Population genetics and disease susceptibility: characterization of central European haplogroups by mtDNA gene mutations, correlation with D loop variants and association with disease

Mitochondrial (mt)DNA haplogroups in a German control group ( n = 67) were characterized by screening mitochondrial coding regions encompassing most of the ND, tRNA and cyt b genes. We used a PCR-SSCP screening approach followed by direct sequencing of polymorphic mtDNA fragments. Five major mtDNA lineages, diverging in at least nine different haplogroups, could be defined by characteristic polymorphic sites in mitochondrial genes. Additional sequencing of two hypervariable segments (HVS-I and II) of the non-coding displacement (D) loop in all control subjects revealed that certain D loop variants were strongly correlated with lineages and haplogroups, while others represented hotspots occurring frequently in different haplogroups. The existence of identified lineages and haplogroups received support from data in the literature, obtained by use of different approaches. Subsequently, we investigated four disease groups for association with these haplogroups: (i) LHON patients ( n = 55) carrying at least one of the primary/intermediate LHON mutations at nt 3460, 11778, 14484 and/or 15257; (ii) patients suffering from Wolfram or DIDMOAD syndrome ( n = 8); (iii) MELAS patients (n = 9); (iv) a group of children, who died from ‘sudden infant death syndrome’ (SIDS) ( n = 9). The distribution patterns among the haplogroups of the disease groups (LHON, DIDMOAD and SIDS) differed considerably from the control population. LHON and DIDMOAD were significantly under-represented in the most frequent German haplogroup D C, but were concentrated in a mtDNA lineage defined by polymorphisms at nt 4216+11251+16126. As this lineage diverged into two precisely defined haplogroups, LHON and DIDMOAD could be assigned to the two haplogroups separately. Strikingly, SIDS was often found in association with two rare German haplogroups. MELAS patients were equally distributed among German haplogroups and, moreover, did not reveal any accumulation of specific D loop variants. We conclude that certain European mtDNA haplogroups define a genetic susceptibility basis for various disorders.


INTRODUCTION
Attempts to identify genes responsible for penetrant single gene disorders have been extraordinarily successful during the last decade and, presumably, will lead to the detection of monogenic defects in most of the corresponding common disorders within the next few years. The major challenge for medical genetics in the near future, however, appears to be the identification of genes contributing to heredity in disorders which are not transmitted in a simple Mendelian fashion (1). Common disorders like hypertension, Alzheimer's and Parkinson's disease or diabetes mellitus may be oligo-or even polygenic. While in chromosomal DNA problems in defining specific genes and gene mutations that play an important role in this concerted action are multiple and complex, the other human genome, i.e. mtDNA, offers the possibility of approaching the problems from another, better defined perspective.
Mitochondria are the power stations of the cell, providing energy for every specialized cell function in the form of ATP or heat. Essential parts of the oxidative phosphorylation apparatus are encoded by the mitochondrial genome. Therefore, severe defects of the mitochondrial genome lead to disease (2). mtDNA has already been fully sequenced (3). It exhibits a few interesting features which make it a suitable tool for phylogenetic and pathogenetic studies. The mitochondrial genome is exclusively maternally inherited and demonstrates no recombination events, but does exhibit high mutation and mutation fixation rates. The study of mtDNA sequence variation from around the world has led to defined population-specific mtDNA lineages which can be *To whom correspondence should be addressed. Tel: +49 89 3068 2670; Fax: +49 89 3068 3911; Email: diabetes@lrz.uni-muenchen.de + Both authors contributed equally to this work traced back to an African origin of the mitochondrial gene pool of modern Homo sapiens (4)(5)(6).
