Refining the phenotype of the THG1L (p.Val55Ala mutation)-related mitochondrial autosomal recessive congenital cerebellar ataxia

Abstract

Roughly 40 genes have been linked to autosomal recessive (AR) ataxia syndromes. Of these, at least 10 encode gene products localizing to the mitochondrion. tRNA-histidine guanylyltransferase 1 like (THG1L) localizes to the mitochondrion and catalyzes the 3′–5′ addition of guanine to the 5′-end of tRNA-histidine. Previously, three siblings with early onset cerebellar dysfunction, developmental delay, pyramidal signs, and cerebellar atrophy on brain magnetic resonance imaging (MRI) were reported to carry homozygous V55A mutations in THG1L. Fibroblasts derived from these individuals showed abnormal mitochondrial networks when subjected to obligatory oxidative phosphorylation. A carrier rate of 0.8%, but no THG1L V55A homozygotes, was found in a cohort of 3,232 unrelated Ashkenazi Jewish individuals, and no homozygotes were found in Exac or gnomAD. This variant is reported with an allelic frequency of 0.02% in Exac, and is not listed in gnomAD. A similar phenotype was recently reported for another, homozygous variant p.L294P was reported with a similar, but more severely affected phenotype [Shaheen et al. (2019); Genetics in Medicine 21: 545–552]. Here, we report two additional Ashkenazi Jewish patients, carrying the same homozygous V55A mutation. We present bioinformatic analyses of the V55A mutation demonstrating high conservation in metazoan species. We refine the clinical and radiological phenotype and discuss the uniqueness of the clinical course of this novel mitochondrial AR ataxia in comparison to the diverse molecular etiologies and clinical phenotypes of other known mitochondrial AR ataxias.

1 |. INTRODUCTION

tRNA-histidine guanylyltransferase 1 like (THG1L) catalyzes the 3′–5′ addition of guanine to the 5′-end of tRNA-histidine. First identified in yeast, THG1L protein was subsequently found to localize to the mitochondrion in HeLa cells. Expression of THG1L—also known as induced in high glucose-1—is transcriptionally upregulated in cultured renal mesangial cells exposed to high glucose and in patients with diabetic nephropathy (Murphy et al., 2008). Several lines of evidence support a mitochondrial role for THG1L. THG1L shRNAi knock down cells show decreased Nuclear respiratory factor-1 (NRF-1), Cytochrome b (cyto B), and ATP synthase subunit 6 (Atp6) expression, as well as decreased Tfam activity (Hickey et al., 2011). Renal-derived HK2 cells expressing doxycycline-inducible IGH1 shRNAi constructs show diminished baseline oxygen consumption rates and reserve capacity; and exogenous overexpression THG1L results in increased numbers of fused mitochondria in HeLa cells (Hickey et al., 2011).

Recently, three siblings from a single Ashkenazi Jewish family were reported to carry homozygous c.164T>C, p.V55A mutations in THG1L. All three individuals displayed cerebellar ataxia, dysarthria, developmental delay, pyramidal signs, and cerebellar hypoplasia versus atrophy on brain magnetic resonance imaging (MRI). The p.V55A variant is reported with an allelic frequency of 0.0001669 in Exac with no homozygotes. The authors calculated a carrier rate of 0.8% in the Ashkenazi population based on a cohort of 3,232 unrelated Ashkenazi individuals. There were no homozygotes in either group. While subtle phenotypes were demonstrated in cultured cells, including abnormal mitochondrial networks in patient fibroblasts subjected to obligatory oxidative phosphorylation (by culture in glucose-free media), enzyme activity assayed using recombinant protein was not affected in vitro (Edvardson et al., 2016). In total, per American College of Medical Genetics (ACMG) 2015 criteria (Richards et al., 2015), the presented data would qualify as “strong” or “moderate” evidence of pathogenicity. Of note, work from another group has also identified a similar phenotype in a patient with a different THG1L allele (c.881T>C, p.(L294P)) (Shaheen et al., 2019), though it is unclear whether this variant was homozygous or heterozygous.

2 |. PATIENTS AND METHODS

2.1 |. Patient 1

Patient 1 is now a 4-year-old girl. She is the first child and first pregnancy (conceived by follistim injection) for her unrelated American parents, who are of Ashkenazi Jewish heritage. The pregnancy was unremarkable. She was born by spontaneous vaginal delivery at 39 weeks and 6 days gestation with a birthweight of 5 pounds 15 oz and noted to be vigorous at birth.

