Characterisation of Repeat and Palindrome Elements in Patients

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ONLINE MUTATION REPORT J Med Genet: first published as 10.1136/jmg.40.7.e86 on 1 July 2003. Downloaded from Characterisation of repeat and palindrome elements in patients harbouring single deletions of mitochondrial DNA A Solano, J Gámez, F J Carod, M Pineda, A Playán, E López-Gallardo, A L Andreu, J Montoya ......

J Med Genet 2003;40:e86(http://www.jmedgenet.com/cgi/content/full/40/7/e86)

ingle deletions of mitochondrial DNA (mtDNA) were the Key points first pathogenic mutations to be identified in human mtDNA. In a seminal paper, Holt et al1 reported the pres- S • We report the molecular findings in a series of 18 ence of single deletions of the mitochondrial genome in patients in whom we have identified single deletions of patients presenting with mitochondrial myopathies, and since their mitochondrial DNA (mtDNA). then, the field has experienced enormous progress. To date, 97 different deletions have been reported in MITOMAP,the main • From a clinical point of view, patients fall into two cat- international database for mtDNA related disorders (www- egories: chronic progressive external ophthalmoplegia .mitomap.org), and most of these deletions are associated (CPEO) and Kearns-Sayre syndrome (KSS). with two clinical presentations: chronic progressive external • After mapping the deletion breakpoints, we report nine ophthalmoplegia (CPEO) and Kearns-Sayre syndrome (KSS). novel deletions varying in size and heteroplasmy levels, Here, we report the molecular characterisation of a series of 18 in which direct tandem repeat elements are involved. patients in whom we have identified single deletions of Also, several palindrome sequences within or near the mtDNA. These patients were, as expected, diagnosed with deletion breakpoints were identified in three patients, CPEO and KSS. Deletions were identified and measured by and in one of these flanking direct repeats were not Southern blot analysis and were mapped by long polymerase found. chain reaction (PCR) to locate the deletion breakpoint. We • These findings further expand our knowledge of the report nine novel deletions and defined their characteristics in mitochondrial genotype in those neuromuscular disor- terms of sequence of the tandem repeat, presence of ders produced by single deletions of mtDNA. palindrome sequences, length of the deleted molecule, and

heteroplasmy level in muscle. http://jmg.bmj.com/ (5′-CGGATACAGTTCACTTTAGCTACCCCCAAGTG-3′) using METHODS LA-Taq (Takara Shuzo, Japan). The long PCR product was Patients sequenced using internal primers in an automatic sequencer The 18 patients included in this study were clinically (ABI Prism 310, Perkin-Elmer, CA) using Big-Dye chemistry. diagnosed with CPEO (10 patients) and KSS (eight patients), The deletion breakpoints were identified by comparing with and there was no evidence of maternal inheritance. CPEO was 2 the reference sequence for the human mtDNA. defined by the presence of ophthalmoplegia, ptosis, and proximal limb weakness. Patients with KSS presented with on October 1, 2021 by guest. Protected copyright. the invariant triad of: (1) onset before 20 years, (2) progressive RESULTS external ophthalmoplegia; and (3) pigmentary retinopathy, Southern blot analysis identified the presence of the so called plus at least one of heart block, cerebellar syndrome, or a pro- 4977 nt “common deletion” in six out of 18 patients (33%). In tein concentration in cerebrospinal fluid above 100 mg/ml. all patients the deletion was found only in muscle (it was not Muscle biopsies were performed after informed consent. found in blood). The classic 13 bp tandem repeat flanking the deletion breakpoint was confirmed by specific PCR and the per- Molecular genetic studies centages of deleted genome in muscle ranged from 6% to 68%. Blood DNA was extracted from peripheral blood cells by con- Twelve patients harboured a single deletion other than ventional methods, and muscle DNA was obtained from 10 mg 4977. Sequence analysis of the long PCR product allowed us to of muscle after treatment with proteinase K and extraction map the deletion breakpoints. Three out 12 patients had pre- with phenol/chloroform/isoamyl alcohol. Southern blot analy- viously reported deletions3–5 and nine were novel. Table 1 sis was performed using 5 µg of total blood or muscle DNA by shows the proportion of mutated genomes in muscle from standard procedures. Each sample was digested with PvuII to those patients without 4977 as well as the length of the linearise the mtDNA and was loaded on a 0.7% agarose gel. deleted mtDNA. These novel deletions ranged from 2.3 to 9.4 After electrophoreses and transfer to a nylon membrane, the kb with percentages of heteroplasmy in muscle from 7% to membrane was hybridised with a 1.3 kb PCR generated probe 86%. Fig 1 shows the nucleotide sequence involved in the encompassing the 16S RNA and ND1 genes (nt positions deletion event. In most cases, these were direct tandem 3130–4407),2 which was labelled with digoxigenine (DIG- repeats ranging from 5 to 15 bp and there was a high High Prime, Boehringer-Mannheim, Mannheim, Germany). Those samples in which single deletions were identified by Southern blot analysis were submitted to long PCR to map the ...... deletion breakpoints. Briefly,the whole mitochondrial genome Abbreviations: CPEO, chronic progressive external ophthalmoplegia; was amplified using the following primers: forward (5′- KSS, Kearns-Sayre syndrome; mtDNA, mitochondrial DNA; PCR, TCTATCACCCTATTAACCACTCACGGGAGCT-3′), and reverse polymerase chain reaction

