Kirsi Huoponen

Clinical expression of Leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background.

Leber hereditary optic neuropathy (LHON) is due primarily to one of three common point mutations of mitochondrial DNA (mtDNA), but the incomplete penetrance implicates additional genetic or environmental factors in the pathophysiology of the disorder. Both the 11778G-->A and 14484T-->C LHON mutations are preferentially found on a specific mtDNA genetic background, but 3460G-->A is not. However, there is no clear evidence that any background influences clinical penetrance in any of these mutations. By studying 3,613 subjects from 159 LHON-affected pedigrees, we show that the risk of visual failure is greater when the 11778G-->A or 14484T-->C mutations are present in specific subgroups of haplogroup J (J2 for 11778G-->A and J1 for 14484T-->C) and when the 3460G-->A mutation is present in haplogroup K. By contrast, the risk of visual failure is significantly less when 11778G-->A occurs in haplogroup H. Substitutions on MTCYB provide an explanation for these findings, which demonstrate that common genetic variants have a marked effect on the expression of an ostensibly monogenic mtDNA disorder.

Identification of SLC7A7, encoding y+LAT-1, as the lysinuric protein intolerance gene.

Lysinuric protein intolerance (LPI; OMIM 222700) is a rare, recessive disorder with a worldwide distribution, but with a high prevalence in the Finnish population; symptoms include failure to thrive, growth retardation, muscle hypotonia and hepatosplenomegaly. A defect in the plasma membrane transport of dibasic amino acids has been demonstrated at the basolateral membrane of epithelial cells in small intestine and in renal tubules and in plasma membrane of cultured skin fibroblasts from LPI patients. The gene causing LPI has been assigned by linkage analysis to 14q11-13. Here we report mutations in SLC7A7 cDNA (encoding y+L amino acid transporter-1, y+LAT-1), which expresses dibasic amino-acid transport activity and is located in the LPI region, in 31 Finnish LPI patients and 1 Spanish patient. The Finnish patients are homozygous for a founder missense mutation leading to a premature stop codon. The Spanish patient is a compound heterozygote with a missense mutation in one allele and a frameshift mutation in the other. The frameshift mutation generates a premature stop codon, eliminating the last one-third of the protein. The missense mutation abolishes y+LAT-1 amino-acid transport activity when co-expressed with the heavy chain of the cell-surface antigen 4F2 (4F2hc, also known as CD98) in Xenopus laevis oocytes. Our data establish that mutations in SLC7A7 cause LPI.

mtDNA Variation in the South African Kung and Khwe—and Their Genetic Relationships to Other African Populations

The mtDNA variation of 74 Khoisan-speaking individuals (Kung and Khwe) from Schmidtsdrift, in the Northern Cape Province of South Africa, was examined by high-resolution RFLP analysis and control region (CR) sequencing. The resulting data were combined with published RFLP haplotype and CR sequence data from sub-Saharan African populations and then were subjected to phylogenetic analysis to deduce the evolutionary relationships among them. More than 77% of the Kung and Khwe mtDNA samples were found to belong to the major mtDNA lineage, macrohaplogroup L* (defined by a HpaI site at nucleotide position 3592), which is prevalent in sub-Saharan African populations. Additional sets of RFLPs subdivided macrohaplogroup L* into two extended haplogroups-L1 and L2-both of which appeared in the Kung and Khwe. Besides revealing the significant substructure of macrohaplogroup L* in African populations, these data showed that the Biaka Pygmies have one of the most ancient RFLP sublineages observed in African mtDNA and, thus, that they could represent one of the oldest human populations. In addition, the Kung exhibited a set of related haplotypes that were positioned closest to the root of the human mtDNA phylogeny, suggesting that they, too, represent one of the most ancient African populations. Comparison of Kung and Khwe CR sequences with those from other African populations confirmed the genetic association of the Kung with other Khoisan-speaking peoples, whereas the Khwe were more closely linked to non-Khoisan-speaking (Bantu) populations. Finally, the overall sequence divergence of 214 African RFLP haplotypes defined in both this and an earlier study was 0.364%, giving an estimated age, for all African mtDNAs, of 125,500-165,500 years before the present, a date that is concordant with all previous estimates derived from mtDNA and other genetic data, for the time of origin of modern humans in Africa.

