Mitochondrial genome variation in male LHON patients with the m.11778G > A mutation

Maternally transmitted pathogenic variants of mitochondrial DNA (mtDNA) may lead to dysfunction of the oxidative phosphorylation system and the development of mitochondrial diseases, a heterogeneous group of usually multi-organ disorders that can occur at any age. Symptoms usually affect post-mitotic tissues with high energy demands, such as muscles and nerves (Greaves et al. 2012; Schon et al. 2012). The m.11778G > A point mutation was one of the first mtDNA pathogenic variants to be associated with human disease (Wallace et al. 1988). It was described in patients with hereditary predisposition to blindness, a disease known as Leber hereditary optic neuropathy (LHON) (Wallace et al. 1988).

LHON is one of the most peculiar mtDNA diseases. Although mitochondrial disorders are generally rare, LHON is the most common in this group (Chinnery et al. 2000; Kim et al. 2018). Sudden, painless and progressive loss of vision is the only symptom in the majority of cases. This clinical presentation exclusively limited to a single tissue results from retinal ganglion cell damage and loss, and characteristic degeneration of the optic nerve, however, the cause(s) for this phenomenon are still under study (Koilkonda and Guy 2011; Yu-Wai-Man et al. 2011). Over 90% of LHON cases are caused by one of three mtDNA point mutations, m.3460G > A, m.11778G > A or m.14484 T > C, in genes encoding subunits 1, 4 and 6, respectively, of respiratory chain complex I (Mackey et al. 1996; Tońska et al. 2010; Piotrowska et al. 2015; Caporali et al. 2017) and the m.11778G > A variant is the most frequent LHON mutation (accounting for 70–90% of all cases), associated with disease worldwide (Lott et al. 2013; Meyerson et al. 2015; Kim et al. 2018). Although necessary, their presence is not sufficient for disease development, implying that additional modifying factors, genetic, epigenetic and/or environmental, must exist (Yu-Wai-Man et al. 2011; Giordano et al. 2014; Piotrowska et al. 2015; Meyerson et al. 2015; Bianco et al. 2016; Caporali et al. 2017). Besides incomplete penetration, LHON also shows gender bias as symptoms affect ~50% of men and only ~10% of women who carry one of the three most common LHON mutations (Yu-Wai-Man et al. 2009; Tońska et al. 2010; Koilkonda and Guy 2011; Piotrowska et al. 2015; Meyerson et al. 2015).

One of the most frequently studied and well known modifying genetic factors of LHON are mtDNA haplogroups that are sets of specific common mitochondrial genetic variants segregating together and reflecting mtDNA evolutionary history. Increased risk of vision loss was observed when the m.14484 T > C mutation was present on the background of haplogroup J1, m.11778G > A on J1 and J2 and m.3460G > A on K, whereas haplogroup H had a protective effect in the case of m.11778G > A (Brown et al. 1997; Man 2004; Carelli et al. 2006; Hudson et al. 2007; Piotrowska et al. 2015; Meyerson et al. 2015; Caporali et al. 2017). These observations compellingly suggested that one or more common mtDNA variants defining these haplogroups act synergistically with primary LHON mutations to modify the risk of developing the disease.

The above-mentioned associations were found in European populations, however, studies on mtDNA variation show that there are significant differences between distinct populations in the frequency of individual mtDNA variants and haplogroups, highlighting the necessity for carefully designed, ethnically matched case-control studies. Thus extrapolation of results from one population to another might cause serious errors. With the advent of next-generation sequencing (NGS) methods, high-throughput analysis of whole genomes, including mitochondrial ones, was enabled, allowing for powerful and sophisticated screening for genetic modifiers of hereditary traits. In this study, we use NGS to investigate the mtDNA variation in male Polish LHON patients with the m.11778G > A mutation to confirm previously reported or to find novel mitochondrial genetic modifying factors. We found that haplogroup K and mtDNA variants defining it, m.3480A > G, m.9055G > A, m.11299 T > C and m.14167C > T, might increase the risk of LHON in male patients with the m.11778G > A mutation in the Polish population.