Besides being useful for phylogenetic studies, there is growing evidence that certain mtDNA clusters are associated with distinct disorders (7)(8)(9)(10)(11)(12)(13). Since most of these studies have been carried out on Caucasians, the exact definition of Caucasoid-specific mtDNA lineages and haplogroups could have important implications for an understanding of the relationship between mutability of the mitochondrial genome and disease. However, most mtDNA haplotype studies have employed RFLP analysis with a limited number of restriction endonucleases. In other studies, dealing primarily with phylogenetic aspects, only the most polymorphic region of the genome, the displacement (D) loop, has been sequenced, without any relation to mitochondrial gene polymorphisms. We used another more differentiating approach. Employing SSCP optimized for mutation detection in mtDNA fragments (14) and direct sequencing of polymorphic fragments, we analyzed wide regions of the mtDNA in a group of healthy German subjects. Here we report that the bulk of the German population can be described by certain well-defined mtDNA lineages which are characterized by homoplasmic gene polymorphisms while, even within these haplogroups, sequencing of the D loop resulted in many different haplotypes. Distribution of various disease groups among these haplogroups revealed that some of the established mitochondrial gene haplogroups predispose to disease.

Mitochondrial gene polymorphisms in the German population
mtDNA haplogroups of 67 healthy German control subjects were characterized by PCR-SSCP followed by direct sequencing of polymorphic PCR fragments. In this ongoing study we have screened, so far, ∼60% of ND, tRNA and cyt b genes and, additionally, small parts of COX, ATPase and rRNA genes. Using this approach, a total of 43 polymorphic sites compared with the Cambridge reference sequence (3) could be identified in our control group (Table 1). These were 27 polymorphic sites within ND genes, five in the cyt b gene, eight tRNA variants, one rRNA variant and two polymorphisms in non-coding regions (variants at nt 574 and 5580). The variants at nt 3423, 11335, 11447, 14199, 14272 and 15326 were detected in all 67 controls and were, Variants in coding regions were detected either by PCR-SSCP and subsequent direct sequencing or by RFLP methods; D loop polymorphisms were identified by sequencing of HVS-I and HVS-II segments.
The insertion variant at nt 574 indicates heteroplasmic C stretches. Control subjects were grouped according to their lineage association. The conservation of amino acids or nucleotides is indicated as high (H), medium (M) or low (L). In the case of an amino acid exchange, the conservation of the specific amino acid, in the case of a silent or a tRNA/rRNA mutation, the conservation of the nucleotide is given. +, base exchange; -, no base exchange; n.d., not detected; NC, non-coding region therefore, likely to represent errors in the reference sequence or very frequent polymorphisms. Only a few variants lead to an amino acid exchange at a highly conserved position (variants at nt 3448, 3394 and 12634) or are localized at highly conserved sites in tRNAs (variants at nt 12308 and 10463). In addition to our SSCP screening approach, the frequencies of seven further polymorphisms (nt 4136, 4216, 4336, 4917, 7028, 13708 and 15257) were investigated directly by RFLP methods ( Table 1). Analysis of the whole sequence data set (a total of 50 polymorphic sites) revealed that our German control population could be described and characterized by five major mtDNA lineages (Fig. 1 (4%), was defined by a C→T exchange at nt 15904 in the tRNA Thr gene. Two 7028(+) German controls were not further classified since no coding region variants have been identified so far. Among the 7028(-) subjects (46%, 31/67), representing the most frequent lineage D C , only six rare sub-branches, D C 3341 , D C 3338 , D C 5580 , D C 10463 , D C 14905 and D C 4336 , could be defined using our sequence data set. Twenty three subjects were identical to the Cambridge reference sequence, at least with respect to the gene fragments investigated here. It should be noted, however, that this latter branch D C was defined per exclusionem, i.e. by subtracting the other haplogroups from the Cambridge reference sequence.

D loop variants and correlation with mitochondrial gene mutations
The sequences of two hypervariable segments of the control region, HVS-I and HVS-II, were determined in all control subjects. Sequencing of a total of 674 bp revealed 90 variants (Table 1) corresponding to 13% of variable sites. As can be seen in Figure 1, many D loop variants were associated with defined lineages or haplogroups, which means that they were haplogroup specific. An A→G exchange at nt 263 and an insertion of one C at nt 311 were found in all subjects, indicating either frequent polymorphisms in the German population or errors in the reference sequence. With respect to the entire data set (coding and D loop regions), we observed 65 different haplotypes in 67 German controls. All control subjects characterized by at least one coding region polymorphism could be distinguished from one another by additional D loop polymorphisms. Only two pairs of control subjects belonging to haplogroup D C (subjects 731/765 and 742/764) shared identical D loop sequences.