Mitochondrial DNA Diversity in Indigenous Populations of the Southern Extent of Siberia, and the Origins of Native American Haplogroups

In search of the ancestors of Native American mitochondrial DNA (mtDNA) haplogroups, we analyzed the mtDNA of 531 individuals from nine indigenous populations in Siberia. All mtDNAs were subjected to high‐resolution RFLP analysis, sequencing of the control‐region hypervariable segment I (HVS‐I), and surveyed for additional polymorphic markers in the coding region. Furthermore, the mtDNAs selected according to haplogroup/subhaplogroup status were completely sequenced. Phylogenetic analyses of the resulting data, combined with those from previously published Siberian arctic and sub‐arctic populations, revealed that remnants of the ancient Siberian gene pool are still evident in Siberian populations, suggesting that the founding haplotypes of the Native American A‐D branches originated in different parts of Siberia. Thus, lineage A complete sequences revealed in the Mansi of the Lower Ob and the Ket of the Lower Yenisei belong to A1, suggesting that A1 mtDNAs occasionally found in the remnants of hunting‐gathering populations of northwestern and northern Siberia belonged to a common gene pool of the Siberian progenitors of Paleoindians. Moreover, lineage B1, which is the most closely related to the American B2, occurred in the Tubalar and Tuvan inhabiting the territory between the upper reaches of the Ob River in the west, to the Upper Yenisei region in the east. Finally, the sequence variants of haplogroups C and D, which are most similar to Native American C1 and D1, were detected in the Ulchi of the Lower Amur. Overall, our data suggest that the immediate ancestors of the Siberian/Beringian migrants who gave rise to ancient (pre‐Clovis) Paleoindians have a common origin with aboriginal people of the area now designated the Altai‐Sayan Upland, as well as the Lower Amur/Sea of Okhotsk region.

Electron transfer properties of NADH: Ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON)

We report the electron transfer properties of the NADH: ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. TheNDI/3460 mutation exhibits 80% reduction in rotenone‐sensitive and ubiquinone‐dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, theND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations: alsoK m for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with theND4 mutation, the rate of oxidation of NAD‐linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH‐dependent dehydrogenases and Complex I, which may result in fast (‘solid state’) electron transfer from the former to the latter.

Identification of an X-Chromosomal Locus and Haplotype Modulating the Phenotype of a Mitochondrial DNA Disorder

Mitochondrial DNA (mtDNA) mutations are a major cause of human disease. A large number of different molecular defects ultimately compromise oxidative phosphorylation, but it is not clear why the same biochemical defect can cause diverse clinical phenotypes. There is emerging evidence that nuclear genes modulate the phenotype of primary mtDNA disorders. Here, we define an X-chromosomal haplotype that interacts with specific MTND mutations to cause visual failure in the most common mtDNA disease, Leber hereditary optic neuropathy. This effect is independent of the mtDNA genetic background and explains the variable penetrance and sex bias that characterizes this disorder.

Epidemiology and penetrance of Leber hereditary optic neuropathy in Finland

We have performed an entire-population-based survey of the epidemiology and penetrance of Leber hereditary optic neuropathy (LHON) in Finland – a country that is among the best-studied genetic isolates in the world. During our long-term clinical follow-up period since 1970, we have so far identified 36 LHON families in Finland, comprised of almost 1000 family members. Counting the unaffected family members has been possible thanks to accessible genealogical records, and this has improved the accuracy of our penetrance figures by minimizing the sample bias. Our results, although confirming some well-known features of LHON, indicate that the overall penetrance of LHON is lower than previously estimated, and that affected females have a higher incidence of affected offspring compared to the unaffected females. The prevalence of LHON in Finland is 1:50 000, and one in 9000 Finns is a carrier of one of the three LHON primary mutations.

