Perrault syndrome (PRLTS) is suspected in a female with bilateral sensorineural hearing loss (SNHL), ovarian dysfunction that may occur in a mild (primary ovarian insufficiency) or severe (ovarian dysgenesis) form and a normal 46,XX karyotype [1]. In males PRLTS is manifested by SNHL and their fertility is generally considered normal but this matter should be regarded with caution due to scarcity of males with PRLTS and data from the animal studies [2, 3]. SNHL is usually the initial expression of PRLTS. In some patients a spectrum of neurologic symptoms develops over time indicating a progressive, age-related neurodegenerative process.

Common neurological abnormalities are motor and sensory neuropathy, muscle weakness and atrophy, hypo- or areflexia, cerebellar ataxia, limited eye movements, nystagmus, dyspraxia, as well as intellectual deficit, developmental delay and seizures [4–7]. In brain magnetic resonance imaging (MRI), nonspecific white matter changes suggestive for cerebral leucodystrophy and cerebellar atrophy are described [5, 6, 8]. Depending on the occurrence of neurologic features, a classification of PRLTS into type I, static and without neurologic involvement and type II with a progressive neurological disease has been proposed [7].

Transmission of PRLTS is autosomal recessive and over the last years five genes causative for PRLTS have been identified. Mutations in HSD17B4 (OMIM *601860; PRLTS1), encoding a peroxisomal enzyme, were found in four PRLTS families [9–12]. Mutations in HARS2 (OMIM *600783; PRLTS2) were detected in three families [4, 13], mutations in LARS2 (OMIM *604544; PRLTS4) in six families [10, 13–15], mutations in CLPP (OMIM *601119; PRLTS3) in seven families [5, 10, 13, 16, 17], and mutations in TWNK (OMIM *606075; PRLTS5) only in four families with PRLTS [6, 10, 13]. All of the four latter PRLTS genes code for mitochondrial proteins. Molecular etiology for PRLTS remains unknown in as many as 55% of the patients, which emphasizes a genetic heterogeneity of the syndrome and shows that novel disease-causing genes still await discovery [10, 13].

Twinkle mtDNA helicase (TWNK), located in the long arm of chromosome 10 (10q24.31), encodes Twinkle, which localizes to mitochondrial nucleoids and forms a hexameric or heptameric ring structures. Twinkle is composed of a primase and helicase domain connected via a linker region, involved in stabilization of the oligomeric complexes of the protein. With the primase activity the protein initiates DNA replication and with the helicase activity unwinds DNA for replication. Twinkle is essential for the replication process of mitochondrial DNA (mtDNA) and lifetime maintenance of human mtDNA integrity [18, 19].

In this study, whole-exome sequencing (WES) was successfully applied to identify the molecular basis of PRLTS in the proband and her sister from a non-consanguineous Polish family. The cardinal manifestations of PRLTS (SNHL and ovarian dysgenesis) in the patients were preceded and dominated by severe and progressive involvement of the nervous system, making the diagnosis not straightforward. Here, we provide a thorough neurological and audiological assessment of both patients that unveil novel features on the phenotypic landscape of PRLTS.

Herein, we report the identification of a distinctive phenotype of PRLTS5 (OMIM #616138), in which the progressive neurologic features, dominating in the phenotype, preceded the diagnosis of SNHL and ovarian dysfunction. Comprehensive analysis of the patients’ phenotype and family history enabled us to establish a clinical suspicion of PRLTS. After applying WES the underlying cause of the disorder has been explained by the detection of compound heterozygous mutations in the TWNK. It is the most recently discovered gene involved in the pathogenesis of PRLTS that heretofore has been reported only in four PRLTS families worldwide.

Pathogenic role of heterozygous TWNK mutations have been first discovered in families with autosomal dominant progressive external ophthalmoplegia (PEOA3; OMIM #609286) [18]. Recessive TWNK mutations are causative for mitochondrial DNA depletion syndrome 7 (MTDPS7; OMIM #271245) also known as infantile-onset spinocerebellar ataxia (IOSCA) [49, 50] and were recently identified in patients with PRLTS5 [6, 10] (Table 3). There is a substantial phenotypic overlap across the conditions resulting from TWNK mutations, particularly in regard to the neurological features. Hearing loss, ataxia, myopathy, neuropathy and ophthalmoplegia have been reported in patients with each of these diseases. In our patients the diagnosis of PEOA3 could be excluded based on the presence of ovarian dysgenesis that has not been described in patients with PEOA3, an earlier age of disease onset (1–2 vs. 2–8 decade of life) and the identification of two TWNK mutations that were not pathogenic in the patients’ parents (heterozygous carriers). In contrast, MTDPS7 begins very early in life, in children below 2 years of age; the course of the disease is severe and includes optic atrophy, intellectual disability and hepatic involvement that were not observed in our patients.

