Background
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.
Patients and methods
Study subjects
Two affected sisters from a Polish non-consanguineous family and their unaffected mother were available for the study. The proband was born at term by Cesarean section after an uneventful pregnancy with 3600 g of body weight and an Apgar score of 1/2/3, indicating severe birth depression. Her psychomotor development was normal until the third year of age, when the motor performance begun to deteriorate progressively and the electromyography (EMG) examination was abnormal (she is unable to run since the age of 15 years). Speech development was age appropriate. At 5 years of age SNHL was diagnosed and pathologic auditory brainstem responses (ABRs) were observed. She has been fitted with hearing aids at the age of 13 but they were of limited benefit. Testing for primary amenorrhea and delayed pubertal development at the age of 15 revealed hypergonadotropic hypogonadism, streak gonads, rudimentary uterus and a normal female karyotype 46,XX. Hormone replacement therapy was introduced. At the age of 16 chronic thyroiditis with elevated levels of anti-thyroid peroxidase antibodies was diagnosed. Nerve conduction studies at the age of 21 showed axonal sensorimotor polyneuropathy. Ophthalmological examination was unremarkable except for impaired eye movements (Table
1).
Table 1
Clinical features and laboratory findings in the affected family members
Sex | F | F |
Age at disease onset, years | 3 | 11 |
Age at examination, years | 27 | 19 |
Disease duration | 24 | 8 |
Sensorineural hearing loss | +(5*) | +(12*) |
Ovarian dysfunction | +(15*) | +(12*) |
Intellectual disability | – | – |
Dementia | – | – |
Epilepsy | – | – |
Cerebellar syndrome | +(3*) | +(11*) |
SDFS | 3 | 2 |
Impaired eyes movement | + | + |
Gaze-evoked horizontal nystagmus | + | + |
Gaze-evoked vertical nystagmus | – | + |
Dysarthria | + | + |
Ataxia | + | + |
Positive Romberg’s test | + | + |
Flaccid paresis | +(?*) | +(?*) |
Muscle weakness | +UL < LL | +UL < LL |
Muscle atrophy | +UL < LL | +UL < LL |
Tendon reflexes UL | Diminished | Diminished |
Tendon reflexes LL | Absent | Absent |
Gait | Steppage | Steppage |
High-arched palate | + | + |
Pes cavus and clawed toes | + | + |
Other features—Hashimoto disease | + | + |
Lactate elevation | n.a. | + |
CK elevation | n.a. | + |
FSH and LH elevation | + | + |
EMG | Axonal, sensorimotor polyneuropathy | Axonal, sensorimotor polyneuropathy |
Abnormal neuroimaging | + | + |
The proband’s sister was born at term with 2850 g of body weight and an Apgar score of 5/6/7. Horizontal nystagmus and imbalance began at the age of 11, walking was gradually deteriorating and sensorimotor polyneuropathy was identified. At the age of 12 SNHL and ovarian dysgenesis with normal female karyotype were diagnosed. From the age of 16 years she has a Hashimoto’s disease. Biochemical studies showed mildly elevated levels of serum lactate and creatine kinase. Metabolic disease screening for organic acids with gas chromatography-mass spectrometry (GS/MS) in urine, amino acids and acylcarnitines with liquid chromatography-tandem mass spectrometry (LC–MS/MS) in dried blood spot and congenital disorders of glycosylation gave normal results. Wilson and Refsum diseases were excluded based on normal blood concentrations of ceruloplasmin and cooper and phytanic acid, respectively.
Neurological and audiological evaluation
Written informed consent was obtained from each participant. The study was approved by the ethics committee at the Institute of Physiology and Pathology of Hearing and performed according to the Declaration of Helsinki. The patients underwent thorough clinical evaluation. Functional impairment was assessed according to spinocerebellar degeneration functional score (SDFS) [
20]. In brain and cervical spine MRI (3T Siemens Magnetom Trio, 12-channel Head Matrix Coil) T1-weighted, T2-weighted and diffusion-weighted images were acquired. In addition, high-resolution 3D structural T1-weighted volumes were acquired using an MPRAGE sequence with 208 sagittal slices and an isotropic resolution 0.9 × 0.9 × 0.9 mm. Sequence parameters were: TR = 1900 ms, TE = 2.21 ms, TI = 900 ms, FA = 9, FOV = 26 × 28.8 cm, matrix = 320 × 290, Pixel bandwidth = 200 Hz/pix, iPAT = 2, TA = 5 min. Brain structures were segmented by Freesurfer version 5.3 (
http://surfer.nmr.mgh.harvard.edu/). The diameter of the vestibulocochlear nerve was evaluated directly and compared with the neighboring facial nerve used as an internal reference. Assessment of the cochlear and vestibular components was based on their visual comparison and with reference to the facial nerve. For comparisons of the cerebrum and cerebellum volumes and their white and gray matters, age- and sex-matched controls [
21‐
23] from the Internet Brain Volume Database funded by The Human Brain Project (
http://ibvd.virtualbrain.org/) were used. A difference above 2.3 of the standard deviation was considered as statistically significant.
