Domínguez‑Ruiz et al. J Transl Med (2019) 17:290 https://doi.org/10.1186/s12967-019-2041-x Journal of Translational Medicine RESEARCH Open Access Perrault syndrome with neurological features in a compound heterozygote for two TWNK mutations: overlap of TWNK‑related recessive disorders María Domínguez‑Ruiz1,2†, Alberto García‑Martínez3†, Marc Corral‑Juan4†, Ángel I. Pérez‑Álvarez3, Ana M. Plasencia5, Manuela Villamar1,2, Miguel A. Moreno‑Pelayo1,2, Antoni Matilla‑Dueñas4, Manuel Menéndez‑González3* and Ignacio del Castillo1,2* Abstract Background: Perrault syndrome is a rare autosomal recessive disorder that is characterized by the association of sen‑ sorineural hearing impairment and ovarian dysgenesis in females, whereas males have only hearing impairment. In some cases, patients present with a diversity of neurological signs. To date, mutations in six genes are known to cause Perrault syndrome, but they do not explain all clinically-diagnosed cases. In addition, the number of reported cases and the spectra of mutations are still small to establish conclusive genotype–phenotype correlations. Methods: Affected siblings from family SH19, who presented with features that were suggestive of Perrault syn‑ drome, were subjected to audiological, neurological and gynecological examination. The genetic study included genotyping and haplotype analysis for microsatellite markers close to the genes involved in Perrault syndrome, whole-exome sequencing, and Sanger sequencing of the coding region of the TWNK gene. Results: Three siblings from family SH19 shared similar clinical features: childhood-onset bilateral sensorineural hearing impairment, which progressed to profound deafness in the second decade of life; neurological signs (spi‑ nocerebellar ataxia, polyneuropathy), with onset in the fourth decade of life in the two females and at age 20 years in the male; gonadal dysfunction with early cessation of menses in the two females. The genetic study revealed two compound heterozygous pathogenic mutations in the TWNK gene in the three affected subjects: c.85C>T (p.Arg29*), previously reported in a case of hepatocerebral syndrome; and a novel missense mutation, c.1886C>T (p.Ser629Phe). Mutations segregated in the family according to an autosomal recessive inheritance pattern. Conclusions: Our results further illustrate the utility of genetic testing as a tool to confirm a tentative clinical diagno‑ sis of Perrault syndrome. Studies on genotype–phenotype correlation from the hitherto reported cases indicate that patients with Perrault syndrome caused by TWNK mutations will manifest neurological signs in adulthood. Molecular and clinical characterization of novel cases of recessive disorders caused by TWNK mutations is strongly needed to *Correspondence: menendezgmanuel@uniovi.es; ignacio.castillo@salud.madrid.org † María Domínguez-Ruiz, Alberto García-Martínez and Marc Corral-Juan contributed equally to this work and they must be considered as joint first authors 1 Servicio de Genética, Hospital Universitario Ramón y Cajal, IRYCIS, Carretera de Colmenar km 9.100, 28034 Madrid, Spain 3 Department of Neurology, Servicio de Neurología, Hospital Universitario Central de Asturias, Avenida Roma sn, 33011 Oviedo, Spain Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Domínguez‑Ruiz et al. J Transl Med (2019) 17:290 Page 2 of 10 get further insight into the genotype–phenotype correlations of a phenotypic continuum encompassing Perrault syndrome, infantile-onset spinocerebellar ataxia, and hepatocerebral syndrome. Keywords: Perrault syndrome, Hearing impairment, Polyneuropathy, Ataxia, Premature ovarian failure, TWNK, Mitochondrial DNA helicase Background approximately 60% of individuals with PRLTS [2], which Perrault syndrome (PRLTS) is an autosomal recessive indicates that its genetic bases are still far from being genetic disorder that is characterized by the association completely understood. of sensorineural hearing impairment and ovarian dys- The number of cases with mutations in each of the six genesis in females [1, 2]. Affected males may have only an known genes is still small to establish definite genotype– apparently non-syndromic hearing impairment. In some phenotype correlations. So far, all patients with PRLTS cases, males and females present with neurological signs, due to mutations in HSD17B4 and TWNK present with including progressive sensory and motor peripheral neu- neurological manifestations (clinical type II) [4, 8, 