She first came to medical attention due to concern for motor, and to a lesser extent, speech/articulation delays. She did not crawl until age 13 months and did not walk independently until age 22 months, being noted from that time to have an unsteady gait. At age 3 years, she climbed stairs alternating with hand held or holding rail and could not yet run or ride a tricycle. At age 3–4 years she has some limited use of spoon and fork, feeds herself well with finger foods. She has had no clear language or social delays. She has never had any loss of developmental milestones or regressions with illness. She has strabismus that has been treated with patching and surgical correction at age 4 years. A modified barium swallow study at age 4 years obtained for concern of coughing with feeds was normal.

Brain MRI at age 3 years revealed a small cerebellum with prominent cerebellar and vermian fissures concerning for white matter and cortical gray matter loss, though hypoplasia could not be excluded. A slightly bulbous appearance of the brainstem was also noted (Figure 1a,b). An electroencephalogram (EEG) obtained for staring spells at the same age showed no frank abnormalities. Neonatal hearing screen was normal.

FIGURE 1.

(a) Magnetization Prepared RApid Gradient Echo I (MP RAGE) sagittal and (b) T2 coronal of Patient 1 brain MRI at the age of 3 years reveal a small cerebellum with prominent cerebellar and vermian fissures concerning for white matter and cortical gray matter loss versus hypoplasia. A slightly bulbous appearance of the brainstem was also noted. (c) T2 weighted sagittal and (d) T2 weighted coronal of Patient 2 brain MRI at the age of 4.3 years demonstrate enlarged cerebellar interfolial spaces with more severe vermian involvement; normal brainstem, basal ganglia and cerebral cortex, and age-appropriate white matter myelination

Detailed neuropsychological assessment at age 4 years 2 months demonstrated general cognitive abilities within expectations for age, with relative vulnerabilities identified in more elaborate language processing and expressive language output. Motor and visuomotor skills generally fell well below expectations for age, though contributions from motor domain and visual challenges were clearly important contributors.

Family history includes a younger brother with torticollis at 9 months of age, a history of extraocular muscle abnormalities requiring surgical correction at age 3 years (precise diagnosis unknown) in the proband’s father, as well as a blighted ovum an anencephalic and a full-term child born to the maternal grandparents. Both parents are of Ashkenazi Jewish heritage (Polish and Lithuanian).

Initial physical examination at age 3 years revealed no dysmorphic features, birthmarks, or somatic abnormalities. Weight was 10th percentile, height 25th percentile, and head circumference 75th percentile. On neurologic exam, she responded to examiner questions in single words and with pointing and followed single-step commands. Cranial nerve examination was notable for strabismus and rare square wave jerks of one eye. She sat well unassisted and was unable to climb into a chair without assistance. Motor, sensory, and reflex exams were normal. Bilateral dysmetria of the upper extremities, truncal titubation on standing, and an unsteady, wide-based gait with mild foot “slapping” were noted.

Extensive clinical laboratory evaluations were undertaken. Plasma amino acid and urine organic acid analyses were normal. Chromosomal microarray revealed the following a copy number variant 95–97 kilobase loss at 7q35: (145995885–146090384/145993894–146091353), occurring within an intron of CNTNAP2. Spinal muscular atrophy screening was negative. Parental screening for Tay Sachs, cystic fibrosis, familial dysautonomia, Mucolipidosis Type 4, glycogen storage disorders, maple syrup urine, and fragile X syndrome were negative prior to conception.

2.2 |. Patient 2

Patient 2 is a 4-year-old daughter of healthy, unrelated Ashkenazi Jewish parents who presented at almost age 4 years for speech/articulation delay and balance difficulties. She was conceived by in vitro fertilization and born by Cesarean section due to breech presentation after an uneventful pregnancy. Development was normal prior to age 2 years when speech delay was first noted. The parents reported gait instability and frequent falls. Her gait worsened during febrile diseases, fatigue, and distraction. Motor, speech, and cognitive skills otherwise progressed without regression. By age 3 years, social skills were felt to be age appropriate. She currently attends a special education school.

