Sequence variation in human succinate dehydrogenase genes: evidence for long-term balancing selection on SDHA

Succinate dehydrogenase (SDH; mitochondrial complex II) is an essential enzyme complex that has dual roles in the Krebs cycle and the electron transport chain (ETC) in mitochondria [1]. SDH is composed of four subunits encoded by the nuclear genes SDHA, SDHB, SDHC, and SDHD. SDHA at chromosome band 5p15 and SDHB at chromosome band 1p35 encode the two catalytical hydrophilic subunits flavoprotein (Fp; 70 kDa) and iron-sulfur (Ip; 35 kDa), respectively. SDHC at chromosome band 1q23 and SDHD at chromosome 11q23 encode the two membrane-spanning hydrophobic subunits, cybL (15 kDa) and cybS (12 kDa), respectively. The SDHA, SDHB, SDHC, and SDHD gene products are encoded by 15, 8, 6, and 4 exons, which span genomic distances of ~38 kb, 35 kb, 50 kb. and 10 kb, respectively [2, 3].

The identification of the SDHD subunit gene as the hereditary paraganglioma type 1 locus (PGL1) has uncovered unexpected links between SDH and tumor susceptibility, and highlighted the role of mitochondria in cancer [4]. Since then, mutations in SDHB, SDHC, and SDHD subunit genes (PGL genes) have been established as an important cause of sporadic and familial paragangliomas [5–10]. The paraganglia specificity of PGL tumors [4] and data from global gene-expression analysis [11], cell biology [12], animal-model studies [13], and gene-environment interaction and population genetics [14] support the hypothesis that constitutive hypoxic stimulation underlies the pathogenesis of PGL.

The role of SDH in disease pathogenesis has been implicated independently through a series of studies on a widely distributed plant and fungal neurotoxin, 3-nitropropionic acid (3-NPA)[15]. Acute food poisoning with 3-NPA, which can lead to central nervous system defects with lifelong disability and to mortality in ~10% of the cases, have been associated with consumption of moldy sugarcanes in China [16]. The neurodegeneration induced by 3-NPA poisoning often involves the basal ganglia, hippocampus, spinal tracts, and peripheral nerves, and the symptoms mimic those of Huntington's disease [17]. 3-NPA irreversibly inhibits SDH, owing to the similarity of the chemical structures of 3-NPA to succinate [18]. It has been suggested that 3-NPA may form a covalent adduct with an arginine residue at amino acid position 345 in the active site of the Fp subunit [19].

Surprisingly, mutations in the major catalytical subunit SDHA have yet to be associated with PGL. Although homozygous mutations in SDHA have been found in Leigh syndrome [20], a severe neurodegenerative disorder of childhood, and with neuromusculopathies, no genetic link between SDHA and paraganglioma susceptibility has ever been established. Current biochemical knowledge on SDH provides very few clues for the phenotypic dichotomy arising from the germline subunit gene mutations. SDHA and SDHB subunits encode the two physically-interacting catalytical subunits, so it is surprising that their mutations would have such different phenotypic consequences [21]. Recently, after identifying cDNA sequences encoding a missense Fp variant containing the Y629F and V657I polymorphisms, Tomitsuka et al [22, 23] proposed that distinct genetic loci encode two Fp variants, namely type I and type II. They reached this conclusion after observing tissue-specific and cell line-specific differential expression of the cDNA variants and PCR amplification from genomic DNA of processed SDHA gene fragments that lacked introns (i.e, a functional SDHA retrogene). However, the genomic location of the retrogene that was proposed to encode the second SDHA gene could not be determined. A retrogene for SDHA is not present in the human genome, according to the March 2006 assembly in The UCSC database. [24] Finally, Briere et al [25] showed the presence of the missense SDHA variants in several different cell types and assumed that these variants originate from two different genes, although they provided no experimental or bioinformatic evidence for the genomic presence of a second SDHA locus. Briere et al [25] suggested that the presence of two SDHA genes in paraganglia prevents tumorigenesis. If Fp were encoded by two different loci, this would indeed have provided a simple explanation for why SDHA mutations would not be associated with PGL susceptibility.

An alternative approach to gain insights into gene function involves analysis of sequence variation in the population. To date, no study has systematically addressed the variation patterns in the SDH subunit genes in normal subjects from different racial or ethnic groups. To gain further insights into the multiple roles of SDH in disease predisposition and to help to integrate the seemingly disparate phenotypic consequences of SDH subunit defects, we examined sequence variation in the complete coding and partial flanking intronic sequences of the four SDH subunit genes in 24 samples from an African American population and 24 samples from a white population. These analyses uncovered an unexpected degree of nucleotide diversity in the SDHA gene.

Our results establish a foundation to understand the selective and demographic forces that have shaped the variation patterns in SDH subunit genes, and have important functional implications. Our findings indicate that the variation pattern in SDHA is characterized by the presence of higher sequence diversity, two common and highly dissimilar haplogroups, and statistical and empirical support for the operation of a balancing selection mechanism. Our data also refute the previous suggestions that the Y629F and V657I variants originate from two distinct genetic loci because these missense variants are encoded by a single, highly polymorphic SDHA gene.