10– ropathy, cerebellar ataxia, and mild intellectual disability 14]. Mutations in CLPP or LARS2 may result in clinical [2, 3]. Accordingly, PRLTS is classified clinically into two types I or II [6, 7, 12, 14–20]. Finally, no neurological types, with or without neurologic manifestations (types manifestations have been reported to date in patients II and I, respectively). PRLTS is a rare disease, although with mutations in HARS2 or ERAL1 [5, 9, 14], but these no accurate data on its prevalence are available. Given are the genes with fewer known cases. Molecular char- that hearing impairment may be the only clinical sign in acterization of novel cases is needed to progress in find- males, underascertainment is likely [2]. ing correlations. Here we report a Spanish familial case of PRLTS is genetically heterogeneous. To date, mutations Perrault syndrome with neurological manifestations and in six genes are known to cause different genetic types of show it is caused by two compound heterozygous muta- the syndrome (Table 1). tions in the TWNK gene, one of which is novel whereas HSD17B4, the first gene whose involvement in PRLTS the other was previously reported in a case of hepatocer- was reported [4], encodes 17β-hydroxysteroid dehydro- ebral syndrome. Implications of these findings in the gen- genase type 4, a peroxisomal multifunctional enzyme otype–phenotype correlations of TWNK mutations are involved in the fatty acid β-oxidation pathway and ster- discussed. oid metabolism. The other five genes code for proteins that play roles in mitochondrial function. HARS2 [5] and LARS2 [6] code for mitochondrial histidyl-tRNA Methods Affected subjects from family SH19 were clinically exam- synthetase 2 and leucyl-tRNA synthetase 2, respectively. ined by the Departments of Pediatrics and Neurology of CLPP [7] encodes an endopeptidase subunit of a mito- Hospital Universitario Central de Asturias. The genetic chondrial ATP-dependent proteolytic complex. TWNK study was performed in parallel by the Service of Genet- [8] encodes Twinkle, a DNA helicase that is essential ics of the Hospital Universitario Ramón y Cajal, and by for the replication of the mitochondrial genome. Finally, the Neurogenetics Unit of the Research Institute Ger- ERAL1 [9] codes for a GTPase that acts as a chaperone mans Trias i Pujol (IGTP) in Badalona (Barcelona). for the 12S mitochondrial rRNA, and so it contributes DNA was extracted from peripheral blood samples to the proper assembly of the 28S small mitochondrial from all members of each family by standard methods. ribosomal subunit. After screening for mutations in Microsatellite markers flanking the genes involved in these six genes, molecular diagnosis is not reached in PRLTS were chosen from the literature [21] and elec- tronic databases [22] (Table 2), and were amplified using fluorescently-labeled primers and PCR conditions as pre- Table 1 Genes known to be involved in Perrault syndrome viously reported. Amplified alleles were resolved by capil- Genetic type MIM entry Gene Gene MIM entry Location lary electrophoresis in an ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems). PRLTS1 233400 HSD17B4 601860 5q23.1 Whole-exome sequencing (WES) was performed on PRLTS2 614926 HARS2 600783 5q31.3 genomic DNA obtained from peripheral blood. Exome PRLTS3 614129 CLPP 601119 19p13.3 capture was performed using SureSelect All Human PRLTS4 615300 LARS2 604544 3p21.31 Enrichment Target Exon (Agilent Technologies) for PRLTS5 616138 TWNK 606075 10q24.31 71 Mb according to the manufacturer’s protocol. Paired- PRLTS6 617565 ERAL1 607435 17q11.2 end, 101-nt long reads were generated on a HiSeq 2000 Domínguez‑Ruiz et al. J Transl Med (2019) 17:290 Page 3 of 10 platform (Illumina Inc.). Read alignment was performed Mutation nomenclature is annotated based on cDNA using BWA (Burrows-Wheeler Aligner) [23]. Variant sequence (GenBank accession number NM_021830.4) calling was performed using a combination of two differ- and according to the current Human Genome Varia- ent algorithms: VarScan [24] and GATK [25]. Identified tion Society rules as implemented by the Mutalyzer 2.0.3 variants were annotated using the Ensembl database [26]. program (Leiden University Medical Center, Leiden, The Variants with a minor allele frequency ≥ 1% in genetic Netherlands). databases including the Exome Aggregation Consor- tium, 1000-Genomes Project and dbSNP, were excluded Results from further analysis. The genetic heterogeneity model Family SH19 