Beware of Leigh Syndrome Pedigrees with MT-ATP6 Mutation Whose Primary Diagnosis is Retinitis Pigmentosa

doi:10.21203/rs.3.rs-3477746/v1

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Beware of Leigh Syndrome Pedigrees with MT-ATP6 Mutation Whose Primary Diagnosis is Retinitis Pigmentosa

https://doi.org/10.21203/rs.3.rs-3477746/v1

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Maternally Inherited Leigh Syndrome (MILS) is a group of systemic diseases caused by defects in mitochondrial oxidative phosphorylation (OXPHOS). We aim to investigate the clinical and genetic characteristics of MILSpedigree with MT-ATP6 mutation who were first diagnosed with RP.

Methods:

This study included a family pedigree consisting of a proband with visual blurring symptoms, his mother with similar symptoms, and a father who was asymptomatic. The medical history was asked, and the best corrected visual acuity (BCVA), intraocular pressure, slit-lamp, wide-field fundus photography, optical coherence tomography (OCT), visual evoked potential (VEP) and whole exome sequencing were performed. In addition, brain Magnetic Resonance Imaging (MRI) and physical examination were performed for the mother and child.

Results:

Whole exon sequencing showed that the proband carried mitochondrial MT-ATP6 gene mutation. The proband's mother carried an 8993T>G heterozygous mutation, with a heterozygous mutation load of 86%, which was inherited from the proband's mother (heterozygous mutation load of 62%). This gene is the primary pathogenic gene of MILS. The patient was diagnosed with MILS after a complete physical examination and imaging examination.

Conclusions:

This study suggests that RP can be the cause of the first visit of MILS patients. Therefore, ophthalmologists should be alert to MILS when they find patients with clinical symptoms of the nervous system in the face of RP patients. MT-ATP6 mutation is one of the causes of RP, and its pathogenic mechanism has not been clearly explained. In the future, the research and intervention of MT-ATP6 mutation may provide a new direction for the diagnosis and treatment of MILS-related RP.

Retinitis pigmentosa

Pedigree study

APT6 mutation

Leigh Syndrome

whole exome sequencing

MILS, also known as subacute necrosis cerebrospinal disease, is characterized by neurological abnormalities, including developmental delay, psychomotor regression, ataxia, seizures, brain stem dysfunction, and peripheral neuropathy[1]. In addition, ocular manifestations may be accompanied by nystagmus, ophthalmic muscle paralysis, strabismus, optic atrophy, and loss of foveal reflex [2–4]. The typical clinical manifestations of patients are neurogenic myasthenia, ataxia, and retinitis pigmentosa (NARP), which can be caused by mutations in mitochondrial DNA and nuclear DNA. The incidence is about 1 in 40,000. At least seven missense mutations of MTATP6 have been found to cause MILS, including 8993T > C, 8993T > G, 9176T > C, 9185T > C, 8597T > C, 9035T > C, and 8936T > A. Patients with the m.8993T > G point mutation had earlier and more severe symptoms than patients with other point mutations of MT-ATP6 associated with MILS. Mitochondrially Encoded ATP Synthase Membrane Subunit 6(MT-ATP6) is part of the F0 domain of mitochondrial ATP synthase [5]. The most common mutation in this gene, m.8993T > G, results in the substitution of highly conserved leucine residues for arginine. Although its therapeutic mechanism is not fully explained, it is clear that the m.8993T > G mutation impairs ATP synthesis due to the abnormal assembly of ATP synthetase. Under electron microscopy, the researchers also observed the mitochondrial ultrastructure destruction of fibroblasts in MILS patients and the reduction of glycolysis capacity and reserves[6–13]. Because of the importance of MILS associated with MT-ATP6 mutation, actively exploring the pathogenesis of this disease and finding effective treatment methods become the key to diagnosing and treating this disease.

In mitochondrial diseases, the mutation load is closely related to clinical symptoms. Most patients with m.8993T > G variation and heterogeneous load less than 60% were asymptomatic. Patients with a heterogeneous load of 75%-90% showed clinical manifestations of NARP, while patients with a heterogeneous load of > 90% showed more severe dysregulated MILS. Existing studies mainly focus on the changes of neurological abnormalities in MILS patients, but do not provide a detailed description of the ocular manifestations, which may lead to insufficient understanding of the disease by ophthalmologists, missed diagnosis in clinical diagnosis and treatment, delay the treatment of patients, and affect the prognosis. Rebecca D. Ganetzky et al. analyzed 218 cases of MT-ATP6-associated MILS reported in the past, among which 72 cases with m.8993T > G mutation had 57 manifestations of RP[14], proving that eye manifestations in this disease can also be a common reason for patients to seek treatment. Therefore, ophthalmologists should be on high alert for MILS when dealing with RP patients and families with neurological changes.

