Anna Rubegni

A second MNGIE patient without typical mitochondrial skeletal muscle involvement

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease caused by mutations in the gene encoding thymidine phosphorylase (TYMP). Clinically, MNGIE is characterized by gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy and leukoencephalopathy. Most MNGIE patients have signs of mitochondrial dysfunction in skeletal muscle at morphological and enzyme level, as well as mitochondrial DNA depletion, multiple deletions and point mutations. A case without mitochondrial skeletal muscle involvement and with a TYMP splice-acceptor site mutation (c. 215–1 G>C) has been reported. Here, we describe an Italian patient with the same mutation and without mitochondrial skeletal muscle involvement, suggesting a possible genotype–phenotype correlation.

Chronic progressive external ophthalmoplegia: A new heteroplasmic tRNALeu(CUN) mutation of mitochondrial DNA

We sequenced all genes of mitochondrial tRNAs of a patient with chronic progressive external ophthalmoplegia with 5% ragged red fibres and 15% COX-negative fibres but without macrorearrangements of mitochondrial DNA (mtDNA). Direct sequencing showed a novel heteroplasmic G>A substitution in position 12316 of tRNA(Leu(CUN)) gene. This change destroys a highly conserved G-C base coupling in tRNA TpsiC branch. By RFLP analysis we could demonstrate different degrees of heteroplasmy in different patients tissues. This alteration, absent in a population of 110 patients with different encephalomyopathies, can be considered pathogenic: it is the tenth tRNA(Leu(CUN)) pathogenic mutation described up to date.

Novel findings associated with MTM1 suggest a higher number of female symptomatic carriers

Highlights • 504 myopathic patients have been screened for MTM1 variants by NGS and CGH array approaches.• Seven novel XLMTM patients and the fifth case of a large Xq28 deletion have been identified.• The identification of two sporadic manifesting female carriers suggests that their number may be underestimated.• Large NGS panels, including the MTM1 gene, are useful tools to identify sporadic female XLMTM patients.• The identification of MTM1 variants, also as incidental findings, complicates genetic counseling.

Pigmentary degenerative maculopathy as prominent phenotype in an Italian SPG56/ CYP2U1 family

SPG56 is an autosomal recessive form of hereditary spastic paraplegia (HSP) associated with mutations in CYP2U1. There is no clear documentation of visual impairment in the few reported cases of SPG56, although this form is complex on clinical ground and visual deficit are extremely frequent in complicated HSP. We report three patients in a consanguineous family harboring the novel homozygous c.1168C>T (p.R390*) in SPG56/CYP2U1, and showing a pigmentary degenerative maculopathy associated with progressive spastic paraplegia. Furthermore, we characterized precisely the ophthalmologic phenotype through indirect ophthalmoscopy, retinal optical coherence tomography and visual evoked potentials. This is the first formal report of pigmentary degenerative maculopathy associated with a CYP2U1 homozygous mutation.

The revolution in migraine genetics: From aching channels disorders to a next-generation medicine

Channelopathies are a heterogeneous group of neurological disorders resulting from dysfunction of ion channels located in cell membranes and organelles. The clinical scenario is broad and symptoms such as generalized epilepsy (with or without fever), migraine (with or without aura), episodic ataxia and periodic muscle paralysis are some of the best known consequences of gain- or loss-of-function mutations in ion channels. We review the main clinical effects of ion channel mutations associated with a significant impact on migraine headache. Given the increasing and evolving use of genetic analysis in migraine research—greater emphasis is now placed on genetic markers of dysfunctional biological systems—we also show how novel information in rare monogenic forms of migraine might help to clarify the disease mechanisms in the general population of migraineurs. Next-generation sequencing (NGS) and more accurate and precise phenotyping strategies are expected to further increase understanding of migraine pathophysiology and genetics.