included four siblings who were born from proposed by Ng et al. [27] was applied in order to iden- non-consanguineous parents (Fig. 1a). The eldest brother tify potential candidate genes. Candidate variants were (subject II:1) died from purulent meningitis at age assessed for their computationally predicted pathogenic- 21 months. The remaining three siblings (two sisters, one ity by SIFT, PolyPhen-2, and MutationTaster [28–30], and brother) shared similar clinical features, including spi- prioritized in accordance with the clinical characteristics nocerebellar ataxia, polyneuropathy, sensorineural hear- shared by affected siblings. Segregation of candidate vari- ing impairment, and gonadal dysfunction in females, as ants was confirmed by Sanger sequencing. detailed below. Primer sequences for PCR amplification of all exons Subject II:2. At age 5 years, she developed a progres- and exon–intron boundaries of the TWNK gene are sive hearing loss leading to profound bilateral deafness listed in Table 3. by age 15. At age 20, her menses ceased, but the subse- quent gynaecological study did not result in an etiologic diagnosis. At age 35 she started to experience difficulties with walking, but she did not visit a neurologist until age Table 2 Microsatellite markers that map close to the genes 43. On examination we confirmed a profound hearing involved in PRLTS impairment, and found fast horizontal and vertical nys- Gene Microsatellite markers tagmus. Standing and gait were unstable, and she was not able to perform tandem gait. Romberg test was positive. HSD17B4 D5S404, D5S494, D5S471, D5S503 She had cavus feet and universal areflexia. Touch and HARS2 D5S2116, D5S658, D5S2010 arthrokinetic sensitivity were diminished in lower limbs CLPP D19S1034, D19S427, D19S406, D19S873 with a distal gradient, whereas the algesic and vibratory LARS2 D3S3624, D3S3582, D3S1581 sensitivities were preserved. Over time, gait became TWNK D10S192, D10S1265, D10S1697 impaired and showed ataxic features. There was also an ERAL1 D17S1878, D17S925, D17S1873 impairment of arthrokinetic sensitivity in upper limbs. Table 3 Primers for PCR amplification of TWNK exons and exon–intron boundaries Exon Sequence of primers Annealing temperature (°C) Amplicon size (bp) 1 (part 1) F: 5′-CTA​AGC​AGC​GAG​GAG​AGG​GGG-3′ 63 543 R: 5′-TCG​CCA​CGT​CCT​TCT​ACA​AT-3′ 1 (part 2) F: 5′-GAG​GAC​GAC​GAG​GAG​ATG​C TGTG-3′ 63 571 R: 5′-CCC​ATG​CCC​CCG​CAA​ATA​C-3′ 1 (part 3) F: 5′-TGC​GTG​GGG​AGT​GGA​TGG​-3′ 63 677 R: 5′-GCT​GGG​TCG​GGG​AAT​AGT​GGT-3′ 1 (part 4) F: 5′-GTG​TGC​GAT​ATC​TGC​GAC​C TG-3′ 63 746 R: 5′-CAT​TGT​CAT​GGC​CCA​AAC​CC-3′ 2 F: 5′-TTT​GGG​CCA​TGA​CAA​TGT​T-3′ 60 467 R: 5′-GGA​GAT​GGG​GGA​GTT​CCT​AC-3′ 3 F: 5′-CTT​C TG​CCT​GGG​GTG​GTC​-3′ 60 348 R: 5′-TCC​ATA​CAT​CCT​GCT​GCC​ATAC-3′ 4 F: 5′-GTA​TGG​CAG​CAG​GAT​GTA​TGGA-3′ 60 413 R: 5′-TGT​TCG​GAT​GGA​CAG​TCA​AGA-3′ 5 F: 5′-CCT​C TC​CCC​ATT​C TT​ATC​ACT-3′ 60 518 R: 5′-AGC​TCA​GGA​CCA​CAG​GAT​AG-3′ F forward, R reverse Domínguez‑Ruiz et al. J Transl Med (2019) 17:290 Page 4 of 10 Fig. 1 Genetic study of family SH19. a Pedigree, haplotype analysis for microsatellite markers and segregation of mutations in the TWNK gene. b Electropherograms of the two heterozygous mutations that were found in TWNK in the family. c Alignment of twinkle orthologous sequences from five model organisms: human (accession number NP_068602.2), mouse (NP_722491.2), cat (XP_003994377.1), chick (NP_001026515.1), and zebrafish (NP_001264527.1). Only stretches aligning to human twinkle residues 614–644 are shown. Asterisks indicate identical residues across all sequences; colons, conserved positions (residues of strongly similar properties); periods, semi-conserved positions (residues of weakly similar properties). The location of the residue affected by the p.Ser629Phe mutation is indicated by an arrowhead Domínguez‑Ruiz et al. J Transl Med (2019) 17:290 Page 5 of 10 Later on we found global hypotony and mild atrophy in Sciences Research Institute Germans Trias i Pujol) the four limbs, as well as dysarthria. Neuroimaging stud- (IGTP), because of the neurological signs that were ies found a thinning of the spinal cord, cerebellar atrophy observed. and signal changes in the cerebellar white matter. In the HURyC, the clinical entity observed in this fam- Subject II:3. At age 3 years, she developed a progres- ily was classified as a syndromic hearing impairment. The sive sensorineural hearing loss leading to profound bilat- association of