David R. Marchington

Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Is the Bottleneck Cracked

Since the development of molecular diagnosis of mtDNA disease, there has been increasing pressure on clinical geneticists for genetic counseling of this uniquely difficult group. However, these advances have revolutionized neither prognostication nor prenatal diagnosis of mitochondrial diseases. This inability to predict risk of affected offspring in mtDNA diseases is largely due to the uniparental inheritance of multiple copies of mtDNA. Recently, however, the prospects for prenatal diagnosis of mtDNA diseases have taken a turn for the better.

Information for genetic management of mtDNA disease: Sampling pathogenic mtDNA mutants in the human germline and in placenta.

Background Families with a child who died of severe, maternally inherited mitochondrial DNA (mtDNA) disease need information on recurrence risk. Estimating this risk is difficult because of (a) heteroplasmy—the coexistence of mutant and normal mtDNA in the same person—and (b) the so-called mitochondrial bottleneck, whereby the small number of mtDNAs that become the founders for the offspring cause variation in dose of mutant mtDNA. The timing of the bottleneck and of segregation of mtDNA during foetal life determines the management options. Therefore, mtDNA heteroplasmy was studied in oocytes and placenta of women in affected families. Results One mother of a child dying from Leigh syndrome due to the 9176T→C mtDNA mutation transmitted various loads of mutant mtDNA to ≤3 of 20 oocytes. This was used to estimate recurrence as ≤5%. She subsequently conceived a healthy son naturally. Analysis of the placenta showed that some segregation also occurred during placental development, with the mutant mtDNA load varying by >10% in a placenta carrying 65% 3243A→G mutant mtDNA. Discussion This is the first report of (a) an oocyte analysis for preconception counselling, specifically, refining recurrence risks of rare mutations and (b) a widely different load of a pathogenic mtDNA mutation in multiple oocytes, apparently confined to the germline, in an asymptomatic carrier of an mtDNA disease. This suggests that a major component of the bottleneck occurs during oogenesis, probably early in the foetal life of the mother. The variable mutant load in placenta implies that estimates based on a single sample in prenatal diagnosis of mtDNA disorders have limited accuracy.

A prevalent POLG CAG microsatellite length allele in humans and African great apes.

The human nuclear gene for the catalytic subunit of mitochondrial DNA polymerase γ (POLG) contains within its coding region a CAG microsatellite encoding a polyglutamine repeat. Previous studies demonstrated an association between length variation at this repeat and male infertility, suggesting a mechanism whereby the prevalent (CAG)10 allele, which occurs at a frequency of >80% in different populations, could be maintained by selection. Sequence analysis of the POLG CAG microsatellite region of more than 1000 human chromosomes reveals that virtually all allelic variation at the locus is accounted for by length variation of the CAG repeat. Analysis of POLG from African great apes shows that a prevalent length allele is present in each species, although its exact length is species-specific. In common chimpanzee (Pan troglodytes) a number of different sequence variants contribute to the prevalent length allele, strongly supporting the idea that the length of the POLG microsatellite region, rather than its exact nucleotide or amino acid sequence, is what is maintained. Analysis of POLG in other primates indicates that the repeat has expanded from a shorter, glutamine-rich sequence, present in the common ancestor of Old and New World monkeys.

Mosaicism for mitochondrial DNA polymorphic variants in placenta has implications for the feasibility of prenatal diagnosis in mtDNA diseases

Women who have had a child with mitochondrial DNA (mtDNA) disease need to know the risk of recurrence, but this risk is difficult to estimate because mutant and wild-type (normal) mtDNA coexist in the same person (heteroplasmy). The possibility that a single sample may not reflect the whole organism both impedes prenatal diagnosis of most mtDNA diseases, and suggests radical alternative strategies such as nuclear transfer. We used naturally occurring mtDNA variants to investigate mtDNA segregation in placenta. Using large samples of control placenta, we demonstrated that the level of polymorphic heteroplasmic mtDNA variants is very similar in mother, cord blood and placenta. However, where placental samples were very small (<10 mg) there was clear evidence of variation in the distribution of mtDNA polymorphic variants. We present the first evidence for variation in mutant load, that is, mosaicism for mtDNA polymorphic variants in placenta. This suggests that mtDNA mutants may segregate in placenta and that a single chorionic villous sample (CVS) may be unrepresentative of the whole placenta. Duplicates may be necessary where CVS are small. However, the close correlation of mutant load in maternal, fetal blood and placental mtDNA suggests that the average load in placenta does reflect the load of mutant mtDNA in the baby. Provided that segregation of neutral and pathogenic mtDNA mutants is similar in utero, our results are generally encouraging for developing prenatal diagnosis for mtDNA diseases. Identifying mtDNA segregation in human placenta suggests studies of relevance to placental evolution and to developmental biology.

Reply to Chinnery et al.