In this work only male cases were studied as men are primarily affected by this disorder. Since LHON shows incomplete penetration and gender bias, women could not be easily enrolled as they very rarely have the disease. The whole mtDNA analysis was performed in a total of 89 Polish male individuals. The patient group consisted of 47 unrelated men (mean age 28.3 ± 9.70) with diagnosed Leber Hereditary Optic Neuropathy and confirmed presence of the m.11778G > A mtDNA mutation. LHON samples included in this study were collected in the Medical University of Warsaw and the Centers for Medical Genetics GENESIS. The control group consisted of 42 unrelated men (mean age 71.0 ± 9.25) treated at the Department of Diagnostics and Microsurgery of Glaucoma, Medical University of Lublin, because of simple senile cataract. LHON (with the presence of any of three most common causal mutations in mtDNA), glaucoma and other intraocular pathologies were excluded. In all cases total DNA collected from peripheral blood was used. Mitochondrial sequence data of control individuals included in this study were extracted from datasets obtained previously in different study (Piotrowska-Nowak et al. 2018) and re-used for the analysis here.

LHON disease was first described almost 150 years ago (Leber 1871). Although our understanding of its pathogenesis has increased remarkably since then, there are still some aspects to be resolved, such as sex bias and incomplete penetration. In search of genetic modifying factors, we investigated whole mtDNA variation in male Polish LHON patients with m.11778G > A primary mutation.

Screening for the presence of other proven pathogenic variants in mtDNA, both point mutations and large deletions, brought negative results, implying that such deleterious changes do not contribute to disease development. Nevertheless, we identified three non-synonymous variants reported previously to associate with LHON (Fauser et al. 2002; Abu-Amero and Bosley 2006; Dai et al. 2018). However, their role in disease development in a synergistic mechanism, as an additional, secondary mutation, in three patients carrying them is debatable since their status was not yet confirmed with functional studies and thus their pathogenicity is unclear. Such variants accompanying primary LHON mutations are reported to be found also in unaffected control subjects, similarly as in one case in this study, and thus are suggested to be rather simple mtDNA variations with neutral effect, but it cannot be completely ruled out that their reported co-occurrence may confer phenotypic variability (Brown et al. 1997; Koilkonda and Guy 2011; Dai et al. 2018).

Haplogroup and SNP association analysis are commonly used methods applied in the search for mitochondrial genetic factors predisposing to or protecting from disease development. The modifying effect of mitochondrial genetic background on the penetrance of LHON mutations is well documented (Brown et al. 1997, 2000; Carelli et al. 2006; Hudson et al. 2007; Yu-Wai-Man et al. 2011; Piotrowska et al. 2015; Caporali et al. 2017). In this study mtDNA haplogroup K was overrepresented in LHON men compared to control individuals. Although the observed frequency difference was on the border of statistical significance, probably due to generally low frequency of haplogroup K in Polish population (about 4%, as reported in the latest large study by Jarczak et al. (2019)) and relatively small sample size in our study groups, subsequent SNP analysis revealed that four variants, markers of haplogroup U8b or K, which derives from U8b (Oven and Kayser 2009), associate with increased risk of LHON and have moderate to high effect size (OR > 2.5). These observations lead us to propose that haplogroup K and its characterizing variants, particularly specific combination of m.3480A > G, m.9055G > A, m.11299 T > C and m.14167C > T variants, may have a negative effect on m.11778G > A mutation and modulate LHON phenotype, its expression and/or penetrance, in male Polish patients. This is a novel finding since so far haplogroup J was commonly reported to associate with m.11778G > A and LHON in European population (Brown et al. 1997; Man 2004; Carelli et al. 2006; Hudson et al. 2007; Yu-Wai-Man et al. 2011; Meyerson et al. 2015). Moreover, so far haplogroup K was associated with increased risk of LHON for the m.3460G > A primary mutation in European patients (Hudson et al. 2007; Yu-Wai-Man et al. 2011; Meyerson et al. 2015), however, in previous studies this mutation was distributed randomly among mtDNA haplogroups (Torroni et al. 1997; Brown et al. 1997; Carelli et al. 2006). Discrepancies between those and our study might result from marked geographic variation of mtDNA haplogroups. However, it is conceivable that haplogroup K may have similar effects when associated with previously reported m.3460G > A and m.11778G > A described here in different populations. Further studies are needed to unravel this question.