clinical signs in the three affected siblings eral deafness by age 13. At age 32, she suffered a traffic was compatible with a diagnosis of autosomal recessive accident that resulted in a traumatic head injury, with Perrault syndrome with neurological manifestations. no sequels. Soon after, her menses ceased. At age 43 she Accordingly, all five subjects in the pedigree were geno- visited the neurologist because of difficulties with walk- typed for 20 microsatellite markers that map closely to ing. We confirmed the profound hearing impairment, the six genes known to be involved in PRLTS (Table 2). and found dysarthria, global hypotony, universal areflexia Haplotype analysis excluded linkage to HSD17B4, and loss of arthrokinetic sensitivity in the feet, which HARS2, CLPP, LARS2 and ERAL1, but showed that the were cavus. There was dysmetria in cerebellar manoeu- three siblings were haploidentical for markers close to vres with lower limbs, and she was unable to perform TWNK (Fig. 1a). Sanger DNA sequencing of all exons tandem gait. Neurophysiologic studies found conduc- and exon–intron boundaries of the TWNK gene revealed tion defects bilaterally in the auditory pathways, in the two potentially pathogenic sequence variants, c.85C>T right visual pathway, and in the somesthetic pathway at (p.Arg29*) and c.1886C>T (p.Ser629Phe) (Fig. 1b), the posterior spinal cords for afferents from both lower which were shared by the three siblings. Segregation and upper limbs. Electroneurography showed symmet- analysis confirmed that the two mutations were in trans, ric demyelinating sensitive polyneuropathy. Brain MRI the father carrying c.85C>T and the mother carrying showed global cerebellar atrophy and signal changes in c.1886C>T (Fig. 1a). the cerebellar white matter, particularly in the middle In the IGTP, the genetic cause of the polyneuropathy cerebellar peduncle. The patient has also been diagnosed that was observed in the affected siblings was investi- with autoimmune hypothyroidism secondary to Hashi- gated by massively-parallel DNA sequencing. WES of moto thyroiditis, vitiligo and increase of liver enzymes of subject II:3, with a ≥ 20× average coverage of 88.7% of unknown cause. the target regions, identified 72,511 variants within 1396 Subject II:4. He was diagnosed with sensorineural candidate genes under a recessive model of inheritance. hearing loss at age 4 years. Progressive ataxia manifested Upon excluding those DNA variants with a minor allele at about age 20. He suffered a syncope at age 29, and was frequency ≥ 1% in known databases, variants were pri- examined by a neurologist for the first time. CT scan and oritized by their predicted deleterious effect, amino acid video-EEG were normal. On examination we found verti- conservation, and associated disease heritability. Six of cal and horizontal nystagmus, universal areflexia and loss these candidate variants were located in genes related of arthrokinetic sensitivity in the feet, which were cavus. to ataxia phenotype including ATM, GALC, SPTBN2, Gait was unstable, and he was unable to perform tandem SYNE1, and TWNK (Additional file 1: Table S1). The gait. Romberg test is positive. Endocrinologic blood tests best candidate variants were identified within the TWNK showed autoimmune hypothyroidism and dysfunction gene, c.1886C>T (p.Ser629Phe), a missense mutation that of the gonadal axis. MRI showed cervical kyphosis, flat- is predicted as pathogenic by all bioinformatic algorithms tening of the spinal cord and cerebellar atrophy (Fig. 2). (SIFT score = 0.04, deleterious; PolyPhen-2 score = 0.96, Neurophysiologic studies confirmed a profound senso- damaging; MutationTaster score = 1, disease-causing) rineural hearing impairment and found a dysfunction of [28–30], and the nonsense mutation c.85C>T (p.Arg29*). somatosensory pathway in the posterior spinal cord and Sanger sequencing confirmed the two sequence variants bulbo-cortical lemniscus. Electroneurography showed and showed the expected pattern of segregation in the mixed sensitive and motor demyelinating and axonal pol- family under a recessive mode of inheritance (Fig. 1a). yneuropathy, including large and small myelinic fibers. Mutation p.Arg29* is expected to result in a very small The patients’ mother (I:2) had been diagnosed with truncated protein lacking both domains of Twinkle, pri- Parkinson’s disease, and the father (I:1) had cavus feet. mase-related and helicase, or in no protein at all because Family history did