To the Editor:We thank Chinnery et al. (1998xGenetic counseling and prenatal diagnosis for mtDNA disease. Chinnery, PF, Howell, N, Lightowlers, RN, and Turnbull, DM. Am J Hum Genet. 1998; 63: 1908–1910Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References1998 [in this issue]) for their appreciation of our article (Poulton et al. 1998xIs the bottleneck cracked?. Poulton, J, Macaulay, V, and Marchington, DR. Am J Hum Genet. 1998; 62: 752–757Abstract | Full Text | Full Text PDF | PubMed | Scopus (66)See all References1998) and for their reiteration of its main points, particularly the need to gather further data prospectively. Our article is in full agreement with all four of their reservations about direct application of current knowledge to clinical practice, and their new data on the 8344 mutation are very similar to the example we cite (Hammans et al. 1993xThe mitochondrial DNA transfer RNA(Lys)A→G(8344) mutation and the syndrome of myoclonic epilepsy with ragged red fibres (MERRF): relationship of clinical phenotype to proportion of mutant mitochondrial DNA. Hammans, SR, Sweeney, MG, Brockington, M, Lennox, GG, Lawton, NF, Kennedy, CR, Morgan-Hughes, JA et al. Brain. 1993; 116: 617–632Crossref | PubMed | Scopus (116)See all References1993). A recent study by White et al. (1998xRecurrence risks for mtDNA mutations at NT8993. White, S, Collins, V, Dahl, H, and Thorburn, D. Muscle Nerve Suppl. 1998; 7: S175See all References1998) that uses an empirical approach generates advice that is very similar to the predictions of our model.We would, however, like to correct two points. First, our figure 2 (Poulton et al. 1998xIs the bottleneck cracked?. Poulton, J, Macaulay, V, and Marchington, DR. Am J Hum Genet. 1998; 62: 752–757Abstract | Full Text | Full Text PDF | PubMed | Scopus (66)See all References1998) refers to levels of mutant mtDNA in ovary and progeny, not in blood (clearly stated in the figure). To clarify the validity of our predictions, we now display the figure, along with the measured levels of mutant mtDNA (fig. 1fig. 1; Marchington et al. 1998xEvidence from human oocytes for a genetic bottleneck in an mtDNA disease. Marchington, DR, Macaulay, V, Hartshorne, G, Barlow, D, and Poulton, J. Am J Hum Genet. 1998; 63: 769–775Abstract | Full Text | Full Text PDF | PubMed | Scopus (84)See all References1998). It is clear that such accurate estimates of the level of mutant mtDNA in ovary only rarely will be available to the genetic counselor; hence, we use the 8344 mutation as an example of a mutation that “generally exhibits less variation between tissues than is seen among some of the other, more common mtDNA mutations” (Poulton et al. 1998xIs the bottleneck cracked?. Poulton, J, Macaulay, V, and Marchington, DR. Am J Hum Genet. 1998; 62: 752–757Abstract | Full Text | Full Text PDF | PubMed | Scopus (66)See all References1998, pp. 755–56).Figure 1Fitting the repeated- and single-selection models to the data on mtDNA rearrangements: idealized plots for patient 1, for predicted percentage mutant in offspring, when 21% mutant mtDNA is in ovary, for repeated sampling (g=15, n=135; top) and for single selection (g=1, n=8; middle). Both reasonably fit the observed distribution (bottom; Marchington et al. 1998xEvidence from human oocytes for a genetic bottleneck in an mtDNA disease. Marchington, DR, Macaulay, V, Hartshorne, G, Barlow, D, and Poulton, J. Am J Hum Genet. 1998; 63: 769–775Abstract | Full Text | Full Text PDF | PubMed | Scopus (84)See all References1998).View Large Image | View Hi-Res Image | Download PowerPoint SlideSecond, our discussion in the section “Models Describing the Mitochondrial Bottleneck” (Poulton et al. 1998xIs the bottleneck cracked?. Poulton, J, Macaulay, V, and Marchington, DR. Am J Hum Genet. 1998; 62: 752–757Abstract | Full Text | Full Text PDF | PubMed | Scopus (66)See all References1998, pp. 754–55) is far from a “recommended acceptance of a proposed simple bottleneck model” or a premature “application to prenatal mitochondrial diagnosis” (Chinnery et al. 1998xGenetic counseling and prenatal diagnosis for mtDNA disease. Chinnery, PF, Howell, N, Lightowlers, RN, and Turnbull, DM. Am J Hum Genet. 1998; 63: 1908–1910Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References1998, p. 1910). We did not recommend acceptance but suggested that “once more data have been collected [such as that described in White et al. 1998xRecurrence risks for mtDNA mutations at NT8993. White, S, Collins, V, Dahl, H, and Thorburn, D. Muscle Nerve Suppl. 1998; 7: S175See all References1998], such estimations will become usable in the medium term; reasonable fits may be more useful to patients than is the quality of information currently issued” (Poulton et al. 1998xIs the bottleneck cracked?. Poulton, J, Macaulay, V, and Marchington, DR. Am J Hum Genet. 1998; 62: 752–757Abstract | Full Text | Full Text PDF | PubMed | Scopus (66)See all References1998, p. 756). We also stated, “Although most clinicians will feel that CVS [chorionic villus sampling] is not yet widely applicable to mtDNA disease, there is clearly an urgent need to collect the human data needed to complete the picture” (Poulton et al. 1998xIs the bottleneck cracked?. Poulton, J, Macaulay, V, and Marchington, DR. Am J Hum Genet. 1998; 62: 752–757Abstract | Full Text | Full Text PDF | PubMed | Scopus (66)See all References1998, p. 756).