Publications by authors named "Alessia Nasca"

21 Publications

  • Page 1 of 1

A novel homozygous MSTO1 mutation in Ashkenazi Jewish siblings with ataxia and myopathy.

J Hum Genet 2021 Feb 22. Epub 2021 Feb 22.

Pediatric Neurology Unit, Hadassah Hebrew University of Jerusalem, 91240, Jerusalem, Israel.

MSTO1 is a cytoplasmic protein that modulates mitochondrial dynamics by promoting mitochondrial fusion. Mutations in the MSTO1 gene are responsible for an extremely rare condition characterized by early-onset myopathy and cerebellar ataxia. We report here two siblings from a large Ashkenazi Jewish family, presenting with a progressive neuromuscular disease characterized by ataxia and myopathy. By whole exome sequencing, we found a novel homozygous missense mutation (c.1403T>A, p.Leu468Gln) in MSTO1. Studies performed on fibroblasts from the index patient demonstrated the pathogenic role of the identified variant; we found that MSTO1 protein level was reduced and that mitochondrial network was fragmented or formed enlarged structures. Moreover, patient's cells showed reduced mitochondrial DNA amount. Our report confirms that MSTO1 mutations are typically recessive, and associated with clinical phenotypes characterized by early-onset muscle impairment and ataxia, often with upper motor neuron signs and varied cognitive impairment.
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http://dx.doi.org/10.1038/s10038-020-00897-4DOI Listing
February 2021

Bi-allelic pathogenic variants in NDUFC2 cause early-onset Leigh syndrome and stalled biogenesis of complex I.

EMBO Mol Med 2020 11 24;12(11):e12619. Epub 2020 Sep 24.

Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK.

Leigh syndrome is a progressive neurodegenerative disorder, most commonly observed in paediatric mitochondrial disease, and is often associated with pathogenic variants in complex I structural subunits or assembly factors resulting in isolated respiratory chain complex I deficiency. Clinical heterogeneity has been reported, but key diagnostic findings are developmental regression, elevated lactate and characteristic neuroimaging abnormalities. Here, we describe three affected children from two unrelated families who presented with Leigh syndrome due to homozygous variants (c.346_*7del and c.173A>T p.His58Leu) in NDUFC2, encoding a complex I subunit. Biochemical and functional investigation of subjects' fibroblasts confirmed a severe defect in complex I activity, subunit expression and assembly. Lentiviral transduction of subjects' fibroblasts with wild-type NDUFC2 cDNA increased complex I assembly supporting the association of the identified NDUFC2 variants with mitochondrial pathology. Complexome profiling confirmed a loss of NDUFC2 and defective complex I assembly, revealing aberrant assembly intermediates suggestive of stalled biogenesis of the complex I holoenzyme and indicating a crucial role for NDUFC2 in the assembly of the membrane arm of complex I, particularly the ND2 module.
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http://dx.doi.org/10.15252/emmm.202012619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645371PMC
November 2020

Homozygous mutations in C1QBP as cause of progressive external ophthalmoplegia (PEO) and mitochondrial myopathy with multiple mtDNA deletions.

Hum Mutat 2020 Jul 11. Epub 2020 Jul 11.

Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano, Italy.

Biallelic mutations in the C1QBP gene have been associated with mitochondrial cardiomyopathy and combined respiratory-chain deficiencies, with variable onset (including intrauterine or neonatal forms), phenotypes, and severity. We studied two unrelated adult patients from consanguineous families, presenting with progressive external ophthalmoplegia (PEO), mitochondrial myopathy, and without any heart involvement. Muscle biopsies from both patients showed typical mitochondrial alterations and the presence of multiple mitochondrial DNA deletions, whereas biochemical defects of the respiratory chain were present only in one subject. Using next-generation sequencing approaches, we identified homozygous mutations in C1QBP. Immunoblot analyses in patients' muscle samples revealed a strong reduction in the amount of the C1QBP protein and varied impairment of respiratory chain complexes, correlating with disease severity. Despite the original study indicated C1QBP mutations as causative for mitochondrial cardiomyopathy, our data indicate that mutations in C1QBP have to be considered in subjects with PEO phenotype or primary mitochondrial myopathy and without cardiomyopathy.
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http://dx.doi.org/10.1002/humu.24081DOI Listing
July 2020

ATPase Domain AFG3L2 Mutations Alter OPA1 Processing and Cause Optic Neuropathy.