not show any other relative with simi- of mRNA degradation through nonsense-mediate decay. lar traits. Mutation p.Ser629Phe has not been reported previously The genetic study was requested in parallel to the Ser- in any database, it affects a highly-conserved residue vice of Genetics of Hospital Universitario Ramón y Cajal (Fig. 1c), and it lies at the C-terminal part of the heli- (HURyC), because of the hearing impairment, and to the case domain, in a region where other mutations causing Functional and Translational Neurogenetics Unit (Health PRLTS have been reported [8, 13]. Domínguez‑Ruiz et al. J Transl Med (2019) 17:290 Page 6 of 10 Fig. 2 MRI images from subject II:4. a T2-weighted coronal slide showing normal brain parenchyma including brain stem, the inner ears and the vestibulocochlear nerves. b T1-weighted transversal slide of the dorsal spine where flattening of the spinal cord can be seen. c T2-weighted sagittal slide showing cervical kyphosis as well as cerebellar and cervical atrophy. d Detail of the sagittal slide to show the cerebellar atrophy more clearly Discussion and it progressed quickly to become profound during Most patients with rare diseases face diagnostic delays, the second decade of life. At that moment, the patients because of the difficulties in noticing the association of received a diagnosis of non-syndromic hearing impair- clinical signs that define their particular disease [31]. In ment. The second clinical sign was the early cessation of Perrault syndrome, diagnosis is further complicated by menses in the two affected females, which took place at the variable expressivity of its clinical features, which ages 20 and 32 years, respectively. However, the possible is related to the age and sex of the patient, and to the association of hearing loss and early amenorrhea was not diversity of genes that are involved. This is well illus- noticed at that time. Neurological signs manifested later trated by the familial case that we report here. Hearing (ages 35 and 43 in the two women, respectively, and age loss was the first manifestation of the disease. It started 20 years in the male). Subsequently, genetic studies were in childhood (ages 3–5 years in the different patients) requested, and only when the molecular results were Domínguez‑Ruiz et al. J Transl Med (2019) 17:290 Page 7 of 10 obtained, the diagnosis of Perrault syndrome with neu- mutations causing AdPEO are missense and affect resi- rological signs could be established. The same situation is dues either within the linker or in close proximity to it in observed in other patients with PRLTS [2]. Neurological the tertiary structure of the protein [40, 41]. They lead to signs can remain absent for years or never manifest. Male either destabilization of the monomers (flexibility of the patients without neurological signs and without affected linker region is diminished) or to defective hexameriza- female relatives will be diagnosed of non-syndromic tion (inhibition of the ring closure or changing the num- hearing loss, with the subsequent risk of having descend- ber of subunits within the ring), which in turn impair the ants with the syndrome. The association of hearing loss, helicase activity and cause replication stalling [40, 41]. On ataxia and polyneuropathy that is found in some male the other hand, recessive mutations include a few trun- patients is also observed in a large list of other neurologi- cating variants, including deletions causing frameshifts, cal diseases [32]. On the other hand, the association of nonsense and splice-site mutations (to our knowledge, clinical signs could be just coincidental and not etiologi- p.Arg29* is the first reported truncating mutation in a cally related [33]. patient with PRLTS). However, the majority of recessive Genetic testing can contribute to clarify the diagnosis, mutations are missense, some of them affecting residues but it is also complicated by the genetic heterogeneity in the primase-related domain, but most of them affect- of PRLTS [34]. In spite of the identification of six genes ing the helicase domain (like p.Ser629Phe in our patients) whose mutations cause PRLTS, genetic testing for these [42]. They result in three different phenotypes: Perrault genes does not elucidate about 60% of the clinically- syndrome type 5 (MIM 606075) [8], infantile-onset spi- diagnosed cases [2]. Although some of the cases might nocerebellar ataxia (IOSCA) [43] and hepatocerebral be just coincidental