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Abstract

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Objective

Diverse and variable clinical features, a loose genotype–phenotype relationship, and presentation to different medical specialties have all hindered attempts to gauge the epidemiological impact of mitochondrial DNA (mtDNA) disease. Nevertheless, a clear understanding of its prevalence remains an important goal, particularly about planning appropriate clinical services. Consequently, the aim of this study was to accurately define the prevalence of mtDNA disease (primary mutation occurs in mtDNA) in the working-age population of the North East of England.

Methods

Adults with suspected mitochondrial disease in the North East of England were referred to a single neurology center for investigation from 1990 to 2004. Those with pathogenic mtDNA mutations were identified and pedigree analysis performed. For the midyear period of 2001, we calculated the minimum point prevalence of mtDNA disease for adults of working age (>16 and <60/65 years for female/male patients, respectively).

Results

In this population, we found that 9.2 in 100,000 people have clinically manifest mtDNA disease, making this one of the commonest inherited neuromuscular disorders. In addition, a further 16.5 in 100,000 children and adults younger than retirement age are at risk for development of mtDNA disease.

Interpretation

Through detailed pedigree analysis and active family tracing, we have been able to provide revised minimum prevalence figures for mtDNA disease. These estimates confirm that mtDNA disease is a common cause of chronic morbidity and is more prevalent than has been previously appreciated. Ann Neurol 2007

The first pathogenic mutations of mitochondrial DNA (mtDNA) were identified in 1988,1, 2 and since then it has become increasingly clear that defects of this diminutive genome are an important cause of neurological disease. However, assessing the true impact of mtDNA disease has been complicated by clinical heterogeneity, the multiplicity of mutations occurring throughout the genome, and the difficulties encountered in establishing a diagnosis. Recent improvements in diagnostic techniques and the opportunity to perform extensive family tracing has led us to reevaluate our previous estimates of mitochondrial disease prevalence,3 focusing particularly on those mutations originating in the mitochondrial genome.

Subjects and Methods

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The North East of England has a relatively stable population of approximately 2.5 million people.4 Over a 15-year period, from January 1990 to January 2004, adults with suspected mitochondrial disease living in this region were referred by neurologists (or other hospital specialists) to the Newcastle Mitochondrial Centre. Comprehensive evaluation of these patients was undertaken including characterization of any mtDNA defect identified. We present data obtained from this cohort of adult patients aged between 16 and 60 (female patients) and between 16 and 65 (male patients) years, using those patients who were alive at the midpoint of 2001 as the numerator to the 2001 census data. Patients outside of these age ranges were excluded from the study because clinical diagnosis of mitochondrial disease in both young and elderly patients is often difficult and referral patterns are less well established. Patients with multiple deletions of mtDNA were excluded by the fact that these defects were the result of primary nuclear DNA mutations (albeit with secondary consequences for mtDNA). The genetic defect was established in mtDNA extracted from muscle, blood, and urine5 using established techniques.6

Ascertainment of Clinically Affected Individuals

Clinically affected individuals were defined as those exhibiting symptoms or signs consistent with the molecular genetic diagnosis but that occur rarely in the general population. Confirmation of the mtDNA defect in each patient was sought but was not required for inclusion, assuming the clinical features were consistent and a pathogenic mutation had been confirmed within the pedigree. This approach was validated by a near 100% rate of positive genetic tests where samples were available.

Signs of clinically manifest disease included chronic progressive external ophthalmoplegia, cerebellar ataxia, seizures, myoclonus, strokelike episodes (strokes of thromboembolic origin excluded), proximal weakness, exercise intolerance, cardiomyopathy, optic atrophy, pigmentary retinopathy, or bilateral deafness.7–9 Diabetes mellitus was considered to represent evidence of mitochondrial disease only in pedigrees carrying mtDNA mutations recognized to cause impaired glucose tolerance, for example, m.3243A>G10 and m.14709T>C.11, 12

To achieve a robust conservative minimum prevalence figure, we did not consider symptoms such as myalgia, fatigue, migraine, dysphagia, gastrointestinal upset, and cataracts, which might have substantial overlap with common medical conditions, as indicative of mtDNA disease. Instead, such patients were regarded as “at risk” for development of mtDNA disease, even when confirmation of the pathogenic mtDNA mutation had already taken place.