mtDNA Haplogroups and Frequency Patterns in Europe

To the Editor: Recently, an article by Simoni et al. (2000), who used (i) SAAP analysis to analyze the population frequencies of mtDNA haplogroups and (ii) AIDA analysis to examine both the frequency and the sequence similarity of truncated mtDNA sequences, appeared in this Journal. The main outcome of their study was that “the overall patterns of mtDNA diversity appear to be poorly significant in Europe.” The raw data comprised 2,619 hypervariable segment I (HVS-I) sequences (denoted as “HVR-I” [hypervariable region I] sequences by Simoni et al. [2000]) that were obtained from 36 regions or populations of Europe, the Near East, and the Caucasus and that were collected from both the literature and unpublished sources. Simoni et al. ostensibly grouped the HVS-I sequences according to haplogroup motifs proposed elsewhere (Richards et al. 1998), and they reported the resulting frequencies for each region/population in table 3 in their study. We have checked the input data displayed in table 3 and have found serious technical errors affecting numerous entries. More critically, the mtDNA categories that they report correspond neither to their own criteria nor to the haplogroup definitions established in the literature (to which they refer). Furthermore, their decision to truncate HVS-I information (and to disregard RFLP information) renders these data inadequate to differentiate even African and East Asian sequences from European sequences in many cases. Inspection of table 3 in the study by Simoni et al. (2000) reveals that (i) the data in the “Galicia” and “Spain: Central” rows have been, in part, crossed-over, (ii) the data in the “Belgium,” “Alps,” and “Turkey” rows have been computed with the use of sample sizes smaller than those reported in table 1 in the same study, (iii) the haplogroup “J” column has been totally randomized, and (iv) the “Other” column is complementary to the last four “superhaplogroup” columns but not to the first 11 haplogroup columns. As for item (iii), almost all positive entries in the haplogroup “J” column have been either displaced or calculated with the use of sample sizes corresponding to nearby rows. Hence, most entries in this column diverge widely from the real haplogroup J frequencies (see the last column of table 1 in the present study). Table 1 Haplogroup J Frequencies According to Simoni et al. (2000), a Crude Default Criterion, and Inference in the Present Study As an example of their haplogroup assignment, Simoni et al. (2000) specifically referred to the motif 16069T–16126C for haplogroup J, but they overlooked the fact that this criterion cannot formally be applied to the sequences in the study by Richards et al. (1996), since these were reported only between 16090 and 16365. This might explain some of the many “0” entries in the haplogroup “J” column of table 3 in the Simoni et al. study (see table 1 in the present study). Simoni et al. should have either adopted the haplogroup J frequencies reported by Richards et al. (1996), excluded these population samples from their study, or trimmed all data to the shortest common segment. In the latter case, by employing the motif 16126C–16294C, one could take the default cluster JT-T (comprising all JT sequences that are not T) as a crude default criterion for haplogroup J (see table 1 in the present study). The discrepancies in haplogroup frequencies are by no means restricted to haplogroup J. Table 2 in the present study shows the marked contrast between published haplogroup frequencies and those assumed by Simoni et al. (2000) for the well-characterized Tuscan, Druze, and Adygei samples (which were typed for RFLPs as well as for HVS-I sequences by Torroni et al. [1996] and Macaulay et al. [1999]). The large differences in frequency for haplogroup H, the most-common European haplogroup, are due to the premise of Simoni et al. (2000) that haplogroup “H contains all sequences . . . that show none of the 22 substitutions considered in this study.” This extreme simplification results, on the one hand, in the dumping of large numbers of haplogroup H mtDNAs mainly into the default category “Other” and, on the other hand, in the inclusion of several non-H sequences within their haplogroup H category. For instance, by their criterion, 10/20 haplogroup H mtDNAs from the Tuscan sample (Torroni et al. 1996) would no longer be scored as “H,” whereas the U sequence 16051G–16309G–16318C would be scored as “H.” In consequence, the haplogroup H category described by Simoni et al. (2000) is bound to be highly polyphyletic in the mtDNA genealogy and does not reflect the spatial patterns of haplogroup H. Table 2 Haplogroup Frequencies, According to Simoni et al. (2000) vs. the Original Studies, in Tuscan, Druze, and Adygei Populations At this point, it is important to clarify what haplogroup classification entails. An mtDNA haplogroup, when properly defined, is a monophyletic clade of the mtDNA genealogy. Originally, high-resolution RFLP analysis (employing 14 enzymes) had been used for identification of clades by signature sites (Torroni et al. 1992, 1993, 1994a, 1994b, 1996; Chen et al. 1995), and current haplogroup nomenclature originated in that context. In retrospect, this approach is indeed quite reliable, although recurrent changes at a few sites, such as 10394 DdeI, may occasionally cause problems. Potential ambiguities can largely be resolved by incorporation of information from other segments of mtDNA sequences or specific positions of the coding regions (Torroni et al. 1997; Brown et al. 1998; Starikovskaya et al. 1998; Macaulay et al. 1999; Quintana-Murci et al. 1999; Schurr et al. 1999). For instance, haplogroup K is now understood to be a clade (as are U1–U6) within haplogroup U. HVS-I data in combination with partial RFLPs can sometimes serve as a satisfactory substitute for a full RFLP analysis (Rando et al 1998, 2000; Kivisild et al. 1999a, 1999b). Unfortunately, HVS-I data alone, which have been produced en masse, often do not contain sufficient information for confident assignment of haplogroup affiliation. The truncation of the HVS-I data to only 13–22 variant positions, as performed by Simoni et al. (2000), yields even poorer results. For example, the motif 16223T–16278T, which was used by Simoni et al. to identify haplogroup X, would transfer most African L1/L2 sequences (Watson et al. 1997; Rando et al. 1998) into the then artefactual category “X.” For Europe, this is relevant insofar as a few L1/L2 sequences are present in Iberia (Rocha et al. 1999), and there even resides an African L1c sequence with the motif 16223T–16278T in the British data (Piercy et al. 1993). In addition, as was previously pointed out (Torroni et al. 1996; Macaulay et al. 1999), one has to be prepared for recurrent mutations in the HVS-I motifs (compare also figs. 4, 5, 8, and 9 of the study by Richards et al. [1998]). For instance, the frequency discrepancy (17.8% vs. 26.7%) for haplogroup X in the Druze sample (see table 2 in the present study) is due to the fact that Simoni et al. did not include four haplogroup X mtDNAs that have mutated to 16223C. Another of the many possible examples of misclassification caused by the use of truncated motifs is illustrated by 16129A–16223T, the motif used by Simoni et al. for classification of haplogroup I mtDNAs. Use of this truncated motif has led them to classify both the Asian haplogroup C mtDNAs (16129A–16223T–16298C–16327T) of the Adygei (6.0%) and the East African haplogroup M1 mtDNA (16129A–16189C–16223T–16249C–16311C–16359C) of the Druze (2.2%) as members of haplogroup I (see table 2 in the present study). The issue of haplogroups only affects the SAAP analysis. However, there are also serious difficulties with the AIDA analysis. Ideally, AIDA should be applied to full DNA-sequence data, but Simoni et al. (2000) included only 22/241 variant positions. One cannot expect that such a truncated data set would show much evidence of geographic patterns within Europe. Most of the haplogroup diagnostic variants in western Eurasian mtDNA are very ancient, and they probably evolved in the Near East and subsequently spread to Europe (Torroni et al. 1998; Macaulay et al. 1999); at any event, they occur throughout western Eurasia. The more recent “rare substitutions,” which have evolved since the earlier dispersals and which Simoni et al. (2000) discarded as “statistical noise,” are precisely those that are most likely to show regional distributions. The exclusion of such mutations severely restricts the capacity to identify phylogeographic units and, thus, is bound to have seriously reduced the power of the approach to detect autocorrelation. Even when haplogroup assignment is done with care, failure to detect significant clines in haplogroup frequencies does not prove the absence of any spatial structure in the mtDNA pool. Such structure would rather be manifest at a phylogenetically finer scale (defined on the basis of more-recent mutations). In any case, one would not expect that meaningful patterns of mtDNA diversity could emerge from analyses based on categories with no demonstrable phylogenetic support.

