Novel mutations of the POLR3A gene caused POLR3-related leukodystrophy in a Chinese family: a case report

POLR3-related leukodystrophy, which includes hypomyelination, hypodontia, and hypogonadotropic hypogonadism (4H syndrome); ataxia, delayed dentition, and hypomyelination (ADDH); tremor-ataxia with central hypomyelination (TACH); leukodystrophy with oligodontia (LO), and hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum (HCAHC), is an autosomal recessive neurodegenerative disorder characterized by onset time ranging from the neonatal period to late childhood and a wide range of severities relating to many systems [1]. The primary clinical features include cerebellar symptoms (i.e., spasticity, ataxia, tremor, and cognitive regression); dental abnormalities (i.e., tooth delay, tooth agenesis, fewer teeth, and abnormal tooth form and arrangement), short stature, dysphagia, hypogonadotropic hypogonadism, and progressive eye abnormalities (e.g., myopia and optic atrophy) [1]. Some rare features have also been reported in other studies (Table 1) [1‐4]. Myopia is seen in almost all patients and short stature occurs in 50% of patients with POLR3-related leukodystrophy. However, dental issues, difficulty swallowing, endocrine features, and aberrant tooth and hormonal abnormities are not always present [2]. Systematic magnetic resonance imaging (MRI) revealed that the combination of hypomyelination with relative T2 hypointensity of the ventrolateral thalamus, optic radiation, globus pallidus, dentate nucleus, cerebellar atrophy, and thinning of the corpus callosum indicate POLR3-related leukodystrophy. Rare characteristics were found in other cases as well (Table 1) [4, 5]. MRI characteristics are the main supporting evidence for diagnosis of POLR3-related leukodystrophy, especially if classic non-neurological features are absent [2, 3, 6‐8].

POLR3-related leukodystrophy is caused by biallelic mutations in POLR3A, POLR3B, POLR1C, and POLR3K (through interaction with POLR3B) genes. These genes are responsible for encoding the two largest subunits of RNA polymerase III (Pol III), which has been hypothesized to be crucial for the synthesis of small RNAs, such as 5SrRNA and transfer RNAs (tRNAs). Mutations of these genes cause abnormal tRNA and non-coding RNA transcription in a cell type and growth state dependent manner, and can impact cellular growth, differentiation, and apoptosis [9, 10]. Patients with POLR3A mutations have a more severe disease course and an unfavorable prognosis compared to cases with POLR3B mutations [2]. For this reason, Bernard et al. hypothesized that POLR3A mutations lead to dysregulation of Pol III and its targets, resulting in decreased expression of certain tRNAs during development and impaired protein synthesis [11]. Previous studies have shown that 14 recessive mutations in the POLR3A gene were found in 19 French-Canadian, Caucasian, and Syrian individuals [11]. However, cases among the Chinese population are still unclear. Most published mutations of POLR3A associated with POLR3-related leukodystrophy [2, 6, 7, 9, 12] have focused on mutations that cause a change of amino acid; studies of splice site mutations and copy number variants are rare. In the present study, we report a female patient with a novel compound heterozygous mutation with an uncommon splice site mutation, c.1771-6C > G and c.2611del of POLR3A. The present study has expanded the current evidence concerning mutations associated with POLR3-related leukodystrophy.