All mutations found in TWNK are missense changes and their location does not explain different clinical manifestations. It has been hypothesized that even slight disturbances to the Twinkle protein, as a consequence of TWNK mutation, may affect its enzymatic activity, DNA binding ability, interaction with subunits or stability and result in a less-effective enzyme [51, 52]. Different bioinformatics tools predicted a deleterious effect of p.Asn399Ser and p.Arg601Gln on the protein function. In the applied 3D model we showed that p.Asn399Ser may affect the oligomeric Twinkle structure, crucial for the enzyme’s ability to unwind the DNA. A consequence of p.Arg601Gln appears to be impaired binding and hydrolysis of ATP, which is of paramount importance for enzyme functioning.

No formal criteria have been elaborated to facilitate accurate recognition of PRLTS. In the first line, karyotype analysis should be performed to exclude Turner syndrome or other abnormalities of the X chromosome as approximately half of females with Turner syndrome (gonadal dysgenesis) suffer from hearing loss [53]. Next, other causes of sensorineural hearing loss (SNHL) and ovarian dysfunction should be considered. Both conditions are genetically heterogeneous and testing of causative mutations in known genes is appropriate. In patients with neurologic involvement, the phenotype of PRLTS may overlap clinically with a mild form of peroxisomal d-bifunctional protein deficiency (DBP type IV; OMIM #261515). Presence of ovarian dysgenesis is considered the major clinical feature differentiating PRLTS from DBP type IV [9]. A comprehensive analysis of WES data provided us with a wealth of information on the genetic constitution of the proband. Except for two TWNK mutations no other mutation has been identified, which alone or in combination with other pathogenic variants could account for the clinical features observed in the proband. After identifying both TWNK mutations in the proband’s sister and confirming their biallelic status we could unequivocally establish the molecular genetic basis of the disease in the studied family. Our results provide further evidence that mutations in TWNK cause PRLTS5 [6, 10].

Neurological features in PRLTS have been observed in patients with mutations either in HSD17B4, CLPP or TWNK genes, i.e. in three out of five known PRLTS genes [5, 6, 9–12, 16, 17]. However, involvement of the nervous system seems to be a constant finding only in patients with TWNK mutations. In contrast to our patients in other individuals with PRLTS5 the neurological problems became noticeable later in life after hearing loss and ovarian dysfunction have been diagnosed [6, 10, 13]. The proband had a subtle atrophy of the cerebellum, a feature previously described in PRLTS patients [9, 10]. Significantly distorted proportion between the cerebellum white and grey matters represents a novel finding in PRLTS patients, which was independently confirmed using two different control groups. Atrophy of cervical medulla, present in both of our patients, was a feature of another PRLTS5 patient, who also shared the TWNK p.Asn399Ser mutation [10].

Audiological examination and neuroimaging studies revealed that hearing loss in PRLTS5 patients has a complex background. Analysis of the hearing threshold together with OAE (absent at higher frequencies) pointed to impairment of the cochlear function. While lack of stapedius reflexes and ABRs, together with normal tympanometry and the presence of OAE, were classical features of auditory neuropathy. Absent ABRs suggested that the defect localizes to the synapse between the hair cells and the auditory nerve. As imaging studies revealed partial atrophy of the vestibulocochlear nerve, their vestibular and cochlear components and the patients manifested peripheral neuropathy, we assume that the disease process affects the vestibulocochlear nerve fibers, particularly their distal parts. It should be also taken into account that absent ABRs may represent a progressed state of the disease, which has begun in the proximal part of the vestibulocochlear nerve as it is observed in other hereditary neurological diseases affecting mitochondrial function such as Friedreich’s ataxia or Charcot–Marie–Tooth (excluded in the proband based on genetic tests) [54, 55].

Partial atrophy of the vestibular nerves was less marked than the atrophy of the cochlear nerve and functionally sufficient to provide normal results of the vestibulo-ocular reflex in caloric tests and to some extend of the rotational testing. However, the number of vestibular nerve fibers discharging synchronously appears insufficient to obtain normal results of VEMP responses. It should be also considered that abnormal or absent VEMPs could be a consequence of the neuropathological process affecting the medial vestibulospinal tract (VEMP descending pathway) as diminished cervical enlargement was found in both patients. Asymptomatic vestibular disorders are commonly observed in patients with auditory neuropathy accompanied by peripheral neuropathy [56].

Our study presents a detailed and systematic assessment of the auditory and nervous systems in patients with PRLTS5, which sheds new insight on the phenotype and disease process of the mitochondrial disorder. We show for the first time in PRLTS patients (1) a complex mechanism of hearing impairment, comprising of cochlear dysfunction and dyssynchronous auditory nerve function, (2) partial atrophy of the vestibulocochlear nerves and their auditory and vestibular parts and (3) aberrant proportion of the cerebellum white and grey matters. Genetic workup provided further evidence on the causative role of TWNK mutations. From the seven known TWNK mutations underlying PRLTS5 one has recurred in our patients and its detection in the second individual with PRLTS5 confirms the pathogenic potential of the variant. The second TWNK mutation has not been reported in the context of any disease and its identification represents a novel genetic association with PRLTS5.