Novel recessive mutations in msto1 cause cerebellar atrophy with pigmentary retinopathy

ABSTRACT Misato 1, mitochondrial distribution and morphology regulator (encoded by the _MSTO1_ gene), is involved in mitochondrial distribution and morphology. Recently, _MSTO1_ mutations

have been shown to cause clinical manifestations suggestive of mitochondrial dysfunction, such as muscle weakness, short stature, motor developmental delay, and cerebellar atrophy. Both

autosomal dominant and recessive modes of inheritance have been suggested. We performed whole-exome sequencing in two unrelated patients showing cerebellar atrophy, intellectual disability,

and pigmentary retinopathy. Three novel mutations were identified: c.836 G > A (p.Arg279His), c.1099-1 G > A (p.Val367Trpfs*2), and c.79 C > T (p.Gln27*). Both patients had compound

heterozygous mutations with a combination of protein-truncation mutation and missense mutation, the latter shared by them both. This survey of two patients with recessive and novel _MSTO1_

mutations provides additional clinical and genetic information on the pathogenicity of _MSTO1_ in humans. You have full access to this article via your institution. Download PDF SIMILAR

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MITOCHONDRIAL DNA MUTATIONS IN KOREAN PATIENTS WITH LEBER’S HEREDITARY OPTIC NEUROPATHY Article Open access 08 March 2024 A NOVEL MUTATION LOCATED IN THE INTERMEMBRANE SPACE DOMAIN OF

_AFG3L2_ CAUSES DOMINANT OPTIC ATROPHY THROUGH DECREASING THE STABILITY OF THE ENCODED PROTEIN Article Open access 15 August 2022 INTRODUCTION Mitochondria are present in most somatic cells

(except for red blood cells) and important in a wide range of cellular processes, such as the production of cellular ATP, calcium homeostasis, and apoptosis [1, 2]. Mitochondrial diseases

are characterized by multiple-organ involvement, especially in tissues and organs with high energy demands. Cerebellar ataxia, peripheral neuropathy, muscle weakness, cognitive disorders,

epilepsy, and small stature have been clinically reported to be common neurological symptoms in mitochondrial diseases [1]. Defects in 36 mitochondrial and 245 nuclear genes have been

reported to be causative of human mitochondrial diseases [2] for which all inheritance patterns (mitochondrial, X-linked, autosomal dominant, and recessive) have been identified. [3]. Misato

1, mitochondrial distribution and morphology regulator, a human ortholog of misato in _Drosophila melanogaster_, is encoded by _MSTO1_ at 1q22. _MSTO1_ knockdown with siRNA caused

mitochondrial fragmentation in human HeLa cells and overexpression of recombinant _MSTO1_ induced the aggregation of mitochondria at the perinuclear region in COS-7 cells, indicating that

_MSTO1_ is critical for mitochondrial distribution [4]. Despite its significance for the integrity of mitochondrial functions, until recently, diseases involving _MSTO1_ mutation were

unknown in humans. However, two groups recently and independently reported that _MSTO1_ mutations caused clinical features reflective of mitochondrial dysfunction [5, 6]. Their inheritance

could be either autosomal dominant or recessive [5, 6]. Specifically, Gal et al. reported a heterozygous missense mutation in _MSTO1_, which caused muscle weakness, short stature, motor

developmental delay, and cerebellar atrophy. Functional analysis of the patients’ fibroblasts revealed that _MSTO1_ was involved in mitochondrial morphogenesis and maintenance by supporting

mitochondrial fusion [5]. Meanwhile, Nasca et al. reported biallelic mutations in _MSTO1_, which caused myopathy, optic atrophy, ataxia, serum creatinine kinase (CK) elevation, and severe

cerebellar hypoplasia [6]. Here, we present novel biallelic mutations in _MSTO1_, which cause global developmental delay, cerebellar hypoplasia, and retinitis pigmentosa. The detailed

clinical features are described and genotype–phenotype correlations are discussed. MATERIALS AND METHODS PATIENTS A series of patients with cerebellar atrophy were collected. Detailed

clinical information was obtained by the clinicians examining the patients. The Institutional Review Board of Yokohama City University of Medicine approved the experimental protocols.

