MME mutation in dominant spinocerebellar ataxia with neuropathy (SCA43)

DISCUSSION

Affected individuals in this family developed a late-onset, motor-predominant axonal polyneuropathy associated with cerebellar signs in all cases but one, presenting with ataxia with mixed afferent and cerebellar features. Pectus carinatum, although not omnipresent, seems to be a distinctive clinical feature. Genetic analyses, including WES performed in 2 affected individuals, identified a variant in the MME gene, p.C143Y, fulfilling several criteria for pathogenicity, including absence in control databases, cosegregation with the phenotype, and predicted damaging effect on the encoded protein.⁵ Although we could not find a second SCA family with the same mutation among the 96 we screened, the recent identification of recessive MME mutations in 10 unrelated individuals from Japan with an essentially identical polyneuropathy, but no cerebral or cerebellar atrophy, supports the causality of the MME mutation in our study family, despite the dominant mode of transmission and the presence of cerebellar involvement.³ In this regard, it is well recognized that mutations in the same gene may lead to different phenotypes and modes of transmission, and such observations are becoming common as next-generation sequencing allows solving a much larger number of cases of genetic disorders, including ataxias.⁶ Experimental functional studies on the p.C143Y MME variant are needed to understand the reason for its dominant effect. While all prediction algorithms we used (SIFT, PolyPhen-2, and PROVEAN) indicated a strong deleterious impact of the p.C143Y variant, the identification of recessive mutations, some leading to complete loss of the MME expression,³ makes haploinsufficiency an unlikely mechanism.

MME encodes neprilysin (NEP), a zinc-dependent metalloprotease that cleaves peptides at the amino side of the hydrophobic residues. NEP is expressed in most tissues, including the central and peripheral nervous systems (CNS and PNS). In the CNS, the expression is almost exclusively neuronal, particularly in axons and synaptic terminals,⁷ while in the PNS, the expression is mostly in Schwann cells,⁸ but NEP has also been shown to be transported along sciatic nerve axons.⁹ Together with the endothelin-converting enzymes (ECE-1 and ECE-2), the erythrocyte surface antigen KELL, and the PEX gene product, NEP belongs to the M13 subfamily of mammalian neutral endopeptidases.¹⁰ These are cell membrane proteins composed of a short N-terminal cytoplasmic domain, followed by a single transmembrane helix, and a large C-terminal extracellular portion composed of 2 major alpha-helical (peptidase M13) domains connected by a linker region. There are 12 cysteine residues in the NEP extracellular portion, all involved in disulfide bridges, 10 of which are also conserved in ECE, KELL, and PEX. Cysteine 143, mutated in our study family, is one of these 10 cysteines. It is localized in the N-terminal peptidase M13 domain and forms a disulfide bridge with cysteine 411 in the linker portion¹⁰ (figure 3).

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Figure 3 Position of the mutated residue

Schematic of neprilysin showing the position of the mutated residue (Cys143) and the disulfide bond with Cys411 that is disrupted by the mutation.

NEP acts on many substrates, including neuropetides such as glucagon, enkephalins, cholecystokinin, neuropeptide Y, substance P, somatostatin, neurotensin, oxytocin, prodynorphin and bradykinin, as well as the amyloid beta (Aβ) peptides accumulated in Alzheimer disease (AD). Because of its activity on Aβ and its higher expression in entorhinal, hippocampal, and cortical areas, NEP has been intensively scrutinized for a possible pathogenic role and as a potential therapeutic target in AD.¹¹ However, although Mme knockout mice and mice exposed to NEP inhibitors have increased brain Aβ levels,¹² they do not develop AD-like pathology. While some studies reported amyloid deposition and neurodegenerative changes in their hippocampus,¹³ a more recent study surprisingly found cognitive improvement in these animals as they age and no amyloid plaques in their brains.¹⁴ No MME mutations are known to cause familial AD, while genetic association studies have provided only limited evidence of association between MME polymorphisms and risk of AD.¹⁵ Furthermore, none of the Japanese CMT2 patients with MME mutations were found to be cognitively impaired after detailed neuropsychological testing, and amyloid PET imaging was negative in the single case in which it was performed.³ Cognitive impairment was also not a feature in our study family. Taken together, these findings indicate that although NEP expression has been reported to decrease with age and in AD, NEP deficiency does not lead to the development of AD, possibly because other enzyme systems degrade excess Aβ and prevent amyloid plaque formation.

The pathogenesis of peripheral neuropathy due to MME mutations is still unclear. It has been hypothesized that NEP has a role in axon–Schwann cell interaction,³ but experimental studies are needed to explore its physiologic role and the consequences of its impaired or loss of function. Equally unknown is the role of NEP in the cerebellum and the reason why recessive loss-of-function mutations do not affect this structure, while the p.C143Y mutation causes cerebellar dysfunction and atrophy. A possible lead comes from the fact that prodynorphin, the precursor of the opioid neuropetides α-neoendorphin and dynorphins A and B (Dyn A and B), is an NEP substrate, and mutations affecting the prodynorphin (PDYN) gene cause SCA23.¹⁶ Of interest, some SCA23 mutations alter the equilibrium between Dyn A and B and raise dynorphin peptide levels, which could be mimicked by altered NEP function.¹⁷ The p.C143Y mutation may have some specific effect on NEP activity on dynorphin peptides, possibly competing with wild-type NEP for substrate affinity and catalytic activity, which eventually triggers cerebellar degeneration. Conversely, as dynorphin is a potent NEP inhibitor,¹⁸ impaired NEP activity might be implicated in SCA23 pathogenesis. Clearly, further research is needed to address these questions. Existing Mme knockout mice should in principle model human recessive polyneuropathy cases, but interestingly they do not develop a severe axonal neuropathy, suggesting important interspecific differences. For this reason, there is a high risk that a mouse model carrying the p.C143Y mutation, obtained by classical knock-in approaches or by genome editing, would also not recapitulate the human phenotype associated with this mutation.

By combining linkage analysis and WES, we found a variant in the MME gene, p.C143Y, that responds to several criteria for pathogenicity, i.e., rarity, cosegregation with the phenotype, predicted deleterious effect, absence of other identified potential pathogenic variants by exome sequencing, and involvement of a gene proven to be mutated in families with a closely related phenotype. While functional studies will be needed to study pathogenic mechanisms, we consider that current evidence suffices to propose this mutation as disease causing in our study family, identifying a novel SCA genotype (SCA43) as well as a novel phenotype associated with a specific MME mutation.