The case was obtained from the Children’s Hospital of Soochow University. The parents were nonconsanguineous and both appeared normal. The little girl had a history of recurrent pneumonia and was the first birth of the parents with a full-term normal delivery and a birth weight of 3000 g. There was no history of asphyxia or injury in the parturition period. Her motor development before 6 months of age appeared to be normal. At 9 months old, she presented with reduced motor ability and required assistance to sit. At the same time, the patient started to show prominent cerebellar signs, including nystagmus, motor ataxia, dysarthria, and spastic tetraplegia. Delayed dentition and development figures, prominent body hair, and hypertonia of both the upper and lower limbs were also observed at 1 year of age. Two febrile seizures with fever occurred at the ages of 1.5 and 2 years. Before 2 years of age, she communicated with her families using facial expressions, gestures, and simple sounds as there were no visual or hearing impairments. When evaluated at the age of 2.5 years, she was admitted to hospital because of severe pneumonia for hyper-breath and poor appetite for 2 days, with aggravated symptoms for a half-day period. The patient underwent a careful physical examination. Short stature was found with a height of 80 cm (≤ − 3 SD), while nutrition and development were within the normal range with a body weight of 15 kg (+ 1 SD). She presented with dysarthria without simple word speaking. In addition, cognitive decline was apparent as she was sometimes not able communicate with her family and neuropsychologic testing also indicated a worsening of her global intelligence quotient (according to the Wechsler Intelligence Scale for Children-Revised, an intelligence quotient of 52 at that time). In addition, spastic tetraplegia, nystagmus, dysarthria, and motor disability were increasing in severity. She could not attain complete head control. Another striking observation was dysphagia. Gastro-esophageal reflux often occurs with tube feeding, indicating decreased visceral smooth muscle mobility. Body examination indicated nystagmus, hypodontia, polytrichia (Fig. 1a and b), ataxia, and spastic tetraplegia. In a previous brain image, we identified an extracerebral space widening at the age of 6 months (Fig. 1c) and further frontotemporal space widening at the age of 11 months with delayed myelination or hypomyelination of white matter in the focal area around the posterior horn of the bilateral lateral ventricles (Fig. 1d–f). Laboratory examination indicated that plasma ammonia, lactate, serum antibody tests for toxoplasma, rubella virus, cytomegalovirus, and herpes simplex virus (TORCH), vitamin B, trace elements, creatine kinase, and thyroid function were normal. Electroencephalogram and electrocardiogram results were negative. The value of auditory brain-stem responses was greater than the threshold line (50 dbnnl) (Table 2). Chest X-ray showed bilateral lung inflammation. Because of recurrent pneumonia, tracheobronchoscopy was performed and an orifice of the right middle bronchus was found to be absent (Fig. 1g), which was first observed in POLR3-related leukodystrophy. Genetic metabolic screening of blood and urine were performed twice and parameters were determined to be within normal range. The results of the abdomen ultrasound examination were negative. Fundus examination was normal without optic atrophy and cataract. Visual acuity was also measured and no myopia was found. The endocrinal profile was not detected because the patient was too young; data regarding motor conduction velocity was also not available. Conventional karyotype analysis revealed a normal 46 XX karyotype.

To achieve an accurate genetic diagnosis, medical exome sequencing was carried out with a Trio sample strategy. A peripheral blood sample was collected from the proband and her parents and genomic DNA was isolated using the High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The medical exome including coding regions and known pathogenic non-coding regions of over 4000 disease-related genes was captured before next-generation sequencing (Amcare Genomic Laboratory, Guangzhou, China). The potential pathogenic variants were filtered by bioinformatics analysis as described previously [12]. Sequencing of 50,902 genomic regions spread over 8,591,731 bp with an average coverage of 274+/− 164× was obtained; the coverage of 99.4% of the sequenced regions exceeded 10× and the coverage of 99.2% of the sequenced regions exceeded 20×. Further analysis revealed two novel mutations of POLR3A in the patient: c.1771-6C > G (NM_007055) adjacent to the mRNA splicing site and c.2611del, which results in early termination of translation (p.M871Cfs*8). The c.1771-6C > G mutation occurs at very low frequency in the population (< 0.001), while the c.2611del mutation is not listed in 1000 Genomes (The 1000 Genome Project Consortium) or The Genome Aggregation Database (gnomAD, Broad Institute). Co-segregation analysis confirmed that the two mutations were inherited from the heterozygous parents of the proband. The father was determined to be the carrier of the c.2611del (p.M871Cfs*8) mutation and the mother was determined to be the carrier of the c.1771-6C > G mutation. Collectively, we identified novel compound heterozygous mutations of the POLR3A gene that caused POLR3-related leukodystrophy in the patient combined with the clinical presentation, MRI brain pattern, and medical exome sequencing (Figs. 1 and 2).