Ann Neurol 2020 07 21;88(1):18-32. Epub 2020 Apr 21.

Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

Objective: Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, with a prevalence of 1:12,000 to 1:25,000. OPA1 mutations are found in 70% of DOA patients, with a significant number remaining undiagnosed.

Methods: We screened 286 index cases presenting optic atrophy, negative for OPA1 mutations, by targeted next generation sequencing or whole exome sequencing. Pathogenicity and molecular mechanisms of the identified variants were studied in yeast and patient-derived fibroblasts.

Results: Twelve cases (4%) were found to carry novel variants in AFG3L2, a gene that has been associated with autosomal dominant spinocerebellar ataxia 28 (SCA28). Half of cases were familial with a dominant inheritance, whereas the others were sporadic, including de novo mutations. Biallelic mutations were found in 3 probands with severe syndromic optic neuropathy, acting as recessive or phenotype-modifier variants. All the DOA-associated AFG3L2 mutations were clustered in the ATPase domain, whereas SCA28-associated mutations mostly affect the proteolytic domain. The pathogenic role of DOA-associated AFG3L2 mutations was confirmed in yeast, unraveling a mechanism distinct from that of SCA28-associated AFG3L2 mutations. Patients' fibroblasts showed abnormal OPA1 processing, with accumulation of the fission-inducing short forms leading to mitochondrial network fragmentation, not observed in SCA28 patients' cells.

Interpretation: This study demonstrates that mutations in AFG3L2 are a relevant cause of optic neuropathy, broadening the spectrum of clinical manifestations and genetic mechanisms associated with AFG3L2 mutations, and underscores the pivotal role of OPA1 and its processing in the pathogenesis of DOA. ANN NEUROL 2020 ANN NEUROL 2020;88:18-32.
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http://dx.doi.org/10.1002/ana.25723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383914PMC
July 2020

New missense variants of NDUFA11 associated with late-onset myopathy.

Muscle Nerve 2019 08 30;60(2):E11-E14. Epub 2019 May 30.

Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

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http://dx.doi.org/10.1002/mus.26511DOI Listing
August 2019

Clinical-genetic features and peculiar muscle histopathology in infantile DNM1L-related mitochondrial epileptic encephalopathy.

Hum Mutat 2019 05 9;40(5):601-618. Epub 2019 Mar 9.

Department of Neurosciences, Unit of Muscular and Neurodegenerative Disorders, Laboratory of Molecular Medicine, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Mitochondria are highly dynamic organelles, undergoing continuous fission and fusion. The DNM1L (dynamin-1 like) gene encodes for the DRP1 protein, an evolutionary conserved member of the dynamin family, responsible for fission of mitochondria, and having a role in the division of peroxisomes, as well. DRP1 impairment is implicated in several neurological disorders and associated with either de novo dominant or compound heterozygous mutations. In five patients presenting with severe epileptic encephalopathy, we identified five de novo dominant DNM1L variants, the pathogenicity of which was validated in a yeast model. Fluorescence microscopy revealed abnormally elongated mitochondria and aberrant peroxisomes in mutant fibroblasts, indicating impaired fission of these organelles. Moreover, a very peculiar finding in our cohort of patients was the presence, in muscle biopsy, of core like areas with oxidative enzyme alterations, suggesting an abnormal distribution of mitochondria in the muscle tissue.
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http://dx.doi.org/10.1002/humu.23729DOI Listing
May 2019

Clinical and Biochemical Features in a Patient With Gene Alteration.

Front Genet 2018 7;9:625. Epub 2018 Dec 7.

Unit of Child Neurology and Psychiatry, Department of Human Neuroscience, Sapienza University of Rome, Rome, Italy.