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Table 1 Molecular characteristics of single mtDNA deletions in patients with CPEO

or Kearns-Sayre syndrome (KSS) (number of mispairings (mp) in non-perfect repeats J Med Genet: first published as 10.1136/jmg.40.7.e86 on 1 July 2003. Downloaded from are shown in parentheses)

% Heteroplasmy Number of bp % Pyrimidines Deleted region Previously Patient (muscle) in repeats in repeats (2) Length (bp) reported

CPEO 1 70 – – 8380–13338 4957 NO 2 29 15 (4 mp) 60 9813–15741 5927 NO 3 7 6 66 7407–13620 6212 NO 4 55 15 (1 mp) 66 9485–13723 4236 Yuzaki et al3 KSS 5 64 15 (1 mp) 62.5 6324–13989 7664 Johns et al4 6 80 10 60 10951–15372 4420 NO 7 77 5 80 10625–13059 2433 NO 8 70 10 80 12112–14422 2309 Rotig et al3 9 86 10 (1 mp) 75 7508–15939 8430 NO 10 69 7 71 7949–14315 6365 NO 11 87 – – 6003–15441 9437 NO 12 50 10 (1 mp) 65 11727–14446 3715 NO

Figure 1 Sequences involved in lost mtDNA. Numbers below sequences define mtDNA positions at breakpoints of the deleted regions (within brackets). Bold indicates direct repeats (perfect or imperfect), while palindromes are shown in italics and are underlined. http://jmg.bmj.com/ on October 1, 2021 by guest. Protected copyright.

proportion of imperfect tandem repeats (50% of cases) which deletion of human mtDNA is a 4977 deletion (the so called were found in those repeats longer than 10 nucleotides. How- common deletion) that ranges from nt position 8470 (in the ever, in two patients (patients 1 and 11) the deleted sequence ATP8 gene) to nt 13447 (in the ND5 gene).67 The deletion is was not flanked by tandem repeat elements. Moreover, several flanked by a 13 bp direct repeat and it represents most patients patients harboured palindrome sequences either within or with CPEO. In our series, we have found a similar frequency of near the deletion breakpoints. These palindromes were found the 4977 deletion (33%) and, as expected, all patients in three patients: patient 8 had a 10 bp tandem repeat plus a 7 presented with CPEO. The presence of direct tandem repeat bp palindrome sequence upstream of the 5′ end of the break- elements flanking the deletion breakpoints has been proposed point; patient 11 did not showed any tandem repeat, but sev- as a genetic criterion to classify mtDNA single deletions. eral palindrome sequences were identified, a 15 bp perfect According to this classification, single deletions may be palindrome anda6bppalindrome within the deletion plus a divided into two groups.8 Class I deletions (representing about 14 bp palindrome 4 nt from the 3′ end of the breakpoint; and 70% of patients) are flanked by direct repeats of variable patient 12 had 14 bp palindrome at the 3′ end plus an 8 bp length, and class II deletions are either not flanked by repeats, palindrome within the deletion at the 5′ end. Interestingly, or if repeats are present, these are located imprecisely relative patient 1 did not have either repeat elements or palindrome to the breakpoints. The mechanism by which single deletions sequences flanking the deletion breakpoint. are produced is not well understood and several explanations have been proposed. Unequal intergenomic crossing over as DISCUSSION well as slipped mispairing are two proposed models explaining Usually, single deletions of mtDNA present with a variable the mechanism of mtDNA deletion during mtDNA range from about 2–8.5 kb and are mostly located in an 11 kb replication,910and the presence of direct repeats flanking the region between the ND5 and ATP8 genes. The most common deletion breakpoints suggest that intramolecular recombinations produced by the repeated sequence are more likely to www.jmedgenet.com Online mutation report 3of4 J Med Genet: first published as 10.1136/jmg.40.7.e86 on 1 July 2003. Downloaded from