Association with disease
Having established the characterization of our healthy central European (German) control group by several major mtDNA lineages segregated into carefully defined genetic sub-branches (haplogroups) and haplotypes (defined by additional D loop variants) we investigated whether distinct disorders cluster in certain lineages, haplogroups or haplotypes. For this purpose we collected our different disease cohorts. These were patients suffering from LHON, MELAS, DIDMOAD and children who had died from SIDS. Table 2 summarizes the distribution of these disease groups among German mitochondrial lineages and haplogroups. 13708 were significantly higher than would be expected from the haplogroup distribution found in our control population (P < 0.001).
To answer the question as to whether the primary LHON mutations were preferentially associated with a distinct haplotype defined by D loop variants we sequenced the two hypervariable D loop regions from 10 representative LHON patients belonging to haplogroup D 4216 13708 . Figure 2 shows that these LHON patients, although belonging to a precisely defined haplogroup, separated into two major branches and eight sub-branches mainly by HVS-I variants. Only two (of five) 11778 carriers and two (of two) 3460 carriers showed identical D loop sequences, while all other LHON patients represented different haplotypes. This pattern strongly suggests three important points. First, LHON is associated with haplogroup D 4216 13708 independent of the nature of the underlying primary LHON mutation. Second, most of the primary LHON mutations must have arisen several times within this haplogroup; for instance, the 11778 mutation at least four times and the 15257 intermediate LHON mutation at least three times; only the association of the 3460 mutation within haplogroup D 4216 13708 could be traced back to one mutational event. Thirdly, disease susceptibility is associated with mtDNA gene variants and not with any specific D loop haplotype, making founder effects unlikely.
DIDMOAD. We were able to demonstrate a statistically significant association between DIDMOAD and a distinct haplogroup (12). Six out of eight patients (75%) were associated with the 4216+11251+16126 lineage, of which five (63%) clustered in haplogroup D 4216 4917 (P < 0.01), with none in D 4216 13708 (Table 2). Although one single DIDMOAD patient carried the polymorphisms 10398 and 12612, he presented as 13708-negative, forming a distinct branch [designated as the 13708(-) sub-cluster]. We also sequenced the two hypervariable D loop regions from all five DIDMOAD patients belonging to the D 4216 4917 haplogroup. Although members of a precisely defined haplogroup, they could be grouped in five different haplotypes, indicating again, as observed in the LHON group, that disease susceptibility lies within the coding region (haplogroup) and is not associated with any distinct D loop haplotype.
MELAS. The haplogroup distribution of nine index patients with MELAS (eight patients with the 3243 mutation, one patient with the 3271 mutation) was investigated to answer the question regarding whether a distinct haplotype background or certain specific D loop variants might play a role in formation and fixation of disease mutations, as previously proposed for the D loop polymorphism 16189 (16,17). Here, we demonstrate that MELAS patients were similarly distributed among the distinct haplogroups, as were the controls ( Table 2): two (22%) belonged to D 4216 4917 , one to D 4216 13708 (11%), another to lineage 12705 (11%) and the remaining five patients were members of haplogroup D C (55%). D loop sequencing of all MELAS patients (data not shown) did not reveal any concentration in a distinct haplotype and showed that the MELAS mutations must have arisen multiple and independent times. Furthermore, we could not detect a higher frequency of the 16189 mutation, as reported by others (16), since only one MELAS patient (11%) harbored this polymorphism (controls 16%).