Mitochondrial Fission Factor (MFF) is part of a protein complex that promotes mitochondria and peroxisome fission. Hitherto, only 5 patients have been reported harboring mutations in , all of them with the clinical features of a very early onset Leigh-like encephalopathy. We report on an 11-year-old boy with epileptic encephalopathy. He presented with neurological regression, epileptic myoclonic seizures, severe intellectual disability, microcephaly, tetraparesis, optic atrophy, and ophthalmoplegia. Brain MRI pattern was compatible with Leigh syndrome. NGS-based analysis of a gene panel for mitochondrial disorders revealed a homozygous c.892C>T (p. Arg298) in the gene. Fluorescence staining detected abnormal morphology of mitochondria and peroxisomes in fibroblasts from the patient; a strong reduction in MFF protein levels and the presence of truncated forms were observed. No biochemical alterations denoting peroxisomal disorders were found. As reported in other disorders affecting the dynamics of intracellular organelles, our patient showed clinical features suggesting both mitochondrial and peroxisomal impairment. High levels of lactate in our case suggested an involvement of the energetic metabolism but without clear respiratory chain deficiency, while biomarkers of peroxisomal dysfunction were normal. We confirm that mutations are associated with epileptic encephalopathy with Leigh-like MRI pattern.
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http://dx.doi.org/10.3389/fgene.2018.00625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292958PMC
December 2018

Neurologic Phenotypes Associated With Mutations in RTN4IP1 (OPA10) in Children and Young Adults.

JAMA Neurol 2018 01;75(1):105-113

MitoLab Team, Unités Mixtes de Recherche Centre National de la Recherche Scientifique 6015-INSERM U1083, Institut MitoVasc, Angers University and Hospital, Angers, France.

Importance: Neurologic disorders with isolated symptoms or complex syndromes are relatively frequent among mitochondrial inherited diseases. Recessive RTN4IP1 gene mutations have been shown to cause isolated and syndromic optic neuropathies.

Objective: To define the spectrum of clinical phenotypes associated with mutations in RTN4IP1 encoding a mitochondrial quinone oxidoreductase.

Design, Setting, And Participants: This study involved 12 individuals from 11 families with severe central nervous system diseases and optic atrophy. Targeted and whole-exome sequencing were performed-at Hospital Angers (France), Institute of Neurology Milan (Italy), Imagine Institute Paris (France), Helmoltz Zentrum of Munich (Germany), and Beijing Genomics Institute (China)-to clarify the molecular diagnosis of patients. Each patient's neurologic, ophthalmologic, magnetic resonance imaging, and biochemical features were investigated. This study was conducted from May 1, 2014, to June 30, 2016.

Main Outcomes And Measures: Recessive mutations in RTN4IP1 were identified. Clinical presentations ranged from isolated optic atrophy to severe encephalopathies.

Results: Of the 12 individuals in the study, 6 (50%) were male and 6 (50%) were female. They ranged in age from 5 months to 32 years. Of the 11 families, 6 (5 of whom were consanguineous) had a member or members who presented isolated optic atrophy with the already reported p.Arg103His or the novel p.Ile362Phe, p.Met43Ile, and p.Tyr51Cys amino acid changes. The 5 other families had a member or members who presented severe neurologic syndromes with a common core of symptoms, including optic atrophy, seizure, intellectual disability, growth retardation, and elevated lactate levels. Additional clinical features of those affected were deafness, abnormalities on magnetic resonance images of the brain, stridor, and abnormal electroencephalographic patterns, all of which eventually led to death before age 3 years. In these patients, novel and very rare homozygous and compound heterozygous mutations were identified that led to the absence of the protein and complex I disassembly as well as mild mitochondrial network fragmentation.

Conclusions And Relevance: A broad clinical spectrum of neurologic features, ranging from isolated optic atrophy to severe early-onset encephalopathies, is associated with RTN4IP1 biallelic mutations and should prompt RTN4IP1 screening in both syndromic neurologic presentations and nonsyndromic recessive optic neuropathies.
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http://dx.doi.org/10.1001/jamaneurol.2017.2065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833489PMC
January 2018

Functionally pathogenic variants in vitro may not manifest a phenotype in vivo.

Neurol Genet 2017 Aug 14;3(4):e162. Epub 2017 Jul 14.

Baylor Research Institute (N.M., B.L., R.S.), Baylor Scott and White Health, Dallas, TX; Unit of Molecular Neurogenetics (A.N., D.G.), Foundation IRCCS Institute of Neurology "Besta," Milan, Italy; Mitochondrial Biology Unit (A.R.), Medical Research Council, Cambridge, United Kingdom; Department of Bioinformatics (B.C.), University of Texas Southwestern Medical Center, Dallas; and Department of Neurology (A.V.), George Washington University School of Medicine, Children's National Health, DC.

Objective: To investigate the genetic etiology of a patient diagnosed with leukoencephalopathy, brain calcifications, and cysts (LCC).