Figure 2 Linear representation of human mitochondrial DNA, and schematic localisation of the reported CPEO and KSS associated deletions. The common region to all of those is also shown (positions 12112 to 13059, dotted rectangle). occur than intergenomic recombinations. Shoffner et al11 breakpoints. Therefore, a combination of these four mecha- proposed a “slip replication” model in which the single paren- nisms, slip replication, illegitimate elongation, pyrimidine tal H strand anneals erroneously with the L strand at the content, and palindrome sequence, could occur. direct repeat site leading to the deletion of the H strand loop. In a recent report, He et al16 described a real time PCR based On the other hand, Buroker et al12 have proposed an method to measure single and multiple deletions of mtDNA. “illegitimate elongation” model in which the elongating D After analysing their own series of patients, they suggested loop strand is displaced by branch migration of the parental H that one region in the ND4 gene (nt 12087–12170) is deleted strand forming base pairs with the complementary L strand, in 82% of patients, while another region in the ND1 gene allowing the liberated D loop strand an erroneous misalign- (3485–3553) is deleted only in 6% of cases, so they proposed ment at direct repeat sites. Elongation of the shortened the simultaneous amplification of these sequences to measure daughter H strand followed by replication of the L strand the proportion of deleted genomes. Our mapping results agree would lead to deletion of the molecule. A recent “pyrimidine with their finding (fig 2) as in all cases 12087–12170 is totally content” hypothesis by Rocher et al13 suggests novel molecular or partially included in the deleted genomes, while 3485–3553 bases for mtDNA rearrangements through the distributive is never deleted. This suggests that a real time PCR based nature of the DNA polymerase gamma, at the level of direct strategy could be a good alternative to Southern blot analysis http://jmg.bmj.com/ repeats. The proposed model suggests the formation of a DNA in the routine diagnosis of patients with single deletions of triple helix between a G rich neosynthesised direct repeat in mtDNA. the heavy strand and the base paired homologous pyrimidine Extensive mapping studies of patients with single deletions rich direct repeat in the light strand. Interestingly, our data are of mtDNA should be performed to clarify the underlying consistent with this hypothesis as our patients had a high mechanisms involved in the generation of the deletion event pyrimidine content in the repeat element (ranging from 60% during mtDNA replication. Although the presence of repeat to 80% with an average of 70%). The three hypotheses require elements seems to explain most cases, there is growing on October 1, 2021 by guest. Protected copyright. the presence of tandem repeat elements which are found in evidence suggesting than mechanisms other than the classic most deletions reported to date as well as in the present series. direct repeat hypotheses could be involved in the pathogen- However, in a few patients, repeat elements are not found. In eses of the mtDNA single deletion phenotype. our series, patients 1 and 11 had no tandem repeats flanking the deletion breakpoint but palindrome sequences either within or near the breakpoint were found in patient 11. ACKNOWLEDGEMENTS Therefore a putative “palindrome sequence” model would This study was supported by grants from the Spanish Ministry of explain the deletion mechanism in those patients in whom Health (FIS 00/0797), Diputación General de Aragón (P032–2000), and Spanish Network for Mitochondrial Disorders (G03–011). AS was repeat elements are not found. This hypothesis is supported by the recipient of a fellowship from the Mexican Government the finding of certain palindrome DNA sequences with the (CONACYT-119894/121963). potential to form DNA hairpin structures that might stabilise the misalignment of direct repeats and have a high frequency 14 of deletion. Palindrome sequences are common in the ...... mtDNA genome. However, in our patients they are located Authors’ affiliations very close to the deletion breakpoint suggesting that they A Solano, A Playán, E López-Gallardo, J Montoya, Departamento de could be involved in the deletion mechanism. Similar findings Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, were reported by Saiwaki et al,15 who identified a patient har- Zaragoza, Spain bouring a 5.3 kb single deletion in whom a 17 bp palindrome J Gámez A L Andreu, Centre d’Investigació en Bioquimica i Biologia Molecular, Hospital Universitari Vall d’Hebron, Barcelona, Spain and 5 bp tandem repeats were found on both sides of the F J Carod, Servicio de Neurología, Hospital Sarah, Brasilia, Brasil breakpoint. M Pineda, Servei de Neuropediatria, Hospital Sant Joan de Déu, Here, we report nine novel deletions that have been mapped Barcelona, Spain to identify the breakpoints (fig 1). Remarkably, in those Correspondence to: Dr A L Andreu, Centre d’Investigació en Bioquimica i patients in whom a direct repeat was not evident, there were Biologia Molecular, University Hospital Vall d’Hebron, P Vall d’Hebron several palindrome sequences near or within the deletion 119–129, 08035 Barcelona, Spain; [email protected]

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