Maintenance of mtDNA is controlled by a homeostatic network, whose effectors are the various components of the mitochondrial replisome, the still largely unknown protein set that forms the mitochondrial nucleoid, and the many enzymes and carrier proteins that provide the mitochondrion with a balanced supply of deoxyribonucleotides, the mtDNA “building blocks”. In principle, abnormalities in each of these players can cause MDS. Eight MDS genes are presently known, but their mutations account for only 60% of the cases. This gap prompted us to adopt exome NGS, an unbiased, systematic approach, to search for the responsible gene in our proband. This new technology carries out the time- and cost-effective sequence analysis of the coding regions of each and every gene. Increasingly powerful in silico filtering strategies are then available to prioritize candidate variants and eventually identify the causative gene. However, since we analyzed only one proband, the number of mutations obtained by exome NGS was still huge, even after the removal of common and synonymous variants, making the identification of the causative mutation a virtually unmanageable challenge. However, since the known consanguinity of the parents and the very low prevalence of the disease made the chance of two mutant alleles derived from distinct ancestors very low, we restricted the number of candidate genes to those contained within the regions of homozygosity obtained by the exome-NGS data. Using this method we identified a new mutation in SUCLA2
gene. This gene encodes for the b subunit of succinyl-CoA ligase, a Krebs cycle enzyme with a yet unexplained role in mtDNA metabolism 
Mutations in SUCLA2
have been reported in only 30 patients (), most of whom originating from a genetic isolate in the Faroe Islands that carry a homozygous G > A transition in intron 4, resulting in the skipping of exon 4 and predicting the synthesis of a truncated protein [20,21]
. In 2005 Elpeleg et al. described for the first time a homozygous deletion/insertion (g.32720del43ins5) in SUCLA2
associated with progressive encephalomyopathy and MDS. The affected members of this small Muslim family presented Leigh-like features with dystonic tetraparesis, alteration in the basal ganglia and deafness 
. Subsequently, Ostergaard et al. described 12 patients born or originating from the Faroe Islands, presenting with encephalomyopathy, hearing loss and mild MMA. Interestingly, only one of these patients had abnormal MRI signals in the basal ganglia 
, but in another study, all 11 affected individuals from the Faroe Islands carrying the same homozygous mutation were hallmarked by Leigh-like lesions of the basal ganglia 
. Three Italian families, each carrying different missense mutations of SUCLA2
, presented with early onset encephalopathy with deafness and alterations in the basal ganglia. Other brain MRI features include cerebral and cerebellar atrophy 
. Both our patients showed alterations in the basal ganglia and PII had also atrophy of cerebellum and a thin medulla oblongata. Similar to other SUCLA2
mutant patients, ours showed severe dystonic tetraparesis with developmental delay and deafness. Feeding problems seem to be an invariant clinical feature of this disease, being present in almost all reported patients (). Our patients showed serious difficulties in feeding and became tube-fed very early in life. Severe and early onset ptosis and ophthalmoparesis were prominent features in our patents, and in the Faroe Islands patients as well 
, but were not reported in the other Italian patients. Methyl-malonic aciduria, which was prominent in previous SUCLA2
mutant patients, was absent or very mild in ours, which in fact prevented earlier diagnosis. The urinary MMA levels measured after the identification of the gene defect showed a modest elevation (value 11 μmol/mmol creatinine, nv: < 5) only in PI.
Laboratory and clinical features of SUCLA2 patients.
Differently from the families reported by Ostergaard et al., the muscle morphology was normal in our patients, showing neither increased variability of fiber diameter, nor scattered hypertrophic fibers, nor lipid accumulation 
, whereas diffuse, partial COX deficiency was histochemically present, together with slight predominance of type I fibers. At the molecular level we identified an unreported mutation in exon 3, a c.308C > A nucleotide change producing an Ala to Asp substitution in position 103. The absence of this variant in SNP databases, the predicted deleterious effect of the substitution and the conservation of the mutant aminoacid residue strongly support the pathogenic role of the Asp103Ala change. Comparing the different mutations described, there is no clinical difference between Italian, Faroese and Muslim patients, suggesting that the corresponding mutations are functionally equivalent ().
MDS ranges from isolated myopathy to fatal liver failure or multisystem involvement 
. However, since most of the causing genes are ubiquitously expressed, the tissue specificity of mtDNA depletion remains unexplained. Encephalomyopathic MDS is typically associated with mutations in SUCLG1
(also reported as SUCLA1
) or in SUCLA2
, coding for subunits of succinyl-CoA ligase, a heterodimeric enzyme, composed of an invariant alpha subunit (encoded by SUCLG1
) and a beta subunit (encoded by SUCLG2
or by SUCLA2
) that determines whether the enzyme is GTP- (G-SUCL) or ATP-dependent (A-SUCL) 
. Thus, the two enzymes, G-SUCL and A-SUCL, catalyze a similar reaction by using different phosphate donors.
The tissue specificity of SUCLA2
mutations can be partly due to the fact that the A-SUCL predominates in the brain and skeletal muscle 
, whereas G-SUCL is predominantly expressed in the liver. This different tissue distribution of the two isoforms is likely to prevent the normal G-SUCL from fully compensating a crippled A-SUCL in critical tissues, namely the brain and skeletal muscle 
The link between impairment of SUCL, a Krebs' cycle enzyme, and mtDNA depletion is unclear; a defect in dNTP supply has been suggested, since both SUCL isoforms are physically associated with the nucleoside diphosphate kinase (NDPK), an enzyme involved in the dNTP salvage pathway during mtDNA synthesis [2,10]
. A recent work in cellular models has confirmed the fundamental role of SUCL (G-SUCL > A-SUCL) in the maintenance of mtDNA, by modulating NDPK activity 
Inherited diseases due to SUCLA2 mutations, as well as many other mitochondrial disorders, are rare and clinically heterogeneous conditions. Our report underlies the difficulty to set up a molecular diagnostic procedure in these disorders. In fact, even if some biochemical and clinical features are suggestive for peculiar genetic forms, such as MMA in SUCLA2- associated MDS (MDS5), these biomarkers can occasionally be normal or overlooked. NGS is a powerful and unbiased procedure, particularly suitable for mitochondrial disorders, which are characterized by high genetic and clinical heterogeneity and relatively loose genotype/phenotype correlation.