Mitochondrial dysfunction in hereditary spastic paraparesis with mutations in DDHD1/SPG28

Mutations in DDHD1 cause the SPG28 subtype of hereditary spastic paraplegia (HSP). Recent studies suggested that mitochondrial dysfunction occurs in SPG28. Here we describe two siblings with SPG28, and report evidence of mitochondrial impairment in skeletal muscle and skin fibroblasts. Patient 1 (Pt1) was a 35-year-old man with spastic paraparesis and urinary incontinence, while his 25-year-old brother (Pt2) had gait spasticity and motor axonal neuropathy. In these patients we identified the novel homozygous c.1429C>T/p.R477* mutation in DDHD1, using a next-generation sequencing (NGS) approach. Histochemical analyses in muscle showed mitochondrial alterations, and multiple mitochondrial DNA (mtDNA) deletions were evident. In Pt1, respiratory chain enzyme activities were altered in skeletal muscle, mitochondrial ATP levels reduced, and analysis of skin fibroblasts revealed mitochondrial fragmentation. It seems possible that the novel nonsense mutation identified abolishes DDHD1 protein function thus altering oxidative metabolism. Qualitative alterations of mtDNA could have a pathogenetic significance. We suggest to perform DDHD1 analysis in patients with multiple mtDNA deletions.

Electron-dense lamellated inclusions in 2 siblings with Kufor-Rakeb syndrome.

Kufor–Rakeb syndrome is a rare form of parkinsonism caused by mutations in the ATP13a2 gene. Although the wild-type protein is normally located in lysosomes, unstable truncated mutants are retained in the endoplasmic reticulum and degraded by proteasomes. A recent report showed that loss of ATP13a2 in human fibroblasts from patients with Kufor–Rakeb syndrome or in mouse primary neurons led to impaired lysosomal degradation capacity. Mutations in the ATP13a2 have recently also been associated with an adultonset form of neuronal ceroid lipofuscinosis in dogs. Pathological observation revealed membrane-bound vacuoles containing heterogeneous material. In 1985 Tom e described cytoplasmic lamellar inclusions in the central and peripheral nervous system in a large family. Later, in the same family, mutations in the ATP13a2 gene were associated with a syndrome of neuronal ceroid lipofuscinosis. Here we report 2 siblings with Kufor–Rakeb syndrome. The clinical findings were previously described. Molecular analysis in both patients detected the homozygous c.G2629A mutation in exon 24 of the ATP13a2 and the c.C1441T heterozygous mutation in exon 9 of the FBX07 gene. After obtaining informed consent, we performed skin and muscle biopsies. Ultrastructural examination showed membranebound electron-dense material of lamellar appearance. The inclusions were observed in skin cells (fibrocytes, endothelial cells, macrophages) and in intermyofibrillar, subsarcolemmal, and endomysial regions of muscle fibers (Fig. 1). Our patients carried a homozygous mutation in the ATP13a2 gene and a heterozygous mutation in the FBX07 gene, the latter responsible for PARK15 syndrome. Because this mutation was also found in their healthy mother, it presumably does not influence the pathological process. Kufor–Rakeb syndrome and neuronal ceroid lipofuscinosis associated with ATP13a2 mutation have different clinical pictures, but they also share several clinical aspects. In particular, some affected members of the family with neuronal ceroid lipofuscinosis showed signs of parkinsonism, and cognitive impairment is reported in both syndromes. To date, the principal difference between the 2 entities is the presence of peculiar storage material in neuronal and extraneuronal cells in ATP13a2-mutation neuronal ceroid lipofuscinosis. Here we have demonstrated accumulation of membrane-bound storage material in muscle and skin cells of Kufor–Rakeb syndrome patients. This material is similar to that reported by Tom e et al in their historical description of a family diagnosed with neuronal ceroid lipofuscinosis, in which Bras recently identified ATP13A2 mutations. In the original report of Tom e, the disease was defined as a familial syndrome with particular lamellated inclusions in neuronal and extraneuronal cells. Indeed, the features of this material were not similar to that usually reported in classical neuronal ceroid lipofuscinosis. In 2010 Paisan-Ruiz detected cytoplasmic membrane-bound inclusions within Schwann, perineural, and smooth muscle cells of the sural nerve of Kufor–Rakeb syndrome patients. In our study inclusions were observed in the cytoplasm of skin cells and in skeletal muscle. Thus, electron-dense lamellated material may be found in neuronal and extraneuronal cells of subjects with homozygous or heterozygous composite ATP13a2 mutations. Our data thus confirm the findings of Paisan-Ruiz. Overall, such observations support the hypothesis that mutations in ATP13a2 may produce a clinical continuum ranging from neuronal ceroid lipofuscinosis to Kufor– Rakeb syndrome. Relevant conflicts of interest/financial disclosures: Nothing to report. Full financial disclosures and author roles may be found in the online version of this article.

