Brief Genetics Report Defining the Importance of Mitochondrial Gene Defects in Maternally Inherited Diabetes by Sequencing the Entire Mitochondrial Genome Alan T.W. Choo-Kang,1 Stephen Lynn,1 Geoffrey A. Taylor,2 Mark E. Daly,1 Sarbpreet S. Sihota,1 Teressa M. Wardell,2 Patrick F. Chinnery,2 Douglass M. Turnbull,2 and Mark Walker1

For any mitochondrial DNA (mtDNA) mutation, the ratio of mutant to wild-type mtDNA (% heteroplasmy) varies across tissues, with low levels in leukocytes and t is well recognized that in some families diabetes high levels in postmitotic tissues (e.g., skeletal muscle). follows a maternal inheritance pattern, and that Direct sequencing is the gold-standard method used to there is an excess of maternal transmission in type 2 detect novel mutations, but can only reliably detect % Idiabetes. As mitochondrial DNA (mtDNA) is passed heteroplasmy >25%, which is rare in leukocytes. There- exclusively down the maternal line, it was postulated that fore, we investigated the role of mtDNA defects in mtDNA defects might contribute to the excess maternal maternally inherited diabetes by first screening for the transmission. This was subsequently confirmed, and a A3243G tRNALeu(UUR) mutation by restriction assay, number of mtDNA defects have been implicated in the followed by sequencing of the entire mitochondrial development of diabetes, as recently reviewed (1–3). An A genome using skeletal muscle derived mtDNA. A total of to G substitution at position A3243G in the tRNALeu(UUR) 28 patients had maternally inherited diabetes either gene is the most commonly reported defect associated ؍ alone (group 1, n 17) or with one or more additional with diabetes. This mutation was first described in relation features of mitochondrial disease, including bilateral to the neuromuscular metabolic MELAS syndrome (mito- sensori-neural deafness and neuromuscular disease chondrial myopathy, encephalopathy, lactic acidosis, and (Three patients (all from group 2 .(11 ؍ group 2, n) strokelike episodes) in which diabetes is a common fea- carried the A3243G mutation. Skeletal muscle mtDNA from eight group 1 patients and six more group 2 ture, and was more recently recognized as a cause of the patients was sequenced. No pathogenic mutations were diabetic subtype of maternally inherited diabetes and found in the group 1 patients, while two patients from deafness (MIDD) (1). This is characterized by bilateral group 2 had mutations at positions 12258 and 14709 in sensori-neural deafness, and diabetes that tends to de- the tRNA serine and glutamic acid genes, respectively. velop during middle age and frequently progresses to We conclude, therefore, that screening for mtDNA mu- insulin therapy. However, autoimmune markers of type 1 tations should be considered in patients with mater- diabetes are nearly always negative. MIDD therefore nally inherited diabetes, but only when additional shares some of the clinical features of both type 1 and type features of mitochondrial disease are present. Diabetes 2 diabetes. 51:2317–2320, 2002 The prevalence of the A3243G mutation in diabetes has been comparatively easy to ascertain and has been shown to account for ϳ1–2% of cases in the general diabetic population (3). However, a much more important question is what is the prevalence of all forms of mtDNA defects in the diabetic population? This has been difficult to address because of several problems related to the complexity of mtDNA biology. First, pathogenic mutations can occur at almost any site throughout the mitochondrial genome, and From the 1School of Clinical Medical Sciences, University of Newcastle upon Tyne, U.K.; and the 2School of Clinical Neurosciences, University of Newcastle hence comprehensive screening requires analysis of the upon Tyne, U.K. entire mtDNA molecule. Second, nonfunctional homoplas- Address correspondence and reprint requests to Dr. Mark Walker, School of mic variants are common and must be distinguished from Clinical Medical Sciences, 4th William Leech Block, The Medical School, Framlington Place, Newcastle upon Tyne, U.K., NE2 4HH. E-mail: functional heteroplasmic defects. Third, mutations may be [email protected]. missed because of variable tissue expression. This is Received for publication 24 January 2002 and accepted in revised form 3 April 2002. because the level of the mutated mtDNA in relation to the S.L. is currently affiliated with the Department of Pediatrics, University of wild-type mtDNA (% heteroplasmy) varies between tis- California, San Francisco, California. sues, being high in postmitotic tissues, such as skeletal % heteroplasmy, ratio of mutant to wild-type mtDNA; ICA, islet cell antibody; MIDD, maternally inherited diabetes and deafness; mtDNA, mito- muscle and brain, and low in rapidly dividing tissues, such chondrial DNA; SSCP, single-strand conformational polymorphism. as blood leukocytes. As blood leukocytes are the usual

