic disorder that causes hyper- substitution of tyrosine for cysteine at amino absorption of dietary iron, leading to acid position 282 in the transcribed increased deposition and various organic (Cys282Tyr); this critically affects an intramo- diseases. Early diagnosis is important if lecular disulphide bond.3 It is possible that the effective treatment is to be applied and the products of MHC class I related are corrected before the onset of involved in iron and that this func- clinical symptoms. Recently, a candidate tion is compromised by a mutation that affects http://mp.bmj.com/ gene has been identified in which a single the structure of the gene product. point mutation shows a very close associ- Over 83% ofhaemochromatosis patients are ation with hereditary haemochromatosis. homozygous for the Cys282Tyr mutation in A polymerase chain reaction method the HFE gene,"4 compared with - 0.2% in using sequence specific primers (PCR- normal controls. The PCR-SSP method de- SSP) is described that, in conjunction scribed here allows rapid and specific typing with a simple DNA extraction method, for this haemochromatosis associated muta- on September 25, 2021 by guest. Protected copyright. would provide a specific diagnostic test or tion. rapid screening procedure for this puta- tive haemochromatosis associated muta- Methods tion. DNA was extracted from 2 ml anticoagulated (7 Clin Pathol:Mol Pathol 1997;50:275-276) blood by a simple salting out method7 and DNA concentration adjusted to 50 Keywords: haemochromatosis; PCR-SSP; HFE gene; ng/pl. rapid screen Polymerase chain reaction (PCR) amplifica- tion was carried out in a reaction volume of 10 gl, consisting of 20 mM ammonium sul- Haemochromatosis is transmitted as an auto- phate, 75 mM Tris-HCl (pH 9.0), 0.01% somal recessive trait with a homozygosity Tween, 1.5 mM MgCl,, 200 iM dNTPs, frequency of 0.005-0.008 and an estimated 1.0 ,uM specific primers (Oswel, Southampton, carrier frequency of 10% among individuals of North European origin. Although a link 1 2 3 4 5 6 7 8 9 1011121314 between haemochromatosis and the human G G A AMG GA AM G G AA leucocyte antigen (HLA) allele, HLA-A3, was Control described over 20 years ago,' a much closer (842 bp) association has now been found with a mutation in a newly recognised gene, assigned nt 845 Specific Histocompatibility the name HFE (the accepted representation for (232 bp) Laboratory, National the haemochromatosis approved Blood Service (Trent by the Genome Database Nomenclature Figure 1 Gel electrophoresis ofPCR productsfrom three Centre), Longley Lane, Committee).2 This gene belongs to the major individuals (I-III). Lanes 1-4 (individual I), nucleotide Sheffield S5 7JN, UK histocompatibility complex (MHC) class I 845GIG, Cys282Tyr -1-; Lanes 6-9 (individual II), nucleotide 845GIA, Cys282Tyr -1+; Lanes 11-14 Accepted for publication related gene family and is situated 4.5 Mb (individual III), nucleotide 845AIA, Cys282Tyr +I+; 26 June 1997 telomeric to HIA-A. The mutation, whereby Lanes S and 10, molecular markers (100 ladder). 276 Short reports