In this study, a group of families with typical RP ocular manifestations were first diagnosed in ophthalmology. To explore the etiology and pathogenesis of patients, exon sequencing was used to detect the genetic information of this family, and the mutation of the MT-ATP6 gene was found, which usually causes MILS. At the same time, RP was only a pathological change in the eye. Therefore, the pathologic changes of MILS were confirmed by extraocular examination, avoiding the missed diagnosis of systemic diseases. Furthermore, it provides a new direction for diagnosing and treating this disease.

2.1 | Ethical compliance

This study followed the tenets of the Declaration of Helsinki. The study and procedures were approved by the Research Ethics Committee of First Hospital of Lanzhou University. This study protocol was reviewed and approved by Research Ethics Committee of First Hospital of Lanzhou University, approval number LDYYLL2023-72.

2.2 | Research object

A total of 3 MILS family members (2 males and 1 female) were admitted to the ophthalmology department of the First Hospital of Lanzhou University in June 2022. Their medical records and related examination reports were collected.

2.3 | Genomic DNA extraction

GenCap Deafness capture kit was used to isolate genomic DNA samples from peripheral blood. Quality control of the obtained DNA were measured with a NanoDrop 1000 spectrophotometer (Thermo Scientific).

2.4 | Sequence analysis

The sequencing was performed using Illumina NovaSeq 6000(San Diego, CA). First, sequences were aligned to the UCSC hg19 human reference genome using BWA. Next, the frequency of corresponding mutations was detected by GATK HaplotypeCaller, then using GATK VariantFiltration to filter variants, and the variants were prioritized based on the following filtering criteria: i) variants with mapping qualities < 30; ii) the Total Mapping Quality Zero Reads < 4; iii) approximate read depth < 5; iv)QUAL < 50.0; v) phred-scaled p-value using Fisher's exact test to detect strand bias > 10.0. Then the data would be transformed to VCF format; variants were further annotated by ANNOVAR and associated with multiple databases, such as 1000 genome, ESP6500, dbSNP, EXAC, Inhouse(MyGenostics), HGMD, and predicted by SIFT, PolyPhen-2, MutationTaster, GERP++. In this course, five steps were used to select the potential pathogenic mutations in downstream analysis: (i) Mutation reads should be more than 5, mutation ratio should be no less than 30%; (ii) Removing the mutation, the frequency of which showed more than 5% in 1000g, ESP6500 and Inhouse database; (iii) If the mutations existed in InNormal database (MyGenostics), then dropped; (iv) Removing the synonymous. (v) After (i),(ii),(iii), if the mutations were synonymous and they were reported in HGMD, left them. When finished above jobs are done, the mutations which were left should be the pathogenic mutations.

3.1｜Clinical characteristics

The proband, a 39-year-old male, came to the first hospital of Lanzhou university's ophthalmology department complaining of poor night vision developed 22 years ago, followed by significant vision loss eight years ago in both eyes. Best-corrected visual acuity (BCVA) was HM/20cm in both eyes. Intraocular pressure (IOP) was 15 mmHg in both eyes. Anterior segment examination of both eyes was unremarkable. Color fundus photographs (Nikon Healthcare Japan, Japan) showed a waxy yellow optic disc with atrophy, arteriolar stenosis, and osteocyte-like pigmentation in the mid-periphery in both eyes （Figs. 1A, 1B). Spectral-domain optical coherence tomography (OCT) (Heidelberg Engineering, Germany) revealed the macular area and peripheral retina of both eyes became thinner, the outer retinal was atrophic, and the pigment epithelium was locally elevated（Figs. 2A, 2B). Flash-VEP (Roland RETIpor, Germany) showed prolonged peak duration and decreased amplitude at 8HZ in both eyes. In addition, four years ago, he began to develop systemic symptoms, such as unsteady walking and shaking hands. Physical examination revealed ataxia and positive signs of bilateral pathology. Brain magnetic Resonance Imaging（MRI）showed evident atrophy of the cerebellum and cerebrum（Figs. 3A, 3B）.