SIDS. Nine children who had obviously died from SIDS were investigated for their association with haplogroups and haplotypes ( Table 2). Three of the SIDS infants (33%) belonged to the 13708(-) side-branch of lineage 4216+11251, previously detected in one single DIDMOAD patient. This sub-cluster has not been found in our controls, since all control subjects carrying variants 4216+11251+12612+10398 also harbored the 13708 polymorphism. Two SIDS infants (22%) were members of the 12705 lineage, both also carrying a heteroplasmic C insertion at nt 574 characteristic for a sub-branch of D 12705 10398 in controls (3%). The children were obviously not related to each other as they were characterized by different D loop variants (data not shown). The heteroplasmic C insertion at nt 574 was also detected in a third SIDS infant, however on the background of lineage 12308+12372. Three SIDS children (33%) belonged to lineage D C . Although the clustering of SIDS in the 13708(-) side-branch as well as the increased frequency of the heteroplasmic polymorphism at nt 574 showed statistical significance (P < 0.005 and P < 0.01 respectively), the number of SIDS children belonging to the respective clusters was too small to provide clear-cut evidence for a disease-predisposing haplogroup.

DISCUSSION
The mitochondrial genome had been fully sequenced 16 years ago (3) and many subsequent publications worldwide have revealed legions of polymorphic sites, as well as populationspecific lineages, haplogroups and haplotypes. Nevertheless, a valid systematic classification remains elusive, although various authors have created their own nomenclature (12,(18)(19)(20). The rich variability within the D loop region compared with the relatively constant constellation within the gene regions provides useful criteria for phylogenetic and pathogenetic studies. Furthermore, the fact that distinct nucleotide positions have different tendencies to mutate has important implications for phylogenetic studies. We therefore suggest a classification of populations based on mtDNA gene variants. Our study demonstrates that the German population can be exactly characterized by a few haplogroups defined by common clusters of homoplasmic mtDNA mutations within the coding regions of the mitochondrial genome. Due to the lower mutability within the gene regions compared with the non-coding D loop, our haplogroup definition is affected with a much lower degree of uncertainty compared with the use of D loop sequences. For example, most of the identified coding region variants (∼70%) occurred at least twice but showed strict haplogroup specificity, while ∼30% of variants occurred only once in our control group. The only exception was the variant at nt 10398, which must have arisen multiple times during evolution as we found it in different haplogroups. In contrast, only ∼25% of D loop polymorphisms could be classified as haplogroup-specific variants and the majority represented highly variable sites or very rare variants. Furthermore, the use of gene mutations leads to a more precise definition, allowing characterization of common extant sub-branches. Nevertheless, as we investigated our control population also with respect to the two hypervariable D loop segments we are able to compare our data with those described in the literature. Comparing our results with those recently obtained by a combined RFLP/sequencing (19,21) and a HVS-I sequencing approach (20) lends support to the existence of the major German lineages and haplogroups (Table 3). In our control population a transition at nt 7028, corresponding to an AluI site at nt 7025 in Torroni et al. (19,21), separates the lineage D C , which is 7028(-) in our nomenclature, from all other lineages. Furthermore, among 7028(+) subjects the major lineages 4216+11251, 12308+12373 and 12705 are all characterized by the transitional variant 073 in our study, a polymorphism also mentioned by Richards et al. (20) as a major separator between the consensus haplogroup (Cambridge Reference Sequence) and four longer branches. The main lineage 4216+11251, which is strongly correlated with the HVS-I variant 16126 (Fig. 1), diverges into haplogroups D 4216 13708 and D 4216 4917 . These well-defined haplogroups were characterized by a number of homoplasmic genes mutations as well as D loop variants. They seem to be consistent with the haplogroups J and T in Torroni's grouping and 2A and 2B in Richard's nomenclature. Both haplogroups were reported to occur only in European populations. However, as mentioned by Richards et al. (20), the ancestral state of haplogroups 2A and 2B, namely the 16126 mutation alone, has only been