On examination at initial presentation, the patient was non-dysmorphic with notably normal biometric parameters, including head circumference. She demonstrated age-appropriate receptive language and followed commands appropriately. She had mild horizontal nystagmus, slow smooth pursuit, severe dysarthria, generalized hypotonia, normal deep tendon reflexes, and nonsustained clonus in her lower limbs. Body and lower extremity tremors were observed on standing. Gait was wide based. She had mild dysmetria and hand tremor leading to graphomotor and fine motor difficulties.

An extensive evaluation was normal including blood lactic and pyruvic acids, amino acids, ammonia, pH, vitamins B1, B6, B12, and E, very long chain fatty acids (VLCFA), alpha-fetoprotein, immunoglobulins, biotinidase, urine amino and organic acid, and ophthalmological and hearing examinations.

Developmental assessment at the age of 4 years revealed a developmental quotient (Veitenhansl et al., 2004) of 75. Brain MRI at the age of 4 years 4 months demonstrated enlarged cerebellar interfolial spaces with more severe vermian involvement, normal brainstem, basal ganglia and cerebral cortex, and age-appropriate white matter myelination (Figure 1c,d).

Repeat examination 1 year following initial presentation revealed a communicative but inattentive and only partially cooperative child with selective mutism. She had brisk reflexes, nonsustained clonus, and a normal plantar response bilaterally. She was unable to cooperate with strength confrontation testing. Mild horizontal gaze evoked nystagmus, slow smooth pursuit, severe dysarthria with scanning speech, and upper extremity tremor and dysmetria were noted. Gait was ataxic with arm posturing. She had graphomotor difficulties.

Improvements in balance, verbal communication, and speech fluency were noted on repeat neurologic exams by the family and treating neurologist, speech, and physical therapists after 3 months of a therapeutic trial with ubiquinol, 30 mg/kg daily.

2.3 |. Exome sequencing

All testings performed for both patients fall within standard clinical care and were thus not performed under a research protocol. Clinical trio whole exome sequencing (WES) and mitochondrial DNA analysis were submitted from the blood samples of Patient 1 and her parents. Samples were processed using the Aligent Clinical Research Exome kit and sequenced by massively parallel sequencing on Illumina HiSeq system with 100 bp paired-end reads. Mean depth of coverage was 109× with a quality threshold of 96.8%.

2.4 |. Bioinformatics

Protein sequence alignments were performed using CLC Sequence Viewer Version 7.7 (https://www.qiagenbioinformatics.com/).

3 |. RESULTS

Trio WES demonstrated homozygous THG1L missense variants c.164T>C; p.V55A in Patient 1, with each parent being a heterozygous carrier. Full sequence analysis and deletion testing of mitochondrial DNA from the same specimen (proband only) were unable to be completed due to suboptimal quality and quantity of mitochondrial DNA obtained from specimen.

Informed by the previous report of Edvardson et al., targeted sequencing of THG1L was performed for Patient 2, revealing the homozygous c.164T>C; p.V55A missense mutation in the proband. Each parent was found to be heterozygous carrier of the same allele.

On our review and others, no c.164T>C homozygotes were reported in Exac or gnomAD (Calvert et al., 2017; Lek et al., 2016). Bioinformatic analysis using multisequence alignments shows striking conservation of the valine-55 residue through yeast (Figure SS1).

4 |. DISCUSSION

The finding of THG1L p.V55A homozygous Ashkenazi Jewish patients with strikingly similar presentations (Table 1) supports the existence of a THG1L-related autosomal recessive (AR) mitochondrial ataxia syndrome. Notably, Edvardson et al. (2016) have found a carrier rate of 0.8% but no THG1L V55A homozygotes in a cohort of 3,232 Ashkenazi Jewish individuals. This variant is reported with an allelic frequency of 0.02% in Exac, and is not listed in gnomAD (Calvert et al., 2017), suggesting that the THG1L p.V55A mutation can be categorized as a founder mutation in the Ashkenazi Jewish population. The experimental evidence of pathogenicity presented by Edvardson et al. can be considered sufficient to support a designation of strong evidence. These data are further supported by the multisequence alignments presented here, which demonstrate conservation of the valine-55 residue throughout metazoan species. Though further research will be required to prove pathogenicity of the variant, together these data support the designation of THG1L p.V55A syndrome as an AR ataxia syndrome.