Ascertainment of Individuals at Risk for Development of Mitochondrial DNA Disease

Although all maternal relatives of a patient with a mtDNA point mutation are at potential risk for development of mtDNA disease, we restricted our study to first-degree relatives of either patients with clinical disease or asymptomatic individuals known to harbor the pathogenic mtDNA point mutation. For example, the mother, siblings, and children of an affected female individual with an mtDNA point mutation would be considered to be at risk for development of mtDNA disease (Fig, A). If one of this person's siblings was also found to carry the mutation, their mother would be implicated as an obligate carrier of the mutation, and those at risk would then include the mother's mother and the mother's siblings (see Fig, B). We have adopted this approach because the majority of known pathogenic mtDNA point mutations have been reported to be transmissible, and proven sporadic mutations are relatively rare. Important exceptions are mutations in the cytochrome b gene, which to date have been reported only as sporadic events.13 No such patients were included in our study. Single large-scale deletions of mtDNA were considered to be predominantly sporadic events with no “at-risk” individuals, although we acknowledge that maternal transmission may sometimes occur.14

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Figure 1. Examples of family tree illustrating “at-risk” ascertainment criteria. (A) Individuals considered to be “at risk” for development of mitochondrial DNA (mtDNA) disease are restricted to first-degree relatives of an affected female individual with a mtDNA point mutation. (B) Expansion of the number of individuals “at risk” if the same mtDNA point mutation is also identified in a sibling of the affected female individual. Circles represent female individuals; squares represent male individuals; filled symbols represent affected individuals; half-filled symbols represent individuals who are asymptomatic but carry the mutation; open symbols represent unaffected individuals.

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Results

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Clinically Affected Adults with Mitochondrial DNA Mutations

The midyear population of adults of working age in the North East government office region for 2001 was 1,535,800.4 Within this age group, pedigrees harboring defined mtDNA defects yielded 141 individuals exhibiting classic features of mtDNA disease. Thus, the minimum point prevalence for clinically manifest mtDNA disease in this population is 9.18 in 100,000 (95% confidence interval, 7.8–10.8/100,000). The m.3243A>G mutation was the most common pathogenic mtDNA mutation identified (40%) (Table) and was also associated with the greatest phenotypic variation. A further 34% of adults with disease (predominantly Leber's hereditary optic neuropathy [LHON]) were affected by the m.11778G>A or m.3460G>A point mutations (Table). Single large-scale deletions of mtDNA were rarely inherited, but despite this, represented 13% of disease cases. The m.8344A>G mutation caused disease (myoclonic epilepsy with ragged red fibers) in a further 4%, whereas the remaining 9% of affected adults harbored 1 of 10 other mtDNA point mutations: m.1624C>T (n = 1), m.4274T>C (n = 1), m.4298G>A (n = 1), m.5816A>G (n = 2), m.7989T>C (n = 1), m.8993T>G (n = 1), m.10010T>C (n = 1), m.12258G>A (n = 1), m.12320A>G (n = 1), and m.14709T>C (n = 3).

Table –. Prevalence Estimates for Mitochondrial DNA Disease in the North East of England
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At Risk

The midyear population of adults and children younger than pensionable age in the North East government office region for 2001 was 2,032,100.4 Limiting our inclusion criteria to first-degree relatives only, we identified 332 clinically unaffected individuals at risk for development of mtDNA disease, which equates to a minimum point prevalence of 16.5 in 100,000 (95% confidence interval, 14.8–18.3/100,000).

To confirm the validity of selection criteria for this group, we evaluated molecular genetic results in asymptomatic adults who had requested predictive genetic testing. Where this was undertaken, 82% of those “at risk” for inheriting the m.3243A>G mutation were positive in at least one tissue. When the analysis was restricted to first-degree relatives confirmed to have no clinical evidence of disease, the m.3243A>G mutation was still present in 75% of cases. An even greater proportion of tests was positive in relatives at risk for inheriting the m.8344A>G and m.14709T>G mtDNA point mutations (94% and 100%, respectively).