Novel POLG mutations and variable clinical phenotypes in 13 Italian patients

POLG gene encodes the catalytic subunit of DNA polymerase gamma, essential for mitochondrial DNA (mtDNA) replication and repair. Mutations in POLG have been linked to a spectrum of clinical phenotypes, resulting in autosomal recessive or dominant mitochondrial diseases. These mutations have been associated with heterogeneous phenotypes, presenting with varying severity and at different ages of onset, ranging from the neonatal period to late adult life. We screened 13 patients for POLG mutations. All patients underwent a complete neurological examination, and in most of cases, muscle biopsy was performed. We detected 15 different variations in 13 unrelated Italian patients. Two mutations were novel and mapped in the pol domain (p.Thr989dup and p.Ala847Thr) of the enzyme. We also report new cases carrying controversial variations previously described as incompletely penetrant or a variant of unknown significance. Our study increases the range of clinical presentations associated with mutations in POLG gene, underlining some peculiar clinical features, such as PEO associated with corneal edema, and epilepsy, severe neuropathy with achalasia. The addition of two new substitutions, including the second report of an in-frame duplication, to the growing list of defects increases the value of POLG genetic diagnosis in a range of neurological presentations.

A series of Greek children with pure hereditary spastic paraplegia: clinical features and genetic findings

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group of neurodegenerative disorders mainly characterized by progressive spasticity of the lower limbs. Adult case series dominate the literature, and there have been only a few studies in children. The purpose of this study is to describe our experience with pediatric HSP in Greece. We report the clinical and genetic findings in our patients and aim to offer insights into the diagnostic difficulties of childhood-onset disease. A series of 15 Greek children affected by pure HSP underwent extensive diagnostic investigations. Molecular analysis included whole exome sequencing (WES) or consecutive screening of candidate genes ATL1, SPAST, REEP1, and CYP7B1. WES performed in three cases yielded previously reported mutations in ATL1 and CYP7B1, and a variant c.397C>T of unknown significance in SPG7. Candidate gene screening performed in the remaining patients identified previously reported mutations in ATL1 (2), SPAST (2), and REEP1 (1), and two novel mutations, c.1636G>A and c.1413+3_6delAAGT, in SPAST. In six cases, the mutations were inherited from their parents, while in three cases, the mutations were apparently de novo. Our data confirm the genetic heterogeneity of childhood-onset pure HSP, with SPG4/SPAST and SPG3A/ATL1 being the most frequent forms. De novo occurrence of HSP does not seem to be uncommon. Candidate gene studies guided by diagnostic algorithms and WES seem both to be reasonable genetic testing strategies.

Additive effect of nuclear and mitochondrial mutations in a patient with mitochondrial encephalomyopathy

We describe the case of a woman in whom combination of a mitochondrial (MT-CYB) and a nuclear (SDHB) mutation was associated with clinical and metabolic features suggestive of a mitochondrial disorder. The mutations impaired overall energy metabolism in the patients muscle and fibroblasts and increased cellular susceptibility to oxidative stress. To clarify the contribution of each mutation to the phenotype, mutant yeast strains were generated. A significant defect in strains carrying the Sdh2 mutation, either alone or in combination with the cytb variant, was observed. Our data suggest that the SDHB mutation was causative of the mitochondrial disorder in our patient with a possible cumulative contribution of the MT-CYB variant. To our knowledge, this is the first association of bi-genomic variants in the mtDNA and in a nuclear gene encoding a subunit of complex II.