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.

Table 1 Summary of clinical features of patients with MSTO1 mutations

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.

Fig. 1
figure 1

Brain MRI of patients. Brain magnetic resonance imaging (MRI) of patient 1 (a, b) and patient 2 (c, d). In patient 1, sagittal fluid-attenuated inversion recovery (FLAIR) imaging at 3 years (a) and sagittal T1-weighted imaging (T1WI) at 8 years (b) show mild cerebellar atrophy. In patient 2, sagittal T1WI at 7 months (c) and 2 years (d) show mildly progressive cerebellar atrophy

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).

Fig. 2
figure 2

Familial pedigrees and schematic representation of MSTO1 and its mutations. (a) Familial pedigrees and MSTO1 mutations. Mutations were annotated based on NM_018116.3. (b) Schematic representation of the MSTO1 protein (570 amino acids) with MSTO1 mutations (lower) and numbers of registered variants in ExAC (upper). In the lower panel, orange and sky-blue ovals are functional domains, namely, Misato segment II tubulin-like domain (6–120 amino acids (aa)) and Tubulin domain (153–346 aa), respectively. Domains were predicted using pfam (http://pfam.xfam.org/). Black and blue arrows indicate reported mutations with dominant inheritance and recessive inheritance, respectively. Red arrows indicate novel recessive mutations in this study. In the upper panel, blue bars in the graph indicate the numbers of nonsynonymous missense variants registered in the ExAC database at every 10 amino acids. Red bars are counts of null variants (nonsense mutations and small deletions/insertions leading to frameshift) in ExAC. Detailed information of variants registered in ExAC is provided in Supplemental Table 3. MAF minor allele frequency

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).

Fig. 3
figure 3

Schematic presentation of the splicing aberration. Splicing aberration was confirmed by RT-PCR for mRNA extracted from lymphoblastoid cells. (a) Schematic representation of genomic and cDNA structure from exons 8–13 of MSTO1. A red circle with lightning indicates the position of the c.1099-1 G > A mutation. TA cloning of cDNA in the patient revealed that c.1099-1 G > A mutation causes abnormal splicing with delG at the very first G of wild-type exon 11. (b) Schematic of the wild-type and mutant transcripts. One-base-pair deletion (delG) is caused by aberrant recognition of the splicing acceptor site (highlighted yellow), leading to frameshift and early protein truncation (p.Val367Trpfs*2), even if aberrant cDNA escapes from NMD

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 patients with biallelic MSTO1 mutations. Our report provides valuable information on the consequences of MSTO1 mutations for human phenotypes.