Informed consent was obtained from the patients’ guardians, in accordance with Japanese regulatory requirements. SAMPLE PREPARATION AND WHOLE-EXOME SEQUENCING Genomic DNA was isolated from

peripheral blood leukocytes using QuickGene 610 L (Wako, Osaka, Japan). It was then captured using the SureSelect Human All Exon v5 (50 Mb) or v6 (60 Mb) Kit (Agilent Technologies, Santa

Clara, CA, USA), and sequenced on an Illumina HiSeq2500 (Illumina, San Diego, CA, USA) with 101-bp paired-end reads. Exome data processing, variant calling, and variant annotation were

performed as previously described [7]. The average read depth of protein-coding regions ranged from 79.6× to 101.7×, and at least from 96.2 to 97.5% of target bases were sequenced by 10 or

more reads. Common single-nucleotide polymorphisms (SNPs) with minor allele frequencies ≥1% in dbSNP 137 and variants observed in more than 5 of our 575 in-house ethnically matched control

exomes were filtered out. Among the remaining rare variants, we focused on amino acid-altering or splicing-affecting variants. Particular attention was paid to mutations in known causative

genes associated with ataxia, cerebellar atrophy, and other neurodegenerative diseases. _MSTO1_ variants were confirmed by Sanger sequencing with an ABI PRISM 3500xl autosequencer (Life

Technologies, Carlsbad, CA, USA) of PCR products using genomic DNA from patients and their parents as a template. CELL CULTURE, RT-PCR, AND TA CLONING Lymphoblastoid cells derived from

patient 1 harboring c.1099 G>A were grown in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum, tylosin, and antibiotic–antimycotic solution in a 5% CO2

incubator. After incubation with dimethyl sulfoxide (as vehicle control) with/without 30 µM cycloheximide (Sigma) to prevent nonsense-mediated mRNA decay (NMD) for 4 h, total RNA was

extracted using RNeasy Plus Mini kit (Qiagen). Four milligrams of total RNA was subjected to reverse transcription using PrimeScript 1st strand synthesis kit with random hexamers (Takara).

cDNA was isolated from lymphoblastoid cells from a patient with a different disease (static encephalopathy of childhood with neurodegeneration in adulthood with _WDR45_ mutation) and the

current patient and PCR-amplified with specific primers (available on request). cDNA was cloned using TA-cloning kit (Takara). The statistical test was calculated by SPSS Statistics version

22.0 (IBM). RESULTS CLINICAL FEATURES Clinical features of the two patients are summarized together with previously described patients in Table 1. Patients 1 and 2 showed similar clinical

features. Both patients were born by cesarean section due to a transient non-reassuring fetal status by fetal heart beat monitoring, and showed small head circumference (31.5 cm (−1.4 S.D.)

in patient 1 and 30.5 cm (−2.7 S.D.) in patient 2) at birth. No perinatal episodes were recognized which could affect their development, but they showed severe growth impairment, hypotonia,

global (especially motor) developmental delay, and pigmentary retinopathy. Both patients showed triangular face and sunken eyes. Brain magnetic resonance imaging (MRI) of patients 1 and 2

showed cerebellar atrophy at 1 year and at 7 months of age, respectively. Patient 1 is a 13-year-old girl who initially showed motor delay at 5 months of age, although she controlled her

head at 3 months. Her hair was thick. She was able to sit and crawl at 1 year and 6 months and 2 years, respectively. She walked with an assisting instrument at 4 years. Her intellectual

development was mild (as she could talk), but her motor skills deteriorated (with loss of the ability to walk at 13 years). She suffered recurrent vomiting and diarrhea since infancy. Brain

MRI studies indicated cerebellar vermis and hemispheres atrophy at 1 year (data not shown, Fig. 1a, b, taken at the age of 3 years and 8 years), but no cerebral atrophy to date. Serum CK was

mildly elevated (430 IU/L) at 9 years of age. Ophthalmologic assessment including funduscopy revealed bilateral retinal pigmentary denaturation, severe hypermetropia, and esotropia. Patient