Synergistic and deleterious effect of haplogroup K-related variants on the pathogenic potential of mtDNA mutation may come from subtle conformational changes shifting the assembly kinetics and stability of respiratory chain complexes, as was shown in LHON mutant cybrid cells belonging to different mtDNA haplogroups (Dudkina et al. 2005; Hudson et al. 2007; Pello et al. 2008; Yu-Wai-Man et al. 2011). Particularly interesting is the m.9055G > A variant which leads to an amino acid change in subunit 6 of ATP synthase and possibly may affect the efficiency of energy production in mitochondria. Although the remaining identified SNPs are synonymous variants, it is worth noting that all localize exclusively in genes encoding subunits of the respiratory chain complex I that is fundamental for proper functioning of the respiratory chain and that is affected by primary LHON mutations, including m.11778G > A. It has been recognized that silent sequence changes can impact the secondary structure or stability of mRNA and thus affect protein expression (Sauna and Kimchi-Sarfaty 2011, 2013) what can be true also in this case. Altogether, a partial complex I defect caused by the m.11778G > A mutation may be enhanced by further, normally subclinical, changes in the functioning of complex I and ATP synthase subunits associated with haplogroup K background. Such complex mtDNA variant interaction may thus impact LHON expression in Polish patients harboring the m.11778G > A mutation, increasing the probability for disease occurrence (Brown et al. 2002).

We also identified one common non-coding variant, m.73A > G, to be associated with increased risk of LHON in men with m.11778G > A in this study. It is an ancestral polymorphism present in multiple mtDNA lineages. It localizes in the main non-coding region of mtDNA referred to as the control region as it covers the main regulatory sequences associated with the replication and transcription of the mtDNA molecule (Anderson et al. 1981; Falkenberg et al. 2007). Therefore a non-coding control region variant, such as m.73A > G, will not directly affect the oxidative phosphorylation system altering energy or ROS production but, by changing a regulatory motif or being adjacent to one, it may impact the replication and transcription of the mitochondrial genome e.g. slightly influencing mtDNA copy number or gene expression (Suissa et al. 2009; Lott et al. 2013; Umbria et al. 2018). This together with other risk factors and m.11778G > A mutation may possibly modulate the LHON phenotype.

DNA sequence variants of a harmful nature are removed from the population through purifying selection. It is therefore assumed that pathogenic variants of mtDNA, even those mildly deleterious, will be rare and more frequently observed on the younger branches of the mtDNA phylogenetic tree (Elson et al. 2004; Pereira et al. 2011; Soares et al. 2013; Wei et al. 2017; Venter et al. 2017; Piotrowska-Nowak et al. 2019). According to the hypothesis “common disease - rare variant” (CDRV), a single rare variant may have mild deleterious effect, but such multiple changes may contribute to the development of the disease in a synergistic way by cumulative effect (Elson et al. 2006; Schork et al. 2009; Pienaar et al. 2017; Venter et al. 2017; Piotrowska-Nowak et al. 2019). Regarding mitochondria this seems particularly attractive because all genes in the mitochondrial genome encode products that are involved in the same basic biochemical process, oxidative phosphorylation. In order to verify this hypothesis, we compared the frequency of rare mtDNA variants between patients and controls in individual, arbitrarily determined, mtDNA regions. Analysis of sequence variation showed increased frequency of rare variants in the region covering genes encoding subunits of respiratory chain complex IV in healthy subjects when compared to LHON patients. This suggests that rare variants may also play a protective role in the development of disease, what was already reported previously (Singh et al. 2018).

Not all variants of low population frequency will actually be harmful, thus we also predicted the pathogenicity of variants in silico using the MutPred algorithm and compared variant cumulative impact between two groups. The usefulness and strength of the MutPred tool in estimating the harmfulness of non-synonymous mtDNA variants has been previously demonstrated (Pereira et al. 2011, 2012; Pienaar et al. 2017; Venter et al. 2017; Piotrowska-Nowak et al. 2019). In this study we did not observe any significant differences in MutPred variant load between patients and controls. However, this can be attributed to the small number of individuals in study groups and thus should be verified in larger cohorts.

The burden of possibly harmful variants was also investigated by analysis of transversions and non-synonymous variants, known to bear deleterious potential. We did not find any significant differences between the studied groups in any of the mtDNA regions tested what implies lack of association between transversion and non-synonymous variation load and risk of LHON associated with m.11778G > A in men in this study.

In summary, in this study we investigated the whole mtDNA variation in male patients with LHON and its association with the m.11778G > A mutation in the Polish population. Most interestingly, we present a possible association between mtDNA haplogroup K and variants in its background and m.11778G > A mutation. Although the presented data are preliminary with a limited sample size not allowing to make firm conclusions, our results indicate possible contribution of novel combination of mtDNA genetic factors to the LHON phenotype, e.g. by increasing the penetrance of the mutation in this mtDNA background. Surprisingly, we did not observe associations previously reported for m.11778G > A and LHON in European populations, particularly for haplogroup J as a risk factor, what implies that mtDNA variation is much more complex. Further investigation in larger cohorts are required to verify these important findings.