Assessment of auditory function consisted of pure-tone and speech audiometry, impedance audiometry, otoacoustic emissions (OAE) and ABRs. Hearing thresholds for air and bone conduction were determined at frequencies 125–8000 and 250–4000 Hz, respectively, using the AC40 clinical audiometer (Interacoustics, Middelfart, Denmark) and the 10/5 dB descending-ascending threshold estimation procedure [
24]. Speech comprehension was tested using monosyllabic Polish words and the AC40 audiometer (Interacoustics) [
25]. Acoustic impedance measurements (tympanograms and stapedius reflex) were performed with the Zodiac 901 instrument (Madsen Electronics, Copenhagen, Denmark). Stapedius reflexes were analyzed for the frequencies 500, 1000, 2000 and 4000 Hz in the ipsi and contralateral modes [
26]. OAE were evoked by standard-click stimuli and 500 Hz tone bursts by using the ILO-292 system (Otodynamics Ltd, Hatfield, United Kingdom) [
27‐
29]. ABRs were recorded using the Integrity V500 system (Vivosonic Inc., Toronto, Canada). The stimuli were 0.1 ms clicks with alternating polarity presented with 90 dB normal hearing level (nHL) intensity at a repetition rate of 11/s. The amplifier bandwidth was 30–1500 Hz and analysis time 12 ms. The number of sweeps required for an averaged response was 1024 [
30].
For evaluation of the vestibular endorgan function Fitzgerald and Hallpike bithermal caloric test with video eye movement recordings (Visual Eyes Micromedical Technologies, Chatham, USA) were used. Sinusoidal harmonic acceleration testing at frequencies 0.01–0.32 Hz was conducted with a Rotational Vestibular Chair System 2000 (Micromedical Technologies, Chatham, USA). Otolith function was measured using air-conducted sound stimulation cervical and ocular vestibular evoked myogenic potentials (cVEMP, oVEMP) at 500 Hz, 95 dBnHL (EclipsVemp, Interacoustics, Assens, Denmark).
Whole-exome and Sanger sequencing
DNA was isolated from blood sample by a standard procedure. WES was performed using SureSelect Target Enrichment (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s protocol. The sample was run on 16% of a lane on HiSeq 1500 using 2 × 100 bp paired-end reads. All bioinformatics analysis was done as described previously [
31]. After primarily CASAVA processing, all reads were aligned to the hg19 reference genome with the Burrows-Wheeler Alignment Tool and analyzed with Genome Analysis Toolkit [
32]. Indel realignment, base quality score recalibration, duplicates elimination as well as SNP/INDEL calling were performed [
33]. The retrieved variants were annotated with ANNOVAR and converted to MS Access format for subsequent manual analyses. Total exon coverage by 20 reads or more was 89% and by 10 reads or more 95.9%. Alignments were inspected with Integrative Genomics Viewer [
34] and analyzed with a pipeline combining protein coding changes, splice site prediction, prevalence in populations, evolutionary conservation and scores from PolyPhen-2 [
35], SIFT [
36] and MutationTaster2 [
37] prediction algorithms of non-synonymous single-nucleotide variants. Sanger sequencing with 3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and BigDye Terminator cycle sequencing kit v. 3.1 (Applied Biosystems) were used to confirm the presence of variants identified by WES.
In silico protein analysis
Homologs of the human Twinkle protein were identified with a PSI-Blast [
38] search (E-value threshold of 0.005) performed against the NCBI non-redundant protein sequence database. The collected 15,000 sequences were initially clustered at 60% sequence identity with cd-hit [
39] and any sequences shorter than 300, longer than 1000 amino acids or described as “hypothetical protein” were removed. The multiple sequence alignment of the twinkle family was derived using MAFFT program [
40].
For preparation of the Twinkle homology model, the crystal structure of the gp4 protein from bacteriophage T7 (Protein Data Bank code1e0j) was selected as a template after analyzing the GeneSilico Metaserver results [
41]. The sequence-to-structure alignment between the Twinkle protein and the template (Additional file
1: Figure S1) was built using the consensus alignment approach and 3D assessment [
42] based on the results of FFAS [
43], HHSearch [
44] and the alignment proposed by Fernandez et al. [
19]. Multiple sequence alignment of the family was also taken into consideration. The 3D model of the protein was built with MODELLER [
45]. A model quality assessment was carried out using ProSA-web server [
46]. Secondary structure elements were predicted with PSI-PRED [
47]. Structure visualization was carried out with PyMOL (
http://www.pymol.org).
Discussion
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].
Authors’ contributions
MO analyzed the data and wrote the manuscript; DO, AP, UL performed genotyping and analyzed the data; MO, IS, GT, KK, TW, MF, HS participated in phenotyping and clinical data collection; DO, MŁ, DP, RP performed computational analysis; RP performed statistical analysis and participated in discussion; All authors read and approved the final manuscript.