optic neuropathy . Finger prick blood testing in Leber hereditary

Individuals from 33 unrelated Australian families with optic atrophy were screened for 10 different single base alterations in mitochondrial DNA (mtDNA) associated with Leber hereditary optic neuropathy (LHON) using direct polymerase chain reaction amplification of blood spots collected on Guthrie cards. This method using blood spots allows easily accessible screening forLHON mtDNA mutations with minimal biohazard risk and reduced expense in the storage and transport of specimens. (BrJ Ophthalmol 1993; 77: 311-312) Leber hereditary optic neuropathy (LHON) commonly presents as a bilateral sequential visual loss which may be associated with Table I mtDNA base changes associated withLHON Department of Ophthalmology D Mackey The Murdoch Institute, Royal Children's Hospital, Melbourne, Australia D Mackey S Nasioulas S Forrest Correspondence to: Dr D Mackey, Department of Ophthalmology, Royal Children's Hospital, Flemington Road, Melbourne, Victoria, 3052, Australia. Accepted for publication 2 February 1993 mt base restriction enzyme sites altered gene mutation *3460 G>A loss of HgaI, AcyI, AhaII, BbiII, HinlI7 ND1 A52T 4160 T>C loss of Alul (with mismatch primer)6 ND1 L285P 4216 T>C creation of NiaIII, NspHI9 ND1 Y304H 4917 A>G creation of MaeI, RmaP ND2 D15ON 5244 G>A loss of HpaII, HapIil' ND2 G259S 7444 G>A loss of Xbal'° Cox ter K *11778 G>A loss of SfaNI, creation of MaeIII12-'4 ND4 R340H 13708 G>A loss of BstNI, Fnu4HI, ApyI, EcoRII, MvaI9 ND5 A458T 15257 G>A loss of AccI'0 CytB D171N 15812 G>A loss of Rsa, Csp6I'0 CytB V356M *There is strong evidence that these mutations are causative. Table 2 PCR primers, products, enzymes, andfragments used in this study PCR fragments Site gene 5' Primer 3' Primer Frag Enzyme size 3460 3275-3295 3553-3573 299 AcyI* 184+115 ND1 (21-mer) (21-mer) 4160 4137-4159 4281-4301 165 AluI* 22+143 ND2 (4158G, 23-mer) (21-mer) 4216 4137-4159 4281-4301 165 NspHI* 83+82 ND2 (4158G, 23-mer) (21-mer) 4917 4680-4700 4980-5000 321 MaeI* 106+90+39+86 ND2 (21-mer) (21-mer) 5244 5195-5212 5526-5546 352 HpaIIt 48+304 ND2 (21-mer) (21-mer) 7444 7353-7373 7650-7670 318 Xbalt 230+88 Cox (21-mer) (21 -mer) 11778 11660-11683 11896-11919 260 SfaNIt 130+130 ND4 (24-mer) (24-mer) MaeIII* 115+131+14 13708 13567-13587 13869-13889 323 Fnu4HIt 139+184 ND5 (21-mer) (21-mer) 15257 15081-15101 15290-15310 230 Acc It 175+55 CytBI (21 -mer) (21-mer) 15812 15749-15769 15919-15939 191 Rsat 65+126 CytBI (21-mer) (21-mer) 5' Primers are complementary to the H strand, 3' primers are complementary to the L strand of mtDNA. Numbering is as in the Cambridge sequence. LHON site is underlined. *Boehringer Mannheim, Indianapolis, IN, USA. tNew England Biolab, Beverly, MA, USA. characteristic optic disc swelling and peripapillary telangiectasia.II Diagnosis of LHON is difficult ifthe affected individual falls outside the classic presentation of a young adult male with a family history of similar visual loss. Fifty per cent of patients presenting with LHON are not men between 18 and 30 years.2 DNA testing for the various single base alterations in mitochondrial DNA (mtDNA) that have been associated with LHON (Table 1) is helpful in the diagnosis even though the aetiological significance of these alterations is not yet clearly defined. Recently, direct polymerase chain reaction (PCR) amplification from Guthrie cards has been used in testing for phenylketonuria3 and cystic fibrosis.4 We used direct PCR amplification of blood from Guthrie cards to detect the described mtDNA base changes ofLHON. Method Informed consent was obtained from patients or, where relevant, parents. Approximately 0 25 ml of blood was placed on a Guthrie card and allowed to air dry. With young children Guthrie cards taken at birth were obtained from the neonatal screening laboratories. A 2-5 mm (approximately 3 p1) disc of dried blood was punched from the card for each PCR fragment amplification, using sterile laboratory methods. Each blood spot was soaked in a solution of 10 p1 1% Triton X-100 (Sigma, St Louis MO), PCR buffer [20 Id 5 xPCR buffer (Perkin Elmer Cetus, Norwalk, CT)] and distilled water to a final total of 100 p1l (including the volume for dNTPs, primers, and enzymes) at 37°C for 1-24 hours in a 15 ml microcentrifuge tube. The eluted Guthrie spot was then boiled for 5 minutes and the appropriate pair of primers' 1 ,ul of each, 10 RI 2 mmol dNTPs, and Taq polymerase 1 [1 taq (PEC) were added. A PCR was run at 94°C for 90seconds, 65°C for 150 seconds, and 72°C for 80 seconds for 25-35 cycles. After -amplification, 16 p1 were then digested with the appropriate enzyme (Table 1). The resultant digest was run alongside the undigested PC-R product on a 3% gel and the fragments compared under ultraviolet light (Table 2). Results More than 100 patients in 33 separate families have been studied by this method and the mutations confirmed by subsequent sequencing of extracted DNA. All but one of the DNA alterations of interest could be detected by restriction enzyme cleavage because they alter recognition sites of one or more enzymes (Table 2). The exception was the 4160 T to C mutation which does not alter any known restriction site. 311 group.bmj.com on June 15, 2017 Published by http://bjo.bmj.com/ Downloaded from