The mother of the proband, a 64-year-old female, complained of vision deterioration in both eyes for over ten years. Best-corrected visual acuity (BCVA) was HM/20cm in both eyes. Intraocular pressure (IOP) was 13 mmHg on the right and 15.5 mmHg on the left. Anterior segment examination of both eyes showed opacification of the crystalline lens. Color fundus photography, Spectral-domain optical coherence tomography (OCT) showed the same as the proband（Figs. 1C, 1D,2C,2D). Flash-VEP showed prolonged peak duration and decreased amplitude at 1HZ in both eyes. The patient had not been aware of systemic symptoms other than ocular symptoms. However, a physical examination revealed ataxia and positive signs of bilateral pathology. Brain MR images showed that her cerebellar atrophy was less severe than the proband's, and cerebral atrophy was not obvious（Figs. 3C, 3D）.

The proband's father, a 68-year-old male, has no ocular symptoms. Best-corrected visual acuity (BCVA) was 0.8 in both eyes. Intraocular pressure (IOP) was 20 mmHg on the right and 15 mmHg on the left. Anterior segment examination of both eyes showed opacification of the crystalline lens. Fundus examination revealed no abnormalities.

3.2｜Identification of mutations in MT-ATP6

Exome sequencing revealed that the proband carried mitochondrial MT-ATP6 gene hetero-variant M. 8993T>G. The proband's mother had the same mutation, and his father showed a normal phenotype. This pedigree was inherited maternally（Figs. 4A, 4B）.

This mutation is a classical pathogenic variant affecting ATP synthesis by reducing ATP synthase assembly, causing systemic diseases due to defective mitochondrial oxidative phosphorylation. However, the neuropathy, ataxia and retinitis pigmentosa caused by MT-ATP6 gene pathogenic variants were broadly consistent with the current clinical manifestations of the patient, which was reported as a pathogenic variant in the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar).

MILS is a severe neurodegenerative disease caused by defective oxidative phosphorylation of mitochondria, which can lead to systemic changes, including RP. Still, it mainly manifests as abnormalities of the nervous system, so patients are often treated in the neurology department. However, if the patient is admitted to the ophthalmology department for vision loss and diagnosed as RP after an ophthalmic examination, the ophthalmologist often ignores the systemic conditions outside the ophthalmic symptoms, delaying the treatment and affecting the prognosis. In this study, a family of patients with "vision impairment" admitted to the ophthalmology clinic was diagnosed with MILS after gene sequencing and neurological consultation. Compared with previous studies, we gave a detailed description of their ocular manifestations and conducted a comprehensive examination, which has important reference significance for future diagnoses of such patients by ophthalmologists. The occurrence of MILS in adult patients is rare and can be hidden. Different neurological syndromes may gradually appear, including typical retinitis pigmentosa, ataxia, epilepsy, mental retardation, developmental delay, etc. [15, 16].In addition to neurological symptoms, cardiac, renal, gastrointestinal, endocrine and other system abnormalities may also occur, so early diagnosis is difficult. Because the systemic manifestations of adult patients in our family were relatively hidden, they did not attract patients' attention. MILS was found after visiting the ophthalmology department after vision loss, reminding us that we should pay attention to the systemic manifestations of RP patients in the future diagnosis and treatment process.

The diagnostic criteria for Leigh syndrome were first proposed by Rahman et al. in 1996 and revised twice in 2014 and 2016[17–20]. The diagnosis of Leigh syndrome requires a combination of the following three criteria: (1)retardation and/or regression of mental and motor development; (2)Imaging changes of the symmetrical brainstem and/or basal ganglia with corresponding clinical manifestations; (3)Defects in the activity of oxidative phosphorylation pathways or pyruvate dehydrogenase complexes, genetic mutations for mitochondrial dysfunction, or elevated lactate in blood or cerebrospinal fluid[21]. However, the brain stem and basal ganglia region in the imaging examination of this pedigree were normal, which did not fully accord with Rahman's diagnostic criteria. Previous reports also found that brain MRI of a small number of MILS patients with MT-ATP6 mutation could show manifestations of cerebral and cerebellar atrophy, consistent with this pedigree's imaging findings [22, 23]. At the same time, the patient had both ataxia and MT-ATP6 gene mutations with mitochondrial dysfunction. Patients with gene mutations and clinical manifestations consistent with mitochondrial dysfunction, which were different from the diagnostic criteria, reminded us that even if they did not fully meet the diagnostic criteria, they still needed to consider whether they were atypical manifestations of the disease.