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TABLE 1 Characteristics of patients in group 1 Age Type and Functional diabetes duration (years) mtDNA Patient Age Database onset current diabetes Criteria for point no. (years) Sex source (years) therapy recruitment ICA Histochemistry mutations Deletions 1 69 F Diabetes 51 Insulin (13) Diabetes across 3 Negative Normal ND ND generations 2 66 M Diabetes 56 Insulin (8) 4 diabetic patients Negative Normal ND ND across 2 generations 3 65 M Diabetes 24 Insulin (41) 3 diabetic patients Negative Normal ND ND across 2 generations 4 62 M Diabetes 57 Insulin (5) 3 diabetic patients Negative Normal ND ND across 2 generations 5 70 F Diabetes 58 Insulin (10) 4 diabetic patients Negative 1–2% negative ND CD detected across 2 fibers generations 6 55 M Diabetes 52 Insulin (2) Diabetes across 3 Negative Normal ND ND generations 7 60 M Diabetes 21 Insulin (39) 3 diabetic patients Negative Normal ND CD detected across 2 generations 8 55 M Diabetes 46 OHA (8) Diabetes across 3 Negative COX-deficient ND CD detected generations fibers CD, common deletion; OHA, oral hypoglycaemic agent; ND, none detected. source of DNA for study, this has important implications tes and no other features of mitochondrial disease (group for mtDNA mutation detection. There is usually no prob- 1, n ϭ 17) and those with maternally inherited diabetes lem detecting known mutations such as the A3243G mu- plus one or more features of mitochondrial disease (group tation, as restriction assays can detect levels of 2, n ϭ 11); these features were defined as bilateral heteroplasmy down to ϳ1%. However, the situation is sensori-neural deafness, myopathy, cerebellar ataxia, non- different for the detection of novel mutations, as the diabetic retinopathy, premature cataracts, stroke-like epi- methods are not as sensitive. Direct sequencing is the sodes before the age of 50 years, and encephalopathy. gold-standard method for mutation detection, but this can Subjects were first screened by restriction digest assay for only reliably detect levels of heteroplasmy Ͼ25%. There- the A3243G mutation, and three patients from group 2 fore, a novel mtDNA mutation could be present at levels of were identified with the mutation and excluded from anything up to 25% in leukocyte DNA and go undetected. further study. A further 11 patients were either excluded As a consequence, this would lead to an underestimation because of contraindications to needle muscle biopsy (n ϭ of the true prevalence of mtDNA defects in conditions 7) or declined to have the procedure (n ϭ 4). Of the such as diabetes. The solution to this problem is either to remaining 14 patients, 8 from group 1 and 6 from group 2 develop more sensitive methods to detect novel mutations underwent needle muscle biopsy. Skeletal muscle samples or to screen a postmitotic tissue in which the levels of were prepared for histology and mtDNA analysis. The mutation will be much higher and therefore amenable to entire mitochondrial genome, including the D-loop, was detection by direct sequencing. In this study, we pursued sequenced using 28 overlapping primer pairs (see Table the second approach to define the role of mtDNA defects A1: online appendix at http://diabetes.diabetesjournals. in maternally inherited diabetes. The specific aim of the org). study was to search for novel mtDNA mutations in pa- Table 1 summarizes the clinical and molecular findings tients with evidence of maternally inherited diabetes, and for the group 1 patients who underwent screening of the to see if there were differences between patients with entire mitochondrial genome. All patients had developed diabetes alone and those with additional features of mito- diabetes in adulthood; all but one were insulin treated, but chondrial disease. all patients were islet cell antibody (ICA) negative. While A questionnaire was used to screen patients attending these features are consistent with mitochondrial-related the Newcastle Diabetes and Audiometry Services, and 28 diabetes, sequencing of the entire mitochondrial genome of 595 patients were identified with clear evidence of in each patient did not reveal any pathogenic point muta- maternal transmission of diabetes. This was defined as tions. The common deletion was detected in muscle transmission through three consecutive generations or mtDNA from three patients, but at very low levels and through two consecutive generations with at least three compatible with age-related changes. affected individuals. In addition, there had to be no evi- Table 2 summarizes the clinical and molecular findings dence of paternal transmission of diabetes. Patients were for the group 2 patients. Like the group 1 patients, those in subdivided into those with maternally transmitted diabe- group 2 displayed many of the clinical features associated