UK), 0.25 jIM control primers, 0.5 U DNA visualised by staining with ethidium bromide polymerase (Advanced Biotechnologies, and photographed under ultraviolet illumina- Epsom, UK), and 50 ng genomic DNA. One tion. Results from three representative indi- duplicate reaction set was specific for the nor- viduals are shown in fig 1. The results demon- Mol Path: first published as 10.1136/mp.50.5.275 on 1 October 1997. Downloaded from mal sequence (845G) and a second reaction set strate that the PCR-SSP method is highly was specific for the mutated sequence (845A); specific for the haemochromatosis associated both reaction sets used a common forward mutation 845G-+A in the HFE gene and that primer (5'-AAGGTGACACATCATGTGA- the method could be used as an aid to diagno- CC-3'), with a reverse primer specific for either sis, or for screening purposes. 845G (5'-CTGGGTGCTCCACCTGGC-3'), or 845A (5'-CTGGGTGCTCCACCT- I am very grateful to Dr Mark Worwood (Department of Hae- GGT-3'), resulting in a PCR product of 232 matology, University ofWales College of Medicine) for his kind an gift of DNA for validation purposes. Primers were designed base pairs. Control primers, which amplified from sequence data for the haemochromatosis gene deposited in 842 base pair fragment of the human growth Genbank (Accession number U60319). hormone gene8 were included in all reactions. Amplification conditions involved an initial 1 Simon M, Bourel M, Fauchet R, Genetet B. Association of denaturation step for one minute at 96°C, fol- HLA-A3 and HLA-B14 antigens with idiopathic haemo- chromatosis. Gut 1976;17:332-4. lowed by five cycles consisting of denaturation 2 Bodmer JG, Parham P, Albert ED, Marsh SGE. Putting a for 25 seconds at 96°C, annealing for 45 hold on 'HLA-H'. Nat Genet 1997;15:234-5. for 30 seconds 3 Feder JN, Guirke A, Thomas W, Tsuchihashi Z, Ruddy DA, seconds at 70°C, and extension Basava A, et al. A novel MHC class I-like gene is mutated in at 720C, then a further 21 cycles where the patients with hereditary haemochromatosis. Nat Genet annealing temperature was lowered to 65°C, 1 996;13:399-408. 4 Jazwinska EC, Cullen LM, Busfield F, Pyper WR, Webb SI, plus four cycles where annealing was for one Powell LW, et al. Haemochromatosis and HLA-H. Nat minute at 55°C, and extension was for Genet 1996;14:249-51. 5 Jouanolle AM, Gandon G, Jezequel P, Blayau M, Campion two minutes at 72°C. A final extension step for ML, Yaouanq J, et al. Haemochromatosis and HLA-H. Nat five minutes at 72°C completed the amplifica- Genet 1996;14:251-2. 6 Beutler E. Genetic irony beyond haemochromatosis: clinical tion. effects of HLA-H mutations. Lancet 1997;349:296-7. 7 Miller SA, Dykes DD, Polesky HF. A simple salting out pro- cedure for extracting DNA from human nucleated cells. Results and discussion Nucleic Acids Res 1988;16:1215. The duplicate reaction sets typing for either 8 Guttridge MG, Burr C, Klouda PT. Identification of HLA- B35, B53, B18, B5, B78 and B17 alleles by the polymerase 845G or 845A were analysed by electrophore- chain reaction using sequence-specific primers (PCR- sis on a 1.5% agarose gel; PCR products were SSP). Tissue Antigens 1994;44:43-6. http://mp.bmj.com/ High resolution single strand conformation polymorphism analysis using formamide and

ethidium bromide staining on September 25, 2021 by guest. Protected copyright.

T Xie, S L Ho, O C K Ma

Abstract Polymerase chain reaction (PCR) amplifica- University Single strand conformation polymor- tion of DNA fragments followed by single Department of phism (SSCP) analysis using ethidium strand conformation polymorphism (SSCP) Medicine, University can be adding analysis is a sensitive method for detecting of Hong Kong, Hong bromide improved by Kong formamide as the denaturant. This gives genetic polymorphisms or mutations. Detec- T Xie higher resolution than previous SSCP tion by autoradiography' or silver staining2 is S L Ho methods; it had 100% sensitivity in the time consuming, costly, and inconvenient. discrimination of 14 PCR samples from Non-isotopic SSCP (cold SSCP) using ethid- Department of Clinical ium bromide staining3" facilitates the rapid Biochemistry two different genes, even for a long O C K Ma fragment close to the upper limit of 250 identification of structural changes in PCR base pairs. This modified procedure is a products. Currently, the denaturant used most Correspondence to: rapid, simple, safe, and yet highly sensi- frequently in cold SSCP is either sodium Dr Ho, University dif- hydroxide4 or methylmercury hydroxide.3 The Department of Medicine, tive method for detecting structural University of Hong Kong, ferences in DNA fragments. former has been reported in some cases to yield Queen Mary Hospital, Hong (T Clin Pathol: Mol Pathol 1997;50:276-278) inconsistent results3; the latter is an extremely Kong. toxic and volatile compound5 and great care Accepted for publication Keywords: single strand conformation polymorphism; has to be taken to avoid skin contact with 14 August 1997 formamide; ethidium bromide methylmercury and inhalation of its aerosol.