Discussion

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We have undertaken a comprehensive epidemiological study of primary mtDNA disease in North East England and can report a minimum point prevalence figure for clinically affected adults of 9.18 in 100,000 people (95% confidence interval, 7.8–10.8/100,000). We have also identified that currently asymptomatic first-degree relatives of patients with mtDNA point mutations are at considerably increased risk for development of mitochondrial disease, the prevalence among this group being 16.5 in 100,000 (95% confidence interval, 14.8–18.3/100,000). This has been confirmed by prospective follow-up of an “at-risk” group harboring the m.3243A>G mutation, of whom approximately 30% developed cardinal features of mitochondrial disease within 5 years (data not shown). These prevalence figures are substantially increased on our previous estimates largely as a result of extensive family tracing of index cases. However, improvements in diagnostic techniques, in particular detection of the m.3243A>G in urinary epithelial cells and whole mitochondrial genome sequencing, and an increased awareness of mitochondrial disease among health professionals working in North East England have contributed significantly to this apparent increase in prevalence. At the time of our previous study, genetic counseling and testing for LHON was well established with large kindreds identified and recorded. This continues to be the case for LHON, but has now also been extended to include other mtDNA point mutations. Consequently, although the prevalence of LHON appears to have altered little since our previous estimates, that of non-LHON mutations has increased. Detailed pedigree analysis and family tracing is important to ascertain the true prevalence of mtDNA disease but is often complicated by variation in the inheritance of different mtDNA mutations and obscuration of maternal inheritance patterns by asymptomatic carriers. Furthermore, insidious progression of symptoms such as ptosis or proximal weakness may go unreported, whereas apparently unconnected disease features in different family members may fail to be associated. Identifying those at risk is particularly important for managing clinical conditions with a long progressive course such as diabetes or cardiomyopathy, where early and sustained intervention can be of benefit.

Comparison with other epidemiological studies is difficult because many of these have confined their analysis to a single mtDNA genotype, specific clinical phenotypes within defined hospital populations, or a combination of the two.15–22 Majamaa and colleagues22 detected a prevalence of clinically affected individuals with m.3243A>G of 5.71 in 100,000 (95% confidence interval, 4.53–6.89/100,000) but did not detect a single individual with m.8344A>G. The reason for the difference from our data is not clear, although differences in the respective genetic backgrounds, population structures, and study design may be important.

Our studies of the m.3243A>G cohort demonstrated no significant variation in the frequency of positive results among mothers, siblings, or children of an affected individual or among the first-, second-, and third-degree relatives who requested predictive genetic testing. Although clinical details of relatives of the Finnish patients were not described, Majamaa and colleagues22 did report that 27 in 33 volunteered blood samples tested for the m.3243A>G mutation had positive results. Although these were from aunts and uncles in addition to mothers and siblings, the positive identification rate of 82% is identical to that obtained in first-degree relatives in the North East of England. Although such results justify offering genetic advice to relatives, the level of heteroplasmy for the m.3243A>G mutation in blood has little or no clinical predictive value.23 This is in contrast with mtDNA extracted from muscle, which offers a correlation between heteroplasmy and the prevalence of clinical features.24 Alternatively, heteroplasmy for the m.3243A>G mutation in urinary epithelial cells has been shown to parallel that in muscle and offers a noninvasive method of assessment.5

To facilitate family tracing and calculation of the “at-risk” group, we have confined this analysis to primary mutations of mtDNA and excluded mutations in nuclear genes that cause multiple deletions (or depletion) of mtDNA. Several such genes have now been described (POLG1,PEO1,TK2,DGOUK,MPV17,SLC25A4, and RRM2B) and are associated with mitochondrial disease in adults and children.25–27 Other recessive nuclear mutations are known to affect structural subunits or assembly of mitochondrial respiratory chain complexes, and again these have not been included in our prevalence figures. Our clinical experience in a tertiary referral center for mitochondrial disease would suggest that approximately 75% of adult-onset mitochondrial disease is a consequence of primary mtDNA mutations, whereas this figure is less than 20% for childhood presentation.

Conclusion

The chronic morbidity associated with mtDNA disease is reflected in the long-term medical and social support provided to these patients. Accurate assessment of mtDNA disease prevalence is therefore extremely important, particularly regarding health resource allocation. Previous studies have focused on one genotype or phenotype and have consequently been hampered by case ascertainment issues or their relevance to other types of mtDNA disease. This study has overcome these difficulties and allowed us to estimate the point prevalence of mtDNA disease in a stable population of working-age adults in the North East of England. Although necessarily conservative, our data provide the highest recorded prevalence figures for mtDNA disease and highlight the risk for disease to relatives, including those who are asymptomatic at diagnosis of the index case.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by a Medical Research Council Clinician Scientist Fellowship (G108/539, R.M.).

We thank EUMITOCOMBAT, the Wellcome Trust, the Muscular Dystrophy Campaign, and the Newcastle upon Tyne Hospitals National Health Service Foundation Trust for their continued support.

References

  1. Top of page
  2. Abstract
  3. Subjects and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References