2318 DIABETES, VOL. 51, JULY 2002 A.T.W. CHOO-KANG AND ASSOCIATES 46 ibts6 H 1 9Daee,C,BDand BND CA, Diabetes, 39 generations 3 in Diabetes generations 2 in (1) Diabetes OHA generations 2 in deafness BND No generations 2 in 60 (21) Diabetes Insulin 50 (17) Insulin 45 Diabetes 44 22 (14) 46 Insulin F Diabetes (5) Insulin 35 61 F OHA Diabetes 38 M 68 Diabetes 14 54 M Diabetes 13 M 65 12 51 11 10 no. Patient 2 group in patients of Characteristics 2 TABLE

3MAdoer 7Isln()3 ibtsi generations 2 in Diabetes 32 (7) Insulin 37 Audiometry M 53 9 with mitochondrial-related diabetes, except one of the six

ϭ patients was ICA positive. None of the patients carried the rlhpgyamcaet gens. agent; hypoglycaemic oral common deletion or major rearrangements. However, we

yas Sex (years) did identify pathogenic point mutations in two patients

Age from group 2. The first functional mutation wasaCtoA substitution at position 12258 in a tRNA serine gene. This mutation has been previously reported (4,11), and was associated with maternally inherited diabetes, sensori- neural deafness, cataracts, and cerebellar ataxia. The second mutation was the previously reported T to C Database

Source substitution at position 14709 in the tRNA glutamic acid gene (5). As observed in our pedigree, previous reports have linked the mutation with the development of diabetes ϭ eeain;BND generations; and myopathy (5,6,12). However, in an Italian pedigree, the clinical expression was heterogeneous with some diabetes (years)

onset affected individuals showing just the MIDD phenotype (6). Age This is the first systematic search for novel inherited mtDNA mutations in maternally transmitted diabetes us- ing mtDNA derived from postmitotic tissue. As mentioned

ϭ above, an alternative approach would be to develop a yeand Type duration diabetes iaea ev efes CA deafness; nerve bilateral therapy current (years) much more sensitive system for mutation detection that could be used to screen peripheral leukocyte DNA sam- ples. Such a system has been reported based upon single- strand conformational polymorphism (SSCP) analysis (7), but the sensitivity of SSCP for mutation detection ranges diagnosed deafness

yas rtrafrrcutetIAHistochemistry ICA recruitment for Criteria (years) between 75 and 97%, even under optimal conditions (8). Age The problem is further compounded by the fact that some mtDNA mutations may simply not be expressed in leuko- cyte DNA. For example, we investigated a patient with a

ϭ mitochondrial point mutation (A to G at position 12320) eeelraai ND ataxia cerebellar that was present at high levels in muscle but was unde- generations 3 in cataracts generations 3 in Myopathy generation 1 in BND generation 1 in Diabetes generations 2 in BND generation 1 in BND tectable in leukocyte mtDNA using the most sensitive method of last-cycle-hot PCR (9). It was these consider- ations therefore that led us to develop the described approach of using the gold-standard method of direct sequencing to screen for mutations in mtDNA from post- mitotic tissue. ϭ

oedtce;N/A detected; None A number of important observations emerge from this study. First, no pathogenic mtDNA mutations were iden- tified in those patients with maternally inherited diabetes

eaieN 179 ND ND T14709C ND ND ND 5 ND Negative ND NA ND Negative Normal ND Positive Normal Negative Normal Negative Normal Negative alone (group 1). Three of these patients were found to have the common deletion, but this was at very low levels and of no functional significance. While we acknowledge that only a comparatively small number of subjects was