2 is a 3-year-old girl who showed hypotonia, multiple arthrogryposis, and difficulty taking nutrition orally at birth. Serum CK was 916 IU/L at 7 days of age. Transient arthrogryposis and

CK elevation were exhibited, but gradually improved until complete recovery at 2 years. She showed global developmental delay: head control at 11 months, sitting at 1 year and 8 months, and

pulling herself up and speaking meaningful words at 2 years. She could speak a few words but could not walk at the age of 3 years. Brain MRI showed atrophy of cerebellar vermis and

hemispheres at 7 months (Fig. 1c), and this atrophy had slightly progressed at 2 years of age (Fig. 1d). The ophthalmologic evaluation led to the possible diagnosis of pigmentary retinal

denaturation. GENETIC ANALYSIS Whole-exome sequencing in the patients revealed biallelic _MSTO1_ mutations. Although the probability of being loss-of-function (LoF) intolerant score of

_MSTO1_ is low (0.0418 based on the Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org/) database), the probability of being LoF intolerant for recessive models score is more

than 0.95 (0.952 based on the ExAC). Segregation of mutations was confirmed by Sanger sequencing of the DNA of the patients and their parents (Fig. 2a; Supplemental Fig. 1). The same

variant (c.836 G > A; p.Arg279His) was found in both families. The results of computational prediction tools for evaluating the c.836 G > A were inconsistent (Supplemental Table 1):

Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/) and MutationTaster (http://www.mutationtaster.org/) indicated that this variant was “possibly damaging” or “pathogenic,” respectively,

whereas SIFT (http://sift.jcvi.org/) indicated that it was “tolerated.” The other mutations were a canonical splice site substitution, c.1099-1 G > A, in patient 1 and a nonsense

mutation, c.79 C > T (p.Gln27*), in patient 2. In the ExAC records, c.836 G > A and c.1099-1 G > A are extremely rare and c.79 C > T is absent (Supplemental Table 1). c.1099-1 G 

> A is located in the acceptor site of intron 10 (Fig. 3a). Reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm aberrant splicing. c.1099-1 G > A resulted

in exon 11 being 1 bp shorter than that in wild-type cDNA (Fig. 3a), being predicted to cause early truncation of the protein (p.Val367Trpfs*2) (Fig. 3b). The intensity of signal peaks

after the splicing mutation increased in the presence of cycloheximide, suggesting that mutant mRNA had been subjected to NMD [8] (Supplemental Fig. 2). The pathogenicity classification of

mutations by American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines indicated that c.836 G > A (p.Arg279His) is likely

pathogenic, and that c.79 C > T (p.Gln27*) and c.1099 G > A (p.Val367Trpfs*2) are pathogenic. Detailed ACMG/AMP criteria are described in Supplemental Table 2. DISCUSSION To date, a

total of eight _MSTO1_ mutations have been reported (including the current mutations) in five unrelated patients: five missense mutations (four recessive and one dominant inheritance), one

nonsense mutation (recessive), and two splice site mutations (recessive) [5, 6]. Although various missense variants throughout _MSTO1_ are registered in ExAC (Fig. 2b; Supplemental Table 3),

most recessive missense mutations are clustered within or just after the tubulin-like domain. p.Val8Met and p.Gln27* are located within the segment II tubulin-like domain [9]. Several

mutations in genes involved in mitochondrial fission (_MEN2_, _OPA1_, _DNM1L_, and _MFF_) cause human neurological diseases: Charcot–Marie–Tooth disease type 2 A, optic atrophy 1, infantile

encephalopathy, and Leigh-like encephalopathy, respectively [10,11,12,13]. A previous study indicated that MSTO1 regulated mitochondrial fission and fusion dynamics, and was involved in

mitochondrial morphogenesis and maintenance [6]. It was expected that the diseases described above were caused by the abnormality of mitochondrial fission regulation. In two previous

studies, clinical features associated with recessive mutations were more severe and had an earlier onset than those associated with a dominant mutation [5, 6] (Table 1). We described two

patients with early-onset global developmental delay, motor developmental delay, growth impairment, and cerebellar atrophy without seizures, who had recessive mutations. These clinical

features were also described previously in patients with recessive _MSTO1_ mutations. In addition, cognitive deficiency was only recognized in our patients. Previous studies and this report