Methods: Whole-exome sequencing was performed on a patient with LCC and his unaffected family members. The variants were subject to in silico and in vitro functional testing to determine pathogenicity.

Results: Whole-exome sequencing uncovered compound heterozygous mutations in , c.328G>A (p.G110S), and c.1045G>A (p.E349K). This gene has previously been implicated in the autosomal recessive leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The p.G110S mutation has been found in multiple patients with LTBL. In silico analysis supported pathogenicity in the second variant. In vitro functional testing showed a significant mitochondrial dysfunction demonstrated by an ∼11% decrease in the oxygen consumption rate and ∼43% decrease in the maximum respiratory rate in the patient's skin fibroblasts compared with the control. EARS2 protein levels were reduced to 30% of normal controls in the patient's fibroblasts. These deficiencies were corrected by the expression of the wild-type EARS2 protein. However, a further unrelated genetic investigation of our patient revealed the presence of biallelic variants in a small nucleolar RNA () responsible for LCC.

Conclusions: Here, we report seemingly pathogenic mutations in a single patient with LCC with no biochemical or neuroimaging presentations of LTBL. This patient illustrates that variants with demonstrated impact on protein function should not necessarily be considered clinically relevant.

Clinicaltrialsgov Identifier: NCT00001671.
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http://dx.doi.org/10.1212/NXG.0000000000000162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511247PMC
August 2017

Recessive mutations in MSTO1 cause mitochondrial dynamics impairment, leading to myopathy and ataxia.

Hum Mutat 2017 08 6;38(8):970-977. Epub 2017 Jun 6.

Molecular Neurogenetics Unit, Foundation IRCCS Neurological Institute Besta, Milan, Italy.

We report here the first families carrying recessive variants in the MSTO1 gene: compound heterozygous mutations were identified in two sisters and in an unrelated singleton case, who presented a multisystem complex phenotype mainly characterized by myopathy and cerebellar ataxia. Human MSTO1 is a poorly studied protein, suggested to have mitochondrial localization and to regulate morphology and distribution of mitochondria. As for other mutations affecting genes involved in mitochondrial dynamics, no biochemical defects typical of mitochondrial disorders were reported. Studies in patients' fibroblasts revealed that MSTO1 protein levels were strongly reduced, the mitochondrial network was fragmented, and the fusion events among mitochondria were decreased, confirming the deleterious effect of the identified variants and the role of MSTO1 in modulating mitochondrial dynamics. We also found that MSTO1 is mainly a cytosolic protein. These findings indicate recessive mutations in MSTO1 as a new cause for inherited neuromuscular disorders with multisystem features.
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http://dx.doi.org/10.1002/humu.23262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575512PMC
August 2017

Not only dominant, not only optic atrophy: expanding the clinical spectrum associated with OPA1 mutations.

Orphanet J Rare Dis 2017 05 12;12(1):89. Epub 2017 May 12.

Unit of Molecular Neurogenetics, Fondazione IRCCS Istituto Neurologico 'Carlo Besta', via Temolo 4, 20126, Milan, Italy.

Background: Heterozygous mutations in OPA1 are a common cause of autosomal dominant optic atrophy, sometimes associated with extra-ocular manifestations. Few cases harboring compound heterozygous OPA1 mutations have been described manifesting complex neurodegenerative disorders in addition to optic atrophy.

Results: We report here three patients: one boy showing an early-onset mitochondrial disorder with hypotonia, ataxia and neuropathy that was severely progressive, leading to early death because of multiorgan failure; two unrelated sporadic girls manifesting a spastic ataxic syndrome associated with peripheral neuropathy and, only in one, optic atrophy. Using a targeted resequencing of 132 genes associated with mitochondrial disorders, in two probands we found compound heterozygous mutations in OPA1: in the first a 5 nucleotide deletion, causing a frameshift and insertion of a premature stop codon (p.Ser64Asnfs*7), and a missense change (p.Ile437Met), which has recently been reported to have clinical impact; in the second, a novel missense change (p.Val988Phe) co-occurred with the p.Ile437Met substitution. In the third patient a homozygous mutation, c.1180G > A (p.Ala394Thr) in OPA1 was detected by a trio-based whole exome sequencing approach. One of the patients presented also variants in mitochondrial DNA that may have contributed to the peculiar phenotype. The deleterious effect of the identified missense changes was experimentally validated in yeast model. OPA1 level was reduced in available patients' biological samples, and a clearly fragmented mitochondrial network was observed in patients' fibroblasts.