ϭ studied, there was clear maternal transmission of the fi o available. not – bers

%CXnegative COX 6% diabetes in each pedigree. Our comprehensive search for mtDNA defects, therefore, allows us to conclude that other mechanisms other than inherited defects of mtDNA must be contributing to the maternal transmission of diabetes in these families. Conversely, five patients with maternally transmitted diabetes plus additional features of mitochondrial disease (group 2) were found to harbor pathogenic mutations; Functional uain Deletions mutations 128 ND C12258A

mtDNA three carried the A3243G mutation, while the other two point carried mutations that were identified by subsequent se- quencing of the mitochondrial genome. However, it is worth noting that the same comprehensive search failed to identify pathogenic mtDNA defects in a further four group 2 patients with the MIDD phenotype (Table 2). To our knowledge, no other mechanism has been described that leads to maternal transmission of both diabetes and deaf- ness. However, sensori-neural deafness, like diabetes, is a

DIABETES, VOL. 51, JULY 2002 2319 MITOCHONDRIAL DIABETES common condition and can arise from a large number of detect rearrangements. DNA (3 ␮g) was added to 2 ␮l10ϫ NEBuffer (New England Biolabs) and 2 ␮l PvuII (10 units/ml; New England Biolabs), and was autosomal-inherited gene defects (10). It is quite possible, ␮ made up to 20 l with dH2O. The DNA was digested for 1.5 h at 37°C using therefore, that these four patients with the MIDD pheno- PvuII. Rearrangements are detected by the generation of different length type were from pedigrees that carried separate defects, fragments. After digestion, the sample was run out on a 0.8% agarose gel, one causing maternally inherited diabetes and the other transferred onto a nitrocellulose membrane, and probed using a labeled probe against the O region (nucleotide position 15782-1289). deafness. H In conclusion, we believe that these observations and those of previous studies allow us to provide the following ACKNOWLEDGMENTS guidance for clinical practice. First, based on our own observations, there is really no case to search for inherited The work was supported by funding from Diabetes U.K. mtDNA defects in pedigrees with maternally inherited diabetes alone. Conversely, we recommend that patients REFERENCES with maternally inherited diabetes plus one or more 1. Van den Ouweland JMW, Lemkes HHPJ, Gerbitz KD, Maassen JA: Mater- additional features of mitochondrial disease should be nally inherited diabetes and deafness (MIDD): a distinct sub-type of referred to a specialist center as they are likely to need diabetes and deafness associated with a mitochondrial tRNALeu(UUR) gene more extensive investigation for mtDNA defects. point mutation. Muscle and Nerve S3:124–130, 1995 2. Gerbitz KD, Gempel K, Brdiczka D: Mitochondria and diabetes: genetic, biochemical and clinical implications of the cellular energy circuit. Dia- RESEARCH DESIGN AND METHODS betes 45:113–126, 1996 Subjects. Patients were recruited from the Newcastle Diabetes and New- 3. Walker M, Turnbull DM: Mitochondrial related diabetes: a clinical perspec- castle Audiometry Services. A questionnaire was sent to patients requesting tive. Diabet Med 14:1007–1009, 1997 information about personal and family history of diabetes and features of 4. Lynn S, Wardell T, Johnson MA, Chinnery PF, Daly ME, Walker M, Turnbull mitochondrial disease, as described in the text. From a total of 1,045 mailed DM: Mitochondrial diabetes: investigation and identification of a novel questionnaires, 595 replies were received, and patients with evidence of mutation. Diabetes 47:1800–1802, 1998 maternal transmission of diabetes were identified. From this initial screen, 28 5. Hanna MG, Nelson I, Sweeney MG, Cooper JM, Watkins PJ, Morgan- patients agreed to undergo further clinical assessment. Between 100 and 150 Hughes JA, Harding AE: Congential encephalomyopathy and adult-onset mg tissue was obtained by quadriceps needle biopsy. Exclusion criteria for myopathy and diabetes mellitus: different phenotypic associations of a muscle biopsy included anticoagulant therapy, extensive peripheral vascular new heteroplasmic mtDNA tRNA glutamic acid mutation. Am J Hum disease and/or neuropathy, and marked obesity. The presence or absence of Genet 56:1026–1033, 1995 sensori-neural deafness independent of age was assessed by pure tone 6. Rigoli L, Priscot F, Caruso RA, Iafuscot D, Ursomannot G, Zuccarello D, audiometry. The study was approved by the Newcastle and North Tyneside Ingenito N, Rigoli M, Barberi I: Association of the T14709C mutation of Joint Ethics Committee. mitochondrial DNA with maternally inherited diabetes and/or deafness in Methods. Frozen sections of muscle were cut at 8 ␮m and stained for an Italian family. Diabet Med18: 333–338, 2001 cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) activity, as 7. Thomas AW, Edwards A, Sherratt EJ, Majid A, Gagg J, Alcolado JC: previously described (4). The remainder of the muscle sample was used for Molecular scanning of candidate mitochondrial tRNA genes in type 2 molecular analysis. The mitochondrial genome, including the noncoding (non-insulin dependent) diabetes mellitus. J Med Genet 33:253–255, 1996 D-loop, was amplified using 28 overlapping primer pairs using the PCR 8. Ravnic-Glavak M, Glavac D, Dean M: Sensitivity of single-stranded confor- (primers listed in Table A1: online appendix at http://diabetes.diabetesjour- mation polymorphism and heteroduplex method for mutation detection in nals.org). The PCR reaction (50 ␮l) was set up as follows: 1.5 ␮l of each primer the cystic fibrosis gene. Hum Mol Genet 3:801–807, 1994 pair (20 ␮mol/l), 1 ␮l DNA (200 ng/␮l), 5 ␮l10ϫ PCR buffer (500 mmol/l KCl, 9. Weber K, Wilson JN, Taylor L, Brierley E, Johnson MA, Turnbull DM,