The PGL genes had much lower nucleotide diversity, which was especially evident in SDHB, suggesting that the SDHB gene product might be under functional constraints that preclude the accumulation of variants. If slightly deleterious variants in PGL genes increase the risk of paraganglioma tumor development, such variants would be eliminated before they reach high frequencies in the population. This potential mechanism might apply especially to SDHB because its mutations are associated with malignancy and early-onset pheochromocytomas that could lead to severe hypertensive crises [37, 38]. In contrast, because there is no evidence that heterozygous mutations in SDHA are associated with a pathologic phenotype, negative selection of deleterious SDHA alleles may operate only when they are in the homozygous state, which often leads to a lethal metabolic syndrome in childhood.

A major finding of our study is the unexpectedly high nucleotide diversity in the SDHA gene in the African American samples. It has been suggested that high local recombination rates may increase SNP density [39]. However, this mechanism is unlikely to contribute to SDHA variant density, because a recent high-resolution recombination map indicates a very low recombination rate at the tip of chromosome 5 short arm, where SDHA is located [40]. It is conceivable that the four SDHA pseudogenes, generated by complete or partial gene duplications, may increase the de novo mutation rate in the SDHA gene through illegitimate recombination or gene conversion during meiosis to increase variant density. However, lack of high nucleotide diversity in the chimpanzee SDHA gene does not suggest that the mutation rate in SDHA is inherently high, even though the chimpanzee genome also contains the duplicated SDHA pseudogenes. Rather, our findings suggest that the high nucleotide diversity of the human SDHA gene is a consequence of persistence of two distinct haplogroups for long periods during human evolution, leading to acquisition of a distinct set of polymorphisms by each haplogroup.

The most important finding of our study is the statistical and empirical support for a balancing selection mechanism on SDHA. A classic example of balancing selection is found at the major histocompatibility complex (MHC) loci [41], where high levels of polymorphisms in the functional MHC genes may confer a selective advantage to the heterozygotes by enabling them to process a wider range of pathogen antigens on T cells. The variation in a few other human genes may also have been shaped by balancing selection. For example, the 5' cis-regulatory region of CCR5, encoding the principal coreceptor for HIV-1 [42], protocadherin alpha gene cluster promoters [43] and the bitter-taste receptor gene, PTC [44], have two major ancient haplotype groups and positive D test statistics, similar to SDHA. However, in contrast to SDHA, these genes did not show significant Tajima D statistics in the African or African American samples. In general, the average Tajima D value is positive in the European American population and negative in the African American population. Positive Tajima D statistics in European Americans are often interpreted to reflect population contraction that occurred during the migration of modern humans out of Africa, whereas negative Tajima D statistics in African Americans may reflect admixture between African and European populations [35]. Thus, evidence of balancing selection on a gene, suggested by statistically significantly positive Tajima D values, is more likely to be confounded by population history in European American samples than in African American samples.

It is conceivable that an environmental factor prevalent in Africa may have contributed to the increased frequency of certain SDHA variants that might have differential roles in the regulation of oxygen homeostasis by the SDH complex. A candidate environmental factor is the neurotoxin 3-NPA and its aliphatic nitrocompounds derivatives. In addition to being a product of certain fungi such as Arthrinium species, 3-NPA and its derivatives are also found in several higher plants. The toxicity of these plants is well established, because their aliphatic nitrocompound contents have been linked to acute and chronic diseases in some domestic animals. Major livestock losses were attributed to plant nitrocompounds in the western United States, Canada and Mexico [45]. Thus, although human toxicity involving moldy sugarcane poisoning have to date been reported only in China, human exposure to 3-NPA and other nitrocompounds might be more common throughout the world than is indicated by the numbe of clinical cases [18, 46]. 3-NPA exposure might be more prevalent in Africa partly because a hot and humid climate promotes the growth of fungi. If certain SDHA variants confer a selective advantage against 3-NPA poisoning by affecting gene expression levels, protein translation efficiency, and/or the binding affinity for 3-NPA, then such variants may provide a survival advantage for their carriers against 3-NPA poisoning. Alternatively, SDH may play a currently unrecognized role against infectious pathogens such as malaria, which are prevalent in Africa. Genetic studies of PGL suggest that inactivation of SDH by subunit mutations inappropriately activates hypoxia-inducible pathways. If the SDHA variants that have increased in frequency during human evolution are hypomorphs or encode Fps that have slight functional deficits, these variants might promote the activation of hypoxia-inducible pathways and help the immune cells to survive better under sustained hypoxic microenvironments of the infected tissues.

Finally, our findings do not support the previous explanations as to why SDHA mutations are not associated with PGL susceptibility because these explanations assume the presence of two SDHA genes in the human genome. Instead, the contrasting patterns of sequence variation between SDHA and the PGL genes suggest the presence of two functionally distinct modules in SDH: one formed by the three closely-associated PGL gene products (PGL module), and the other a loosely-interacting, highly-variable SDHA protein product. This model provides an alternative explanation as to why SDHA mutations do not cause PGL and predicts the following two conditions:

(i) The relative concentration of SDHA protein product is much higher (>two-fold) than the PGL module in the paraganglionic tissues. Thus, even a 50% reduction in SDHA protein levels, as a result of heterozygous mutations, would not compromise the SDH function in paraganglia to initiate tumor formation.

(ii) The physical interaction between the SDHA protein product and the PGL module is loose and kinetically fast during catalysis, thus a mutant SDHA protein product could not irreversibly trap a PGL module to initiate tumor formation.

Our findings demonstrate that the SDHA gene carries a strong signature of balancing selection in the African American population and that PGL and SDHA gene products are subject to distinct selective constraints. Collectively, these data provide new insights into SDH biology and may catalyze further research on the causes and the consequences of the unexpectedly high sequence diversity in the SDHA subunit gene.