Conclusions: This report provides evidence that bi-allelic OPA1 mutations may lead to complex and severe multi-system recessive mitochondrial disorders, where optic atrophy might not represent the main feature.
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http://dx.doi.org/10.1186/s13023-017-0641-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427524PMC
May 2017

Biallelic Mutations in DNM1L are Associated with a Slowly Progressive Infantile Encephalopathy.

Hum Mutat 2016 09 11;37(9):898-903. Epub 2016 Jul 11.

Unit of Molecular Neurogenetics, Fondazione IRCCS Istituto Neurologico 'Carlo Besta', Milan, Italy.

Mitochondria are highly dynamic organelles, undergoing continuous fission and fusion, and mitochondrial dynamics is important for several cellular functions. DNM1L is the most important mediator of mitochondrial fission, with a role also in peroxisome division. Few reports of patients with genetic defects in DNM1L have been published, most of them describing de novo dominant mutations. We identified compound heterozygous DNM1L variants in two brothers presenting with an infantile slowly progressive neurological impairment. One variant was a frame-shift mutation, the other was a missense change, the pathogenicity of which was validated in a yeast model. Fluorescence microscopy revealed abnormally elongated mitochondria and aberrant peroxisomes in mutant fibroblasts, indicating impaired fission of these organelles. In conclusion, we described a recessive disease caused by DNM1L mutations, with a clinical phenotype resembling mitochondrial disorders but without any biochemical features typical of these syndromes (lactic acidosis, respiratory chain complex deficiency) or indicating a peroxisomal disorder.
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http://dx.doi.org/10.1002/humu.23033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108486PMC
September 2016

New genes and pathomechanisms in mitochondrial disorders unraveled by NGS technologies.

Biochim Biophys Acta 2016 Aug 8;1857(8):1326-1335. Epub 2016 Mar 8.

Mitochondrial Biology Unit, Medical Research Council, Cambridge CB2 0XY, UK. Electronic address:

Next Generation Sequencing (NGS) technologies are revolutionizing the diagnostic screening for rare disease entities, including primary mitochondrial disorders, particularly those caused by nuclear gene defects. NGS approaches are able to identify the causative gene defects in small families and even single individuals, unsuitable for investigation by traditional linkage analysis. These technologies are contributing to fill the gap between mitochondrial disease cases defined on the basis of clinical, neuroimaging and biochemical readouts, which still outnumber by approximately 50% the cases for which a molecular-genetic diagnosis is attained. We have been using a combined, two-step strategy, based on targeted genes panel as a first NGS screening, followed by whole exome sequencing (WES) in still unsolved cases, to analyze a large cohort of subjects, that failed to show mutations in mtDNA and in ad hoc sets of specific nuclear genes, sequenced by the Sanger's method. Not only this approach has allowed us to reach molecular diagnosis in a significant fraction (20%) of these difficult cases, but it has also revealed unexpected and conceptually new findings. These include the possibility of marked variable penetrance of recessive mutations, the identification of large-scale DNA rearrangements, which explain spuriously heterozygous cases, and the association of mutations in known genes with unusual, previously unreported clinical phenotypes. Importantly, WES on selected cases has unraveled the presence of pathogenic mutations in genes encoding non-mitochondrial proteins (e.g. the transcription factor E4F1), an observation that further expands the intricate genetics of mitochondrial disease and suggests a new area of investigation in mitochondrial medicine. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
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http://dx.doi.org/10.1016/j.bbabio.2016.02.022DOI Listing
August 2016

Mitochondrial leukoencephalopathy and complex II deficiency associated with a recessive mutation with reduced penetrance.

Mol Genet Metab Rep 2015 Dec;5:51-54

Unit of Child Neurology, Fondazione Istituto Neurologico 'Carlo Besta', IRCCS, Milan, Italy.

Mitochondrial disease involving complex II is rare among respiratory chain deficiencies and its genetic cause remains often unknown. Two main clinical presentations are associated with this biochemical defect: mitochondrial encephalomyopathy and susceptibility to tumors. Only one homozygous mutation has been described in a patient with mitochondrial disorder. We report here two sisters, who presented highly different phenotypes (neurological impairment with leukoencephalopathy vs. asymptomatic status) and harbored the same homozygous mutation, suggesting reduced penetrance.
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http://dx.doi.org/10.1016/j.ymgmr.2015.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695914PMC
December 2015

A novel AIFM1 mutation expands the phenotype to an infantile motor neuron disease.