100 nmol/l Tris-HCl, pH 8.3, 15 mmol/l MgCl2, and 0.01% (wt/vol) gelatin), and Bindoff LA: A new mtDNA mutation showing accumulation with time and 0.25 ␮l AmpliTaq Gold DNA Polymerase (5 units/␮l; Applied Biosystems). The restriction to skeletal muscle. Am J Hum Genet 60:373–380, 1997 reaction conditions were one cycle at 94°C for 12 min, followed by 30 cycles 10. Kalatzis V, Petit C: The fundamental and medical impacts of recent at 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min, and finally 72°C for 8 min. progress in research on hereditary hearing loss. Human Mol Genet The amplified fragments were column purified using the QIA Quick PCR 7:1589–1597, 1998 Purification Kit (Qiagen Ltd, Crawley, U.K.) and then sequenced using a Big 11. Mansergh FC, Millington-Ward S, Kennan A, Kiang A-S, Humphries M, Dye Terminator cycle sequencing kit on a ABI Prism 377 DNA sequencer Farrar GJ, Kenna PF: Retinitis pigmentosa and progressive sensorineural (Appied Biosystems). The sequence data were analyzed using the software hearing loss caused by a C12258A mutation in the mitochondrial MTTS2 packages Factura and Sequence Navigator (Applied Biosystems), and were gene. Am J Hum Genet 64:971–985, 1999 compared with the Cambridge sequence and MITOMAP (MITOMAP 1999). 12. Damore ME, Speiser PW, Slonim AE, New MI, Shanske S, Xia W, Santorelli Long PCR was used to detect large scale deletions. This used the Expand FM, DiMauro S: Early onset diabetes mellitus associated with the mito- Long Template PCR System (Roche Diagnostics, Lewes, U.K.), and products chondrial DNA T14709C point mutation: patient report and literature were run out on a 1% agarose gel. Southern Blot analysis was carried out to review. J Pediatr Endocrinol 12:207–213, 1999

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