Longitudinal analysis of the segregation of mtDNA mutations in heteroplasmic individuals

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Abstract

The mutation load of the pathogenic LHON (Leber hereditary optic neuropathy) mtDNA mutation at nucleotide 3460 has been followed over time in the WBC/platelet fraction from members of a matrilineal pedigree. Longitudinal analysis over a sampling period of five to six years indicates that, in all five heteroplasmic family members, the mutation load decreases at a mean overall rate of approximately 1% per year. There was no change in mutation load in homoplasmic wildtype or in homoplasmic mutant individuals. For the purposes of comparison, a longitudinal analysis of a silent mtDNA polymorphism at nucleotide 14560 was also carried out for members of a second matrilineal pedigree. In contrast to the results for the pathogenic mtDNA mutation, there was no change in the proportion of the silent polymorphism in the WBC/platelet fraction of four family members over a period of seven years. These results indicate that the pathogenic 3460 LHON mutation segregates under negative selection in these cell populations. One possible mechanism through which selection may operate is that, in heteroplasmic individuals, the hematopoietic stem cells are generally homoplasmic, either for the wildtype or for the mutant allele. The homoplasmic mutant stem cells, because of their mitochondrial respiratory chain defect, produce fewer mature WBCs and platelets over time than do the wildtype stem cells. Alternatively, the stem cells may be heteroplasmic and selection may act to favor proliferation of mitochondria with lower levels of the pathogenic mutation in the WBC/platelet cell populations.

Introduction

Leber hereditary optic neuropathy (LHON), which involves an acute or subacute onset of bilateral loss of central vision, is a major cause of inherited blindness in males (reviewed in Ref. [1]). LHON shows strict maternal inheritance in families and the primary etiologic component of LHON is a mutation in the mitochondrial genome (mtDNA). LHON is thus one of a number of mitochondrial diseases [1]. In the majority of LHON patients and family members, the pathogenic mtDNA mutation is homoplasmic; that is, all mtDNA molecules carry the mutant allele. However, a small proportion of LHON patients are heteroplasmic and there are two populations of mtDNA molecules, those which carry the mutant allele and those which carry the wildtype allele [2]. In contrast to LHON, patients with other mitochondrial diseases are almost always heteroplasmic [1].

The inheritance and segregation of human mitochondrial genes are complex processes, partly because of high mtDNA copy numbers/cell (for example, Refs. [3], [4]). The analysis of heteroplasmic pathogenic mtDNA mutations has been important for the analysis of these processes. A thorny area of human mitochondrial genetics is the extent to which inheritance and segregation of pathogenic mtDNA point mutations are influenced by selection. This important issue is rife with results and hypotheses that are in apparent conflict. Problems have arisen, because neither the timespan over which selection acts, nor the level(s) at which it operates – molecular, cellular, or population – have been clarified. When analyzed at the level of population genetics and human evolution, a large fraction of all mtDNA sequence changes are mildly deleterious and subject to negative selection (briefly reviewed in Refs. [1], [5]). As a consequence of their severe phenotypic effects, highly pathogenic mtDNA mutations should segregate under strong negative selection and be eliminated quickly from the population (for example, Ref. [6]). Nevertheless, a model of positive selection has been proposed on the basis of studies of these pathogenic mtDNA mutations in cybrid lines [7]. It was hypothesized that there is preferential replication of mutant mtDNA molecules, because of a compensatory proliferation of mitochondria that are respiration deficient (see also Ref. [2]), and that mitochondria – rather than the mtDNA molecules themselves – are the unit of selection. Other studies of cybrid systems, however, contradict this model [8]. The cybrid analyses, including the dependence of the cybrid cells on mitochondrial respiratory chain function, are apparently complicated by the choice of nuclear genetic background, as well as by the culture conditions and media components. Furthermore, extensive analyses of myoblast cultures show negative selection with elimination of the pathogenic mutations within a relatively few cell doublings [9].

The operation of selection has not been clarified by the in vivo studies that have been reported. Both longitudinal [10], [11], [12] and cross-sectional [13], [14], [15], [16] analyses indicate that some pathogenic mutations increase over time within heteroplasmic individuals, whereas other mutations decrease or stay at the same level. Conflicting trends for the same mutation have been reported (for example, Ref. [14] versus Ref. [16]). Furthermore, there is substantial evidence that a pathogenic mutation can show different patterns of segregation as a function of the tissue that is analyzed [15], [17], [18]. Thus, the mutation load is generally lower in WBC/platelets than in muscle, and it has been proposed that the mutant mtDNA molecules segregate under conditions of negative selection in these blood cell populations [18].

We report here the results of longitudinal analyses of the segregation of heteroplasmic mtDNA mutations. The rationale of this approach is that a significant and consistent change in allele frequency (mutation load) with time is direct evidence for the operation of selection, positive or negative depending upon whether mutation load increases or decreases (for example, chapter 2 of Ref. [19]; see also Ref. [18]).

Section snippets

DNA samples

Venous blood samples were obtained with informed consent and shipped by courier service to UTMB. After standing overnight in a cold room, the WBC/platelet fraction was removed and the cells pelleted by centrifugation. DNA was then isolated by standard procedures of SDS/proteinase K digestion, phenol extraction, and ethanol precipitation. DNA was resuspended in buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and stored at 4°C.

Primer extension assays

Assays of the mutation load at nucleotide 3460 were carried out as

Longitudinal analysis of a mtDNA silent polymorphism

The members of the NWC1 LHON family (pedigree A in Ref. [23]) are homoplasmic for the LHON mutation at nucleotide 3460, but heteroplasmic for a GC:AT silent polymorphism at nucleotide 14560 [24], as designated with the nucleotide coordinates of the Cambridge Reference Sequence [25]. The initial blood samples were obtained in June 1990, the WBC/platelet fractions were used for DNA isolation, and the proportions of the two alleles at nucleotide 14560 were determined through nucleotide sequencing

Discussion

The decrease in mutation load over time indicates that the pathogenic 3460 LHON mutation segregates in the WBC/platelet cell population under negative selection. Although the present report is the most extensive longitudinal study of mtDNA segregation reported thus far, there are two concerns that should be noted before any further interpretation of these results is offered. In the first place, both pedigrees need to be assayed at additional time points to yield a more complete picture of the

Acknowledgements

This study would not have been possible without the cooperation of the LHON families and we extend to them our appreciation. The technical assistance of Iwona Kubacka and Steven Halvorson is gratefully acknowledged. We thank Drs Rob Taylor and Patrick Chinnery (University of Newcastle) for their assistance in obtaining and processing of blood samples from the NWC1 LHON family. This research was supported by grants from the National Eye Institute (RO1 EY10758) and the John Sealy Memorial

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