Eur J Hum Genet 2016 Mar 15;24(3):463-6. Epub 2015 Jul 15.

Neuromuscular and Neurodegenerative Diseases Unit, Children Research Hospital Bambino Gesù, Rome, Italy.

AIFM1 is a gene located on the X chromosome, coding for AIF (Apoptosis-Inducing Factor), a mitochondrial flavoprotein involved in caspase-independent cell death. AIFM1 mutations have been associated with different clinical phenotypes: a severe infantile encephalopathy with combined oxidative phosphorylation deficiency and the Cowchock syndrome, an X-linked Charcot-Marie-Tooth disease (CMTX4) with axonal sensorimotor neuropathy, deafness and cognitive impairment. In two male cousins with early-onset mitochondrial encephalopathy and cytochrome c oxidase (COX) deficiency, we identified a novel AIFM1 mutation. Muscle biopsies and electromyography in both patients showed signs of severe denervation. Our patients manifested a phenotype that included signs of both cortical and motor neuron involvement. These observations emphasize the role of AIF in the development and function of neurons.
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http://dx.doi.org/10.1038/ejhg.2015.141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755377PMC
March 2016

RNASEH1 Mutations Impair mtDNA Replication and Cause Adult-Onset Mitochondrial Encephalomyopathy.

Am J Hum Genet 2015 Jul 18;97(1):186-93. Epub 2015 Jun 18.

Mitochondrial Biology Unit, Medical Research Council, Cambridge CB2 0XY, UK. Electronic address:

Chronic progressive external ophthalmoplegia (CPEO) is common in mitochondrial disorders and is frequently associated with multiple mtDNA deletions. The onset is typically in adulthood, and affected subjects can also present with general muscle weakness. The underlying genetic defects comprise autosomal-dominant or recessive mutations in several nuclear genes, most of which play a role in mtDNA replication. Next-generation sequencing led to the identification of compound-heterozygous RNASEH1 mutations in two singleton subjects and a homozygous mutation in four siblings. RNASEH1, encoding ribonuclease H1 (RNase H1), is an endonuclease that is present in both the nucleus and mitochondria and digests the RNA component of RNA-DNA hybrids. Unlike mitochondria, the nucleus harbors a second ribonuclease (RNase H2). All affected individuals first presented with CPEO and exercise intolerance in their twenties, and these were followed by muscle weakness, dysphagia, and spino-cerebellar signs with impaired gait coordination, dysmetria, and dysarthria. Ragged-red and cytochrome c oxidase (COX)-negative fibers, together with impaired activity of various mitochondrial respiratory chain complexes, were observed in muscle biopsies of affected subjects. Western blot analysis showed the virtual absence of RNase H1 in total lysate from mutant fibroblasts. By an in vitro assay, we demonstrated that altered RNase H1 has a reduced capability to remove the RNA from RNA-DNA hybrids, confirming their pathogenic role. Given that an increasing amount of evidence indicates the presence of RNA primers during mtDNA replication, this result might also explain the accumulation of mtDNA deletions and underscores the importance of RNase H1 for mtDNA maintenance.
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http://dx.doi.org/10.1016/j.ajhg.2015.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4572567PMC
July 2015

A nonsense mutation of human XRCC4 is associated with adult-onset progressive encephalocardiomyopathy.

EMBO Mol Med 2015 Jul;7(7):918-29

Molecular Neurogenetics Unit, Foundation IRCCS Institute of Neurology "Carlo Besta", Milan, Italy MRC Mitochondrial Biology Unit, CB2 0XY, Cambridge, UK