Martine Uittenbogaard et al. found significant mitochondrial ridge defects in fibroblasts of MILS patients with MT-ATP6 mutations and reduced glycolytic capacity and reserve in the patient's fibroblasts, as well as impaired ability to switch to glycolytic after complete inhibition of OXPHOS activity[24]. In addition, an autopsy of MILS children with transmission electron microscopy showed a large number of dilated mitochondria in cells, especially in retinal pigment epithelium, non-pigmented ciliary epithelium and corneal endothelium. Such morphological changes were not common in simple RP[25]. It may have some suggestive significance for studying the pathogenesis of MILs-related RP, which indicates the abnormal structure and function of mitochondria in the cells of MILS patients with MT-ATP6 mutation. Normal rod cells are rich in mitochondria, so the metabolism of the outer retina is active. When the outer retina consumes oxygen, the oxygen of the inner retina diffuses to the outer retina along the concentration gradient. However, when RP occurs, the rod cells may have abnormal metabolism due to the mutation of the MT-ATP6 gene, and the patient shows the symptom of night blindness. Meanwhile, due to abnormal metabolism and reduced oxygen consumption of the outer retina, oxygen in the inner retina cannot spread to the outer layer with a high oxygen environment. The visual impairment or even blindness of patients may be caused by the high oxygen environment of the inner retina, which leads to Oxidative stress and nitrosative stress damage of proteins, lipids and DNA. Finally, the outer layer of the retina is damaged and the cone cells die[26].

There is no specific treatment for RP caused by MILS, and maintaining the patient's sustained energy metabolism balance may be the goal of treating this disease. The primary treatment of this disease is mainly thiamine therapy and a ketogenic diet[27–29]. With the development of stem cell therapy, correcting mutations at the gene level has also become a meaningful solution. Giovanni Manfredi et al. used allotopic expression to transfer mitochondrial DNA encoding gene MT-ATP6 into the nucleus[15, 17]. The proven transfected cells' recovery ability and ATP synthesis were significantly improved after growth in a selective medium[16]. Their research demonstrated the possibility of gene modification to treat mitochondrial diseases. Correction of mutated gene loci by stem cell therapy has become an important research direction. At the same time, proper prenatal diagnosis is necessary due to the heritability of mitochondrial diseases such as MILS.

In conclusion, the significance of this study is that we should not ignore the systemic symptoms of some RP patients except in ophthalmology, especially if the patients have ataxia or neurological symptoms, MILS should be suspected and sent to the neurology department for examination in time. However, the number of patients in this family was limited in this study. In the future, more MILS family patients could be further studied in cooperation with the neurology department to clarify the pathogenic mechanism of MT-ATP6 gene mutation. Intervention and treatment could be carried out through gene correction and stem cell technology.

Ethics approval and consent to participate

This study protocol was reviewed and approved by Research Ethics Committee of First Hospital of Lanzhou University, approval number LDYYLL2023-72. Written informed consent was obtained from all individual participants included in the study.

Consent for publication

Written informed consent was obtained from the patient for the publication of this report and any accompanying images.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Competing interests

The authors declare that they have no conflicts of interest that pertain to this work.

Funding Sources

This study was supported by Health industry management project of Gansu Province under No. GSWSKY-2019-93, the Academic Fund from the Frist Hospital of Lanzhou University under Grant No. ldyyn2018-22.

Author Contributions

Yike Li: Conceptualization, Methodology, Writing Original Draft, Project administration
Yang Liu: Conceptualization, Writing Review and Editing, Supervision, Funding acquisiti Data Availability Statement
Ziyu Du: Ophthalmologic examination, Writing Original Draft；
Kaijin Zhou: Conceptualization, Supervision;
Bin Mao: Conceptualization, Genetics, Methodology;
Lu Zhang: Genetics, Methodology;
AYong Yu: Conceptualization, Writing Review and Editing, Supervision;

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No competing interests reported.

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Beware of Leigh Syndrome Pedigrees with MT-ATP6 Mutation Whose Primary Diagnosis is Retinitis Pigmentosa

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