Our case from the southern district of China displayed severe neurological manifestations and presented with typical childhood onset with various features such as cerebellar symptoms (spasticity and ataxia), cognitive regression, motor decline, and delayed dentition. Brain MRI indicated delayed myelination or hypomyelination of white matter in the focal area around the posterior horn of the bilateral lateral ventricles. Takanashi et al. reported that hypomyelination of the brain often indicates POLR3A mutation, which is associated with leukodystrophy disorders [6]. To verify this, we performed medical exome sequencing and found novel compound heterozygous mutations of the POLR3A gene, reminiscent of other patients. According to the clinical manifestations, we concluded the diagnosis and identified the compound heterozygous variants as the causative variants for the disease in this patient. It is noteworthy that this disease has mostly been reported in European populations, including French-Canadian, Caucasian, and Syrian individuals [2, 7]. Occasional cases have been reported in the Indian population [13‐15]. However, this is the first case reported in a Chinese family.

Neurological impairment of our case started in the infantile period with a decline in motor ability, cognitive impairment, and cerebellar features. Although cerebellar signs of this case became progressively obvious, cerebellar atrophy was not observed, which is likely related to the molecular basis or other factors. Previous studies have found that cerebellar anomalies were more severe in patients with POLR3B defects while the pattern of hypomyelinization was more evident in the MRI of patients with POLR3A mutations [2, 6]. This may be another explanation for our case. Our patient also showed classical extraneurologic features, characterized by hypodontia with delayed tooth eruption and short stature. She also displayed polytrichia, an atypical feature of POLR3-related leukodystrophy, which may be due to aberrant endocrine hormone levels or other reasons. Hypogonadotropic hypogonadism was not detected because she was too young. Previous studies have also shown that the syndrome may or may not be associated with hypodontia and/or hypogonadotrophic hypogonadism in many cases [8, 11]. The case did not show myopia and optic atrophy. This is inconsistent with most cases, which are usually accompanied by myopia [2]. Her dysphagia phenotype was striking. She had obvious difficulty with tube feeding and forceful vomiting occurred frequently. This is likely due to the incoordination of swallowing of cerebellar syndrome, or due to other unpredictable reasons. Bronchodysplasia is another feature first observed in POLR3-related leukodystrophy, suggesting that it was not recognized previously in the POLR3-related leukodystrophy spectrum. Thus, in addition to the classical extraneurological features, abnormal body hair and visceral smooth muscle features should be carefully looked for in patients with POLR3-related. When classical features do not exist, rare manifestations will a clue in the diagnosis of this disorder. Although there is no cure for this disease to date, treatment of manifestations such as seizures, hypogonadotropic hypogonadism, dystonia, and dysphagia can be managed on an individual basis for an improved quality of life and the prevention of complications.

Our case presented with severe manifestation at early onset and diverse manifestations among those of patients with POLR3-related leukodystrophy, which may be a result of the genotype identified in this patient; further analysis is necessary. To date, four genes (POLR3A, POLR3B, POLR1C, and POLR3K) have been reported to be associated with POLR3-related leukodystrophy [11, 16]. Most of the identified mutations are point mutations in the codon region; however, non-coding DNA variants are suspected to account for a substantial portion of undiscovered causes of rare diseases [17, 18]. Minnerop et al. identified mutations in deep intronic regions of POLR3A as a common cause of hereditary spastic paraplegia and cerebellar ataxia, and > 80% of POLR3A mutation carriers presented the same deep intronic mutation (c.1909 + 22G > A), which leads to a novel, distinct, uniform, and severe phenotype [17]. Jay et al. also reported alteration of mRNA splicing in POLR3A causing neonatal progeroid syndrome with severe clinical manifestations [23]. In this study, we identified the c.1771-6C > G (NM_007055) mutation adjacent to the mRNA splice site demonstrating that exploring non-coding genomic regions was helpful in revealing the causes of related hereditary diseases.