We studied two monozygotic twins, born to first cousins, affected by a multisystem disease. At birth, they both presented with bilateral cryptorchidism and malformations. Since early adulthood, they developed a slowly progressive neurological syndrome, with cerebellar and pyramidal signs, cognitive impairment, and depression. Dilating cardiomyopathy is also present in both. By whole-exome sequencing, we found a homozygous nucleotide change in XRCC4 (c.673C>T), predicted to introduce a premature stop codon (p.R225*). XRCC4 transcript levels were profoundly reduced, and the protein was undetectable in patients' skin fibroblasts. XRCC4 plays an important role in non-homologous end joining of DNA double-strand breaks (DSB), a system that is involved in repairing DNA damage from, for example, ionizing radiations. Gamma-irradiated mutant cells demonstrated reduction, but not abolition, of DSB repair. In contrast with embryonic lethality of the Xrcc4 KO mouse, nonsense mutations in human XRCC4 have recently been associated with primordial dwarfism and, in our cases, with adult-onset neurological impairment, suggesting an important role for DNA repair in the brain. Surprisingly, neither immunodeficiency nor predisposition to malignancy was reported in these patients.
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http://dx.doi.org/10.15252/emmm.201404803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520657PMC
July 2015

A novel mutation in TTC19 associated with isolated complex III deficiency, cerebellar hypoplasia, and bilateral basal ganglia lesions.

Front Genet 2014 14;5:397. Epub 2014 Nov 14.

Unit of Molecular Neurogenetics, Foundation IRCCS Istituto Neurologico Carlo Besta Milan, Italy.

Isolated complex III (cIII) deficiency is a rare biochemical finding in mitochondrial disorders, mainly associated with mutations in mitochondrial DNA MTCYB gene, encoding cytochrome b, or in assembly factor genes (BCS1L, TTC19, UQCC2, and LYRM7), whereas mutations in nuclear genes encoding cIII structural subunits are extremely infrequent. We report here a patient, a 9 year old female born from first cousin related parents, with normal development till 18 months when she showed unsteady gait with frequent falling down, cognitive, and speech worsening. Her course deteriorated progressively. Brain MRI showed cerebellar vermis hypoplasia and bilateral lentiform nucleus high signal lesions. Now she is bed ridden with tetraparesis and severely impaired cognitive and language functions. Biochemical analysis revealed isolated cIII deficiency in muscle, and impaired respiration in fibroblasts. We identified a novel homozygous rearrangement in TTC19 (c.213_229dup), resulting in frameshift with creation of a premature termination codon (p.Gln77Argfs*30). Western blot analysis demonstrated the absence of TTC19 protein in patient's fibroblasts, while Blue-Native Gel Electrophoresis analysis revealed the presence of cIII-specific assembly intermediates. Mutations in TTC19 have been rarely associated with mitochondrial disease to date, being described in about ten patients with heterogeneous clinical presentations, ranging from early onset encephalomyopathy to adult forms with cerebellar ataxia. Contrariwise, the biochemical defect was a common hallmark in TTC19 mutant patients, confirming the importance of TTC19 in cIII assembly/stability. Therefore, we suggest extending the TTC19 mutational screening to all patients with cIII deficiency, independently from their phenotypes.
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http://dx.doi.org/10.3389/fgene.2014.00397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231952PMC
December 2014

Novel DYT11 gene mutation in patients without dopaminergic deficit (SWEDD) screened for dystonia.

Neurology 2014 Sep 22;83(13):1155-62. Epub 2014 Aug 22.

From the Parkinson Institute (R.C., G.S., D.V., G.P., S.G.), Istituti Clinici di Perfezionamento, Milan; Unit of Molecular Neurogenetics (C.R., A.N., C.B., B.G.), Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan; IRCCS Santa Maria Nascente Fondazione Don Gnocchi Milano (A.C., A.M.), Milan; Dipartimento di Bioscienze (M.M.-F., M.G.), Università degli Studi di Milano, Milan; Nuclear Medicine (G.M.), IRCCS-Ospedale Maggiore, Milan; and Department of Neurosciences (D.V.), S. Gerardo Hospital, University of Milano-Bicocca, Monza, Italy.

Objective: To test the hypothesis that adult-onset primary dystonia may be the underlying etiology of tremulous patients with clinical diagnosis of Parkinson disease (PD) but without evidence of dopaminergic deficit at nigrostriatal SPECT imaging.

Methods: We retrospectively reviewed clinical and imaging data of patients with clinical diagnosis of PD assessed at our tertiary movement disorder clinic, who underwent dopamine transporter SPECT imaging consecutively between 2002 and 2011. Molecular screening for DYT1, DYT5, DYT6, DYT11, and DYT16 dystonia genes was performed in all cases who met the following criteria at the time of SPECT scan: (1) clinical diagnosis of PD; (2) normal dopamine transporter SPECT; (3) asymmetric rest tremor, with or without postural/kinetic component; (4) ≥ 12-month follow-up; and (5) normal brain MRI. We excluded subjects with (6) overt dystonic features, and (7) head or voice tremor.