found in the Middle East. Our second major lineage, 12308+12372, splits into three haplogroups designated D 12308 11467)3197 , D 12308 11467)16224 and D 12308 4646 , each characterized by several homoplasmic mitochondrial gene mutations. D 12308 11467)16224 is probably consistent with Richard's group 4 and Torroni's group K, mainly because of the HVS-I variants 16224, 16311 and a HinfI restriction site gain at nt 12308. The D loop variant at nt 16270, which is associated with nt 3197, indicates that D 12308 11467)3197 is consistent with groups 5 and U in Richard's (20) and Torroni's (21) studies respectively. Our third sub-cluster of lineage 12308+12372, haplogroup D 12308 4646 , although not equivalent to any specific haplogroup described in other studies, might also belong to Torroni's haplogroup U. Lineage 12705 branched into a 10398(-) and a 10398(+) haplogroup. The latter (D 12705 10398 ) was defined by several further mitochondrial gene and D loop variants; it is likely to be consistent with Richard's group 3A. A sub-branch of D 12705 10398 is defined by a heteroplasmic C insertion at nt 574, described by Torroni et al. (19) as a characteristic part of their haplogroup I. The lineage D C in our study, called haplogroup H in Torroni's nomenclature, is equivalent to group 1 of Richards et al. (20). It is by far the most frequent group and comprises between 40 and 70% of the Human Molecular Genetics, 1997, Vol. 6, No. 11 1843 European population. However, as this group is defined by a substraction of the other group-specific polymorphisms, it remains the most poorly defined group which, obviously, will be subdivided into further groups as our study continues. Examples of this are the rare haplogroups D C 3341 , D C 3338 , D C 5580 , D C 10463 , D C 14905 and D C 4336 , which were 7028(-) and were found infrequently in our control population. D loop sequences of all 31 members of lineage D C (Table 1) revealed 29 different haplotypes. Thus the existence of major and frequent haplogroups within this lineage seems unlikely.
The relative frequencies of the haplogroups described differ slightly within the central European area, as shown in Table 3. Haplogroups D 4216 13708 and D 4216 4917 were found with a frequency of 7 and 12% respectively in our German control group. The distribution of both haplogroups, however, differs considerably across the Middle East and Europe (20,21,23,24). For instance, Torroni's group T (our haplogroup D 4216 4917 and Richards' group 2B) was not detectable in Turkey or Iceland (20), but was found with higher frequencies in the Middle East (12%), in Sardinia, Spain, Portugal (all 13%) (20,22) and, to the remarkably high degree of 75%, in a Ladin speaking isolate in Val Gardena in the northern part of Italy (24). It should be noted here that two of our DIDMOAD index patients belonging to haplogroup D 4216 4917 were of Turkish origin (12). Richard's haplogroup 2A (our haplogroup D 4216 13708 ) also varies widely within European populations, ranging from 2% in the Basques to 22% in Cornwall (20). Sub-clusters of lineage 12308+12372, averaging 7% in European countries (19,20), could not be found among Asians or Native Americans (20). Lineage 12705 is also found to a large degree in the Middle East (24%) and in Turkey (18%), but only to a much lesser extent in several European populations (20). We presume that part of the reported differences in the central European mitochondrial haplogroup distribution is due to uncertainties produced by hypervariable hotspots in the D loop sequences.
Recently Zischler et al. (6) described an integration of a mitochondrial D loop fragment into human chromosome 11, where it presumably evolves as a chromosomal pseudogene ('fossil') and, therefore, more slowly than its mitochondrial counterparts. The common ancestor of the insertion and the contemporary mitochondrial gene pool probably originated shortly before the genesis of a common ancestor of the current mitochondrial gene pool. When compared with this nuclear 'fossil', the Cambridge reference sequence is found to contain 25 base exchanges. Of the mitochondrial lineages and haplogroups described here, only the 12308+12372 and 12705 lineages share several additional positions with the fossil sequence, namely an A at nt 16129, a C at nt 16249, a T at nt 16278, a C at nt 16311, a C at nt 16263 and a C at 16356. As pointed out by Torroni et al. (21), haplogroup U (our haplogroup D 12308 4646 ) is the only one that Europeans share with Africans. Thus the 12308+12372 and, probably, 12705 lineages seem to be the oldest ones, originating possibly in Africa and expanding into the Middle East and Europe (21).
