4. Mutation spectrum of SLC25A46 and genotype-phenotype correlation

#### 4.1. Mutation spectrum of SLC25A46

homogenous SLC25A46 mutations in an additional nine patients (age range, 7 days to 28 years) from five unrelated families who presented with neurological phenotypes similar to the core features of HMSN6B. Among these nine patients, eight had optic atrophy (the exception was a patient with an age of onset 23 years) and eight had cerebellar atrophy (the exception was a 28-year-old patient without remarkable cerebellar atrophy or axonal neuropathy) (Table 1) [19, 21, 23, 26]. Beyond the key clinical features of optic atrophy, peripheral neuropathy, and cerebellar atrophy, the presently documented population of 17 patients with HMSN6B (or an HMSN6B-like condition) exhibited other clinical symptoms sporadically, including ataxia, hypotonia, myoclonus, dysmetria, nystagmus, speech difficulties, abnormal brain imaging, and elevated lactic acid (Table 1). The clinical manifestations, medical examination findings, and differential diagnoses for these patients were strongly suggestive of a progressive mitochondrial

A recent study reported the identification of SLC25A46 loss-of-function mutations in four patients from two unrelated families with a diagnosis of severe congenital PCH, leading to very early mortality [22]. Then, two independent groups reported an additional seven patients from three unrelated families with severe PCH associated with truncating mutations of

PCH is a rare, heterogeneous group of prenatal onset neurodegenerative disorders, mainly (but not exclusively) affecting the cerebellum and pons. The current PCH classification scheme includes 10 distinct PCH subtypes defined by clinical features and genetic etiology. PCH1 is distinguished from the other PCH subtypes by its association with spinal muscular atrophy due to spinal motoneuron degeneration; it often leads to early death. All patients with obvious loss-of-function SLC25A46 genotypes in the literature suffered severe lethal congenital PCH, presenting with the phenotypic hallmarks of cerebellar and brainstem degeneration as well as spinal muscular atrophy, respiratory failure, early death, occasional optic nerve atrophy, and axonal neuropathy. Based on these features, SLC25A46-associated PCH could be classified as PCH1, and perhaps a new PCH1 subtype, PCH1D, clinically distinguished from other PCH1 subtypes (mutations in VRK1, EXOSC3, and EXOSC8 are associated with PCH1A, PCH1B, and PCH1C, respectively) [25]. However, the most severe clinical presentation associated with SLC25A46 mutations is probably not restricted to PCH. A homozygous SLC25A46 mutation that resulted in the complete absence of the protein was identified recently in a terminally ill child with progressive brain lesions consistent with those seen in Leigh syndrome (Table 1) [20].

Cerebellar and brainstem atrophy are shared phenotypic features of PCH, Leigh syndrome, and most variant SLC25A46-related HMSN6B cases. Meanwhile, optic nerve and peripheral nerve axonal pathology are seen consistently in HMSN6B. Features that are prominent in lateronset cases might be overlooked or not assessed in neonatally lethal cases. Thus, SLC25A46-

To sum up, SLC25A46-related neurological disease has high clinical heterogeneity. Patients with biallelic SLC25A46 mutations show high phenotypic variability with respect to age of

related PCH or Leigh syndrome could be extreme forms of HMSN6B.

disorder.

3.2. SLC25A46-related PCH and Leigh syndrome

SLC25A46 (Table 1) [24, 25].

76 Recent Advances in Neurodegeneration

The SLC25A46 gene, located on chromosome 5q22.1, spans approximately 27 kb and is composed of eight exons. The main protein isoform has 418 amino acids and is encoded by a 1257-nucleotide-long open reading frame. Since SLC25A46 mutations associated with neurological disease were first reported in 2015, more than 28 patients with various mutations from 16 unrelated families have been diagnosed genetically, most by whole-exome sequencing, leading to the discovery of a total of 18 pathogenic mutations in the last 2 years (Figure 2). Of these, 50% are missense mutations; 16.7% are nonsense mutations; 11.1% are splice variants; and 22.2% are micro-deletions, insertions, or duplications. The mutation sizes range from a single nucleotide polymorphism to a 2.4-kb deletion. Although some mutations have been found in all exons except exons 2, 6, and 7, 50% of the mutations are located in exon 8, the largest exon, which accounts for half of the SLC25A46 open reading frame (Figure 2). Although there is no suspected mutation hotspot site, the c.1081C>T variant was observed in 3 of 16 independent families (Table 1). The identification and genetic diagnosis of additional cases in the future may reveal a SLC25A46 mutation pattern.

#### 4.2. Genotype-phenotype correlation

A systemic genotype-phenotype analysis of all available cases indicates that phenotype severity correlates strongly with the magnitude of SLC25A46 protein level reduction caused by each

Figure 2. Schematic diagram of reported pathogenic SLC25A46 variants. Exons 1–8 are represented by blue blocks. Mutations are color coded as follows: red, nonsense and missense mutations that would be expected to destabilize the protein; blue, micro-deletions/insertions/duplications; orange, splice-site mutations; and black, regular missense mutations.

mutation. As shown in Table 1, very severe SLC25A46-related disease has been linked to mutations that yield markedly reduced SLC25A46 levels, including homozygous or compound heterozygous nonsense mutations, (c.691C>T, p.Arg231\*; c.736A>T, p.Arg246\*; c.42C>G, p.Tyr14\*), a splice site variant (c.462+1G>A), and a micro-deletion (g.chr5: 110738771\_11074670del). In addition, Wan et al. and Janer et al. verified that the three missense mutations c.1022T>C (p.Leu341Pro), c.998C>T (p.Pro333Leu), and c.425C>T (p. Thr142Ile) destabilize the protein without nonsense-mediated mRNA decay, causing a marked loss of SLC25A46 function. Such protein-depriving mutations lead to severe clinical symptoms of PCH or Leigh syndrome. In contrast, missense mutations of SLC25A46 that are associated with normal or mildly reduced protein levels tend to result in a relatively mild phenotype. For instance, stable expression of the p.Gly249Asp mutant protein produces low-severity manifestations of optic atrophy spectrum disorder [17, 22] (Table 1). Thus, the more stable, functional, and abundant the mutant protein, the less severe the clinical manifestations.

In conclusion, the main molecular causes of SLC25A46-related neurological disease appear to be SLC25A46 loss of function or deficiency. The recessive inheritance pattern observed in all pathogenic SLC25A46 mutation-affected individuals and families thus far contrast with the dominant inheritance pattern observed with other mitochondrial dynamic genes, including OPA1, MFN1/2, and DRP1, for which haplo-insufficiency and dominant negative effects are observed [8, 36, 37].