Leber hereditary optic neuropathy: clinical and molecular genetic findings

Abstract. Leber hereditary optic neuropathy (LHON) is a maternally inherited disease characterized by acute or subacute painless central visual loss usually in young adults, predominantly in males. Except for optic atrophy, LHON patients are usually otherwise healthy. Occasionally, LHON is associated with neurological, cardiac, and skeletal changes. The clinical course of LHON has several stages. Peripapillary microangiopathy is present from the beginning. Microangiopathy disappears as the disease progresses towards the end stages. Simultaneously, the retinal nerve fiber layer fades from view, first papillomacular nerve fiber bundles, and months later, the whole nerve fiber layer becomes atrophic. At the end stage the centrocecal scotoma is large and absolute. Loss of vision is usually permanent, but spontaneous recovery can occur. Despite a few attempts, no effective treatment to prevent or halt LHON has been found. Several mitochondrial DNA (mtDNA) mutations are associated with LHON, but the pathogenic processes leading to optic nerve atrophy are largely unknown. About 15% of the families are heteroplasmic, i.e., both mutant and wild type mtDNA coexist within an individual. The level of heteroplasmy between different tissues can vary markedly. mtDNA mutations are not sufficient to cause visual loss in LHON, since not all individuals harboring a pathogenic LHON mutation express the disease. There are additional genetic and/or environmental precipitating factors, but thus far they are unknown.

The spectrum of mitochondrial DNA mutations in families with Leber hereditary optic neuroretinopathy.

The mitochondrial complex I genes were sequenced in seven Leber hereditary optic neuroretinopathy (LHON) families without the ND4/11778 and ND1/3460 mutations. Four replacement mutations restricted only to LHON families were found, one in the ND1 gene at nt 4025, and three in the ND5 gene at nt 12811, 13637, and 13967. The mutations did not change evolutionarily conserved amino acids suggesting that they are not primary LHON mutations in these families. They may be considered as secondary LHON mutations serving as exacerbating factors in an appropriate genetic background. A complex III mutation, cyt b/15257, has been suggested to be one of the primary mutations causing LHON. Its presence was determined for 23 Finnish LHON families, and it was detected in two families harboring the ND4/11778 mutation. Similarly, complex IV mutation COI/7444 was screened in Finnish LHON families, and it was found in one family carrying the ND1/3460 mutation.