There also exists some uncertainty about the tree position and the value of the A→G exchange at nt 10398. While Torroni et al. (19,21) suggested that the presence or absence of this DdeI site at nt 10394 separates all Caucasian mtDNAs into two major groups, our data suggest this polymorphism as being a hotspot occurring in different haplogroups. Otherwise, the haplogroups D 4216 13708 and D 4216 4917 would not share variants 4216+11251+16126 as an ancestral lineage and, furthermore, the major lineages 11251+4216+16126, 12308+12372 and 12705 must have arisen at least twice during evolution. A further possibility is that 10398 is indeed very ancient, but underwent back-mutation in certain haplogroups.
Most of the D loop variants characteristic of certain haplotypes have also been described in the skeleton network recently published by Richards et al. (20), summarizing the Caucasian HVS-I sequence data from DiRienzo and Wilson (22), Piercy et al. (23) and Pult et al. (25). Thus even at the level of the HVS-I fragment the Caucasian population can be identified by a limited number of variants. On the other hand, several positions within the D loop sequences represent mutational hotspots. Furthermore, most of the D loop positions investigated in the present and other studies (>80%) remain unaffected. Thus D loop positions seem not to be randomly affected by mutational events. Rather, a selection of mutational sites seems to be taking place. If so, this would have important implications for phylogenetic studies, because widely used methods such as maximum parsimony analysis (22,(26)(27)(28) and pairwise difference analysis (22,29) usually assign an equal weight to all nucleotide exchanges, as recently stated by Torroni et al. (21). Hence, for further phylogenetic studies the single D loop positions would have to be weighted individually.
The disease groups were chosen for several reasons. The LHON and MELAS groups consisted of patients routinely diagnosed in our laboratory. They represented typical maternally transmitted disorders with specific homoplasmic (most LHON patients) or heteroplasmic (all MELAS patients) mtDNA point mutations. Diagnostic studies 3 years ago on the LHON group had revealed that the bulk of the LHON patients were associated with a cluster of so-called secondary LHON mutations (9). By extending these studies to a group of healthy German subjects we found the frequency of haplogroup D 4216 13708 to be significantly higher in our LHON cases compared with controls (12,15), irrespective of the nature of the primary/intermediate LHON mutations. D loop sequencing of 10 LHON patients belonging to this haplogroup revealed that the 11778, 14484 and 15257 LHON mutations have arisen several times independently within haplogroup D 4216 13708 (Fig. 2). The accumulation of LHON on this haplogroup background is unlikely to be the result of founder mutations, thus haplogroup D 4216 13708 seems prone to formation or expression of LHON mutations. The observation that another disorder exhibiting optic atrophy in combination with diabetes mellitus is concentrated in haplogroup D 4216 4917 (12) makes disease susceptibility at the level of lineage 4216+11251+16126 likely. DIDMOAD has been proposed to be a mitochondrial-mediated disease (30). However, it lacks maternal transmission and is thus unlikely to be commonly caused by severe mtDNA mutations (12,31,32). It has recently been mapped to chromosome 4p (33).
Children who have died from SIDS have been included in a mtDNA screening program and haplotype analysis for the following reasons. SIDS is the most common cause of death in infants from the first week to the first year of age, however, despite extensive global research the reasons and the pathomechanisms triggering the fatal event remain generally unknown. In a relatively high proportion SIDS is preceded by one or more so-called 'near miss for SIDS' or 'apparent life threatening events (ALTE)' which are characterized by respiratory arrest, cyanosis, increased temperature with massive sweating, etc. Obviously ALTE symptoms can be related to alterations in coupling of the respiratory chain and the oxidative phosphorylation apparatus. Furthermore, certain mtDNA mutations might affect coupling (2) and this could have pronounced effects, especially in the mitochondria-rich brown adipose tissue, which regulates thermogenesis in the newborn during the first year of life. Although a detailed mtDNA screening for disease mutation by our group (unpublished results) did not reveal any clear-cut evidence for SIDS-causing mtDNA mutations, our haplogroup analysis showed a different distribution compared with our control population. First, the SIDS infants accumulated in a side-branch of 4216+11251, characterized by additional variants at nt 10398 and 12612, a haplogroup not found in 67 German controls. Second, 3/9 SIDS children carried a heteroplasmic C insertion at nt 574 while only two control subjects (3%) carried this polymorphism, in both cases on a haplogroup D 12705 0195 background. These unstable C stretches in the non-coding region have been previously discussed as predisposing to somatic mutations (19), as also suggested for unstable stretches of C residues around nt 16189 (34). Whether this variant plays a role in susceptibility for SIDS needs further investigation.