TABLE 1.

Clinical features of THG1L patients

Kindred/patient/citation	Age at onset	Ataxia	Dysarthria	Motor delay	Pyramidal signs	Cognitive impairment	Opthalmologic findings	Seizure	Brain MRI
1/A/Walker et al. (Unpublished)	3 years, F	+	+/−	+	-	Very mild	Strabismus	-	Small cerebellum with prominent cerebellar and vermian fissures
2/A/Walker et al. (Unpublished)	3 years, 9 months, F	+	+	+	+	Mild	-	-	Enlarged cerebellar interfolial spaces with more severe vermian involvement
3/A/Shaheen et al. (2019)	18 months, M	U	U	U	U	Severe	Optic atrophy	+	Diffuse cerebral and cerebellar atrophy, diffuse thinning of the corpus callosum
4/A/Edvardson et al. (2016)	9 months, M	+	+	+	+	Mild	U		Vermian hypoplasia
4/B/Edvardson et al. (2016)	1.5 years, F	+	+	+	+	Mild	U		Vermian hypoplasia
4/C/Edvardson et al. (2016)	2.5 years, F	+	+	+	+	Very mild	U		Vermian hypoplasia

Abbreviations: M, male; F, female; U, unknown.

It remains unclear whether the p.L294P THG1L variant reported by Shaheen and colleagues constitutes a more severe phenotype of the same syndrome or an allelic disorder. Similar to many of the early onset AR mitochondrial ataxias, this patient was affected with a severe, multisystem disease with cardiac, gastrointestinal, hematologic abnormalities in addition to marked developmental delay, epilepsy, and cerebral atrophy in addition to cerebellar hypoplasia/atrophy.

Mitochondrial dysfunction is a frequent cause of cerebellar disorders. Of the roughly 40 known AR ataxia syndromes (which feature ataxia as a primary feature), 10 are linked to genes functioning in the mitochondrion (Bertini, Zanni, & Boltshauser, 2018) (Table S1). Although AR mitochondrial ataxias are a clinically heterogeneous group of disorders, the vast majority of patients demonstrate multisystem involvement and a progressive course characteristic of primary mitochondrial disorders—which by definition arise from genetic perturbations of oxidative phosphorylation—more broadly. Intriguingly, while none of the genetic associations reported to date involve structural components of the electron transport chain, deficiencies in multiple complexes of the electron transport chain have been demonstrated in a majority of AR mitochondrial ataxias (7 of 10) (Table S1).

Further research and reporting of the natural history of THG1L-related disease will be required to determine whether the phenotype is more consistent with a classical AR mitochondrial ataxia (multisystem disease and demonstrable oxidative phosphorylation defects) or whether it will be better described as a nonprogressive congenital cerebellar ataxia syndrome, with early onset and subsequent stabilization, which is more rarely seen in the larger AR mitochondrial ataxia group, specifically in alpha-mitochondrial processing peptidase-related disease (Bertini et al., 2018). Given the risk of the former, close monitoring of multiple systems—particularly cardiac, liver, and auditory functions—is warranted in these patients. We would therefore recommend genetic evaluation with at least a next-generation sequencing panel covering these genes and, preferably, WES in any child presenting with congenital ataxia, as positive genetic results would alert physicians to the need for surveillance of systems for treatable disease manifestations (cardiac, liver, etc.).

5 |. CONCLUSIONS

Homozygous p.V55A THG1L mutations present with a clear phenotype of congenital nonprogressive cerebellar ataxia and cerebellar hypoplasia versus atrophy within the broader spectrum of the AR mitochondrial ataxias. Unlike most mitochondrial disorders, all reported patients demonstrate monosystemic involvement without laboratory evidence of impairment in mitochondrial function. Intriguingly, a single reported case of homozygous p.L294P mutations presents with a more fulminant, multisystem disorder more reminiscent of classical oxidative phosphorylation disease. It is unclear whether this represents a spectrum of the same syndrome or an allelic disorder. While no case of THG1L p. V55A-associated ataxia, including those reported here, has been tested for oxidative phosphorylation activity, it remains possible that a defect is present. Further study will be required to understand how perturbations of mitochondrial function produce the associated spectrum of hereditary ataxia syndromes.