Results: Twenty-three subjects were eligible for molecular analysis. Positive family history for tremor or PD was present in 45% of probands. We found one patient with a novel heterozygous frameshift mutation in the DYT11 gene (c.1058-1062 delCACCA/p.Gln352fsX376). Electrophysiologic study of tremor revealed that the main contributor was 5- to 6-Hz pseudo-rhythmic myoclonus, primarily involving extensor muscles. In 2 brothers, we found a missense variant in the DYT5 gene (c.334A>G; p.Thr112Ala) of uncertain pathogenicity in humans.

Conclusion: Our findings provide further support to the hypothesis that adult-onset monogenic dystonia may underlie a "PD look-alike" clinical phenotype. In addition to dystonic tremor, pseudo-rhythmic myoclonus may be mischaracterized as "rest tremor."
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http://dx.doi.org/10.1212/WNL.0000000000000821DOI Listing
September 2014

VARS2 and TARS2 mutations in patients with mitochondrial encephalomyopathies.

Hum Mutat 2014 Aug 24;35(8):983-9. Epub 2014 Jun 24.

Unit of Molecular Neurogenetics, Fondazione IRCCS (Istituto di Ricovero e Cura a Carattere Scientifico) Istituto Neurologico "Carlo Besta", Milan, Italy.

By way of whole-exome sequencing, we identified a homozygous missense mutation in VARS2 in one subject with microcephaly and epilepsy associated with isolated deficiency of the mitochondrial respiratory chain (MRC) complex I and compound heterozygous mutations in TARS2 in two siblings presenting with axial hypotonia and severe psychomotor delay associated with multiple MRC defects. The nucleotide variants segregated within the families, were absent in Single Nucleotide Polymorphism (SNP) databases and are predicted to be deleterious. The amount of VARS2 and TARS2 proteins and valyl-tRNA and threonyl-tRNA levels were decreased in samples of afflicted patients according to the genetic defect. Expression of the corresponding wild-type transcripts in immortalized mutant fibroblasts rescued the biochemical impairment of mitochondrial respiration and yeast modeling of the VARS2 mutation confirmed its pathogenic role. Taken together, these data demonstrate the role of the identified mutations for these mitochondriopathies. Our study reports the first mutations in the VARS2 and TARS2 genes, which encode two mitochondrial aminoacyl-tRNA synthetases, as causes of clinically distinct, early-onset mitochondrial encephalopathies.
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http://dx.doi.org/10.1002/humu.22590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140549PMC
August 2014

The isolated carboxy-terminal domain of human mitochondrial leucyl-tRNA synthetase rescues the pathological phenotype of mitochondrial tRNA mutations in human cells.

EMBO Mol Med 2014 02 10;6(2):169-82. Epub 2014 Jan 10.

Department of Radiology, Oncology and Pathology, Sapienza University of Rome, Rome, Italy.

Mitochondrial (mt) diseases are multisystem disorders due to mutations in nuclear or mtDNA genes. Among the latter, more than 50% are located in transfer RNA (tRNA) genes and are responsible for a wide range of syndromes, for which no effective treatment is available at present. We show that three human mt aminoacyl-tRNA syntethases, namely leucyl-, valyl-, and isoleucyl-tRNA synthetase are able to improve both viability and bioenergetic proficiency of human transmitochondrial cybrid cells carrying pathogenic mutations in the mt-tRNA(Ile) gene. Importantly, we further demonstrate that the carboxy-terminal domain of human mt leucyl-tRNA synthetase is both necessary and sufficient to improve the pathologic phenotype associated either with these "mild" mutations or with the "severe" m.3243A>G mutation in the mt-tRNA(L)(eu(UUR)) gene. Furthermore, we provide evidence that this small, non-catalytic domain is able to directly and specifically interact in vitro with human mt-tRNA(Leu(UUR)) with high affinity and stability and, with lower affinity, with mt-tRNA(Ile). Taken together, our results sustain the hypothesis that the carboxy-terminal domain of human mt leucyl-tRNA synthetase can be used to correct mt dysfunctions caused by mt-tRNA mutations.
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http://dx.doi.org/10.1002/emmm.201303198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3927953PMC
February 2014