The association of different disorders with certain mitochondrial haplogroups seems to us the most important result of our study. If this can be confirmed by further studies it will have unforeseen consequences and implications for genetic research in general. If the idea of disease-susceptibile haplogroups holds true, populations with, for example, higher frequencies of haplogroups D 4216 13708 and D 4216 4917 should show an increase in the penetrance of distinct disorders. On the other hand, populations with higher frequency of haplogroup D C should be relatively protected from such diseases.
Is there any rational explanation for the association of certain mtDNA haplogroups and diseases? mtDNA encodes several peptide subunits necessary for optimal functioning of the respiratory chain and the oxidative phosphorylation apparatus in the mitochondrion, responsible for the essential production of ATP in all organs and cells, especially in those with high energy demand such as brain, heart and skeletal muscle. It is therefore not surprising that single mtDNA gene mutations cause functional deficiencies in the corresponding gene products, thus leading to insufficient function of the energy-providing machinery and, ultimately, to monogenic mitochondrial diseases of the brain, heart, skeletal muscle and neuroendocrine system (2). To our knowledge there are only a few publications dealing with the role of polymorphic mitochondrial genetic backgrounds in cellular function and individual cellular fitness. In 1991 Dionne et al. (35) correlated the mtDNA RFLP pattern of atheletes with maximal oxygen uptake and response to endurance training and found that distinct sequence variation clusters were associated with different levels of physical fitness. Recently Vergani et al. (36) showed that the LHON 11778 mutation leads to a deficiency in ATP production, especially when associated with the polymorphic site at nt 4216. This study supplied, for the first time, a functional basis for the assumption that lineage 4216+11251 might increase the risk of expression and penetrance of LHON and, possibly, other disorders. Thus the disease susceptibility of certain haplogroups might be caused by relative differences in energy production due to clusters of synergistically working point mutations, as already proposed by others (10,11,13). Furthermore, certain haplogroups (or specific mtDNA variants) might be more prone to the formation of disease-causing mutations; this possibility has been discussed for the heteroplasmic C stretches at nt 574 (19) which were detected with high frequency in our SIDS group. A third possibility is that different mtDNA mutation clusters might generate different amounts of oxygen radicals, with their respective consequences on DNA, protein and lipid structures. However, up to now evidence for any such explanation is weak; obviously, further studies are needed.

Glossary
As several mitochondrial lineages and sub-clusters are population specific, we used more informative and distinctive terms for the haplogroups characterized in our study. Thus the 'D' in, for example, our term D 4216 13708 defines a German population; furthermore, the upper mtDNA position indicates the branching site defining the mtDNA lineage, while the lower mtDNA position characterizes the sub-cluster or mitochondrial haplogroup. Mitochondrial haplotypes are defined by a cluster of recurrent D loop variants belonging to one defined haplogroup.

Subjects
Our control group consisted of 67 healthy German blood donors who were collected randomly with respect to age and sex. All eight DIDMOAD patients have been described previously (12). The LHON group consisted of 55 patients clinically defined by bilateral optic neuropathy. All nine MELAS patients presented with typical clinical features of this syndrome. LHON and MELAS patients were analyzed for the respective disease-causing mtDNA mutations during routine diagnosis in our laboratory. Cardiac muscle probes from nine SIDS children were provided by the Department of Forensic Medicine, Munich.

DNA extraction
Total DNA was prepared from tissue (SIDS disease group) or blood cells by standard proteinase K digestion at 65_C for 2-4 h and NaCl extraction. DNA was precipitated using isopropanol at -20_C, washed in 70% ethanol, vacuum dried and resuspended in 10 mM Tris-HCl, 1 mM EDTA. DNA concentration was assayed spectrophotometrically at 260 nm.