indicated that the cerebellum and limb muscles showed more susceptibility to impaired mitochondrial fission rather than the cerebral cortex and cardiac muscle [5, 6]. Mutations of _DNM1L_

and _OPA1_, which are known to be associated with mitochondrial fission, cause different human diseases [11, 13]. Inhibition of _Drp1_ (a mouse ortholog of human _DNM1L_) by transfecting a

dominant negative mutant _Drp1_ disturbs dendritic development in cultured Purkinje cells [11], suggesting that several mitochondrial fission proteins play important roles in cerebellar

development. Haploinsufficiency of _OPA1_ is related to human optic atrophy and the expression of _OPA1_ is most abundant in the retinal ganglion cells [13], indicating that the tissue

susceptibility can be associated with the expression level of genes. _MSTO1_ is expressed widely in various tissues based on BioGPS (http://biogps.org/) and GeneCard (www.genecards.org).

Therefore, _MSTO1_ mutations may lead to not only the disorder of cerebellum and skeletal muscles, but also other organs. Our unrelated patients share a missense mutation (c.836 G > A,

p.Arg279His), together with a truncating mutation: c.79 C > T (p.Gln27*) in patient 1 and c.1099-1 G > A in patient 2. Notably, one individual with homozygous c.836 G > A

(p.Arg279His) is registered in ExAC. This might be suggestive of residual MSTO1 function that is somehow retained by the missense variant (Supplemental Figure 3) [14]. Therefore,

homozygosity for p.Arg279His may occur among the individuals classified as controls (removed patients with severe pediatric disease). p.Arg279His only combined with a protein-truncating

mutation can cause an affected status, which is supported by the lack of individuals with homozygous protein-truncating variants in ExAC. In conclusion, we have described two unrelated

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  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the individuals and their families for their participation in this study. We also thank Nobuko

Watanabe and Mai Sato for technical assistance. We are also thanks Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. This work was supported by grants from

Research on Measures for Intractable Diseases, Comprehensive Research on Disability Health and Welfare, the Strategic Research Program for Brain Science (SRPBS), the Practical Research

Project for Rare/Intractable Diseases, the Initiative on Rare and Undiagnosed Diseases from the Japan Agency for Medical Research and Development, a Grant-in-Aid for Scientific Research on

Innovative Areas (Transcription Cycle) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Grants-in-Aid for Scientific Research (A and B), Grant-in-Aid for

Young Scientists (B), Challenging Exploratory Research from the Japan Society for the Promotion of Science, the fund for Creation of Innovation Centers for Advanced Interdisciplinary

Research Areas Program in the Project for Developing Innovation Systems from the Japan Science and Technology Agency, grants from the Ministry of Health, Labor and Welfare, and the Takeda

Science Foundation. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan Kazuhiro Iwama, Chihiro

Ohba, Satomi Mitsuhashi, Satoko Miyatake, Atsushi Takata, Noriko Miyake, Takeshi Mizuguchi & Naomichi Matsumoto * Department of Pediatrics, Graduate School of Medicine, Yokohama City

University, Yokohama, Japan Kazuhiro Iwama & Shuichi Ito * Department of Child Neurology, National Center Hospital, National Center of Neurology and Psychiatry, Tokyo, Japan Toru Takaori

 & Akihiko Ishiyama * Niigata Prefecture Hamagumi Medical Rehabilitation Center for Disabled Children, Niigata, Japan Ai Fukushima * Department of Pediatrics, Epilepsy Center,

Nishi-Niigata Chuo National Hospital, Niigata, Japan Jun Tohyama * Clinical Genetics Department, Yokohama City University Hospital, Yokohama, Japan Satoko Miyatake & Shuichi Ito *

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K., Takaori, T., Fukushima, A. _et al._ Novel recessive mutations in _MSTO1_ cause cerebellar atrophy with pigmentary retinopathy. _J Hum Genet_ 63, 263–270 (2018).

https://doi.org/10.1038/s10038-017-0405-8 Download citation * Received: 05 October 2017 * Revised: 04 December 2017 * Accepted: 10 December 2017 * Published: 16 January 2018 * Issue Date:

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