associations of signs and not true syndrome [44], although the last two disorders are now PRLTS [33], it seems clear that other genes remain to grouped under a unique code (MIM 271245). In fact, the be identified. Moreover, establishing genotype–pheno- three diseases constitute a phenotypic continuum rang- type correlations with the existing data is still hampered ing from the relatively milder form (PRLTS5) to the most by the small number of cases carrying mutations in each severe form (hepatocerebral syndrome, with acute liver gene (Table 4). Neurological signs have been reported failure). Hearing impairment is reported in patients of in patients with PRLTS and mutations in the HSD17B4, the three conditions, and ovarian insufficiency may be TWNK, CLPP and LARS2 genes [4, 7, 8, 10–16, 20; and also a shared feature if not masked by a premature lethal- this work]. For CLPP and LARS2, there are also reports of ity. Therefore, classification of cases depends on the age patients without neurological signs [6, 7, 12, 14, 17–19], of onset and severity of other clinical signs. In IOSCA like all the patients with mutations in HARS2 or ERAL1 and hepatocerebral syndrome, the neurological signs who have been reported to date [5, 9, 14]. However, it manifest in early childhood [42–44], whereas in PRLTS5 would be premature to define genotype–phenotype cor- the onset takes place much later in life [8, 12–14], as also relations on these bases, as the picture could change after shown in patients of family SH19. In hepatocerebral syn- careful follow-up of the already reported patients over drome, the liver is severely affected [44]. Of note, subject the years, and after novel reports of cases with mutations II:3 of family SH19 shows an increase of liver enzymes of in HARS2 or ERAL1, still underrepresented. Moreo- unknown cause. The existence of this phenotypic con- ver, different mutations in some of the six PRLTS genes tinuum is also supported by the spectrum of mutations may result in other diseases. For example, mutations in in the TWNK gene. Whereas there is no overlap between LARS2 have been reported to cause hydrops, lactic aci- the mutations causing dominant or recessive phenotypes, dosis, sideroblastic anemia, and multisystem failure in a this overlap does exist among the recessive diseases [42]. female newborn [35]. Also, different mutations in TWNK For example, mutation p.Tyr508Cys has been reported have been reported in autosomal dominant progressive in cases of IOSCA (it is a founder mutation in Finland) external ophthalmoplegia (AdPEO, MIM 609286) and in [43], but also in hepatocerebral syndrome [44, 45]. More- three autosomal recessive disorders. over, mutation p.Arg29* has been reported in a case The TWNK gene codes for the 684-aa monomer of of hepatocerebral syndrome [45] and in PRLTS5 (this the mitochondrial DNA helicase twinkle, which local- work). It is remarkable that a child with hepatocerebral izes to the mitochondrial matrix and is thought to play syndrome is compound heterozygous for p.Arg29* (also a key role in mtDNA replication [36, 37]. The enzyme is seen in PRLTS5) (this work) and p.Tyr508Cys (frequent a ring-shaped homohexamer, each monomer containing in IOSCA) [45]. It is clear that clinical and genetic char- a primase-related domain (residues 79–346), a helicase acterization of additional patients, like those reported domain (residues 384–635) and a linker region between here, is needed to progress in understanding the geno- these two domains [38]. The linker region is criti- type–phenotype correlations in subjects with mutations cal for hexamerization and helicase activity [39]. Most in TWNK. Domínguez‑Ruiz et al. J Transl Med (2019) 17:290 Page 8 of 10 Table 4 Overall identified mutations causing Perrault syndrome Gene (accession number) Mutation References DNA level Protein level HSD17B4 (NM_000414.3) c.46G>A p.Gly16Ser [12] c.244G>T p.Val82Phe [12] c.298G>T p.Ala100Ser [11] c.587C>T p.Ala196Val [10] c.650A>G p.Tyr217Cys [4] c.1704T>A p.Tyr568* [4] 12-kb deletion (exons 10–13) [10] HARS2 (NM_012208.3) c.598C>G p.Leu200Val [5] c.1010A>G p.Tyr337Cys [14] c.1102G>T p.Val368Leu [5] CLPP (NM_006012.2) c.21delA p.Ala10Profs*117 [16] c.270+4A>G [7] c.425C>T p.Pro142Leu [16] c.430T>C p.Cys144Arg [12] c.433A>C p.Thr145Pro [7] c.439T>A p.Cys147Ser [14] c.440G>C p.Cys147Ser [7] c.484G>A p.Gly162Ser [16] c.624C>G p.Ile208Met [17] c.685T>G p.Tyr229Asp [15] Deletion of several exons [16] LARS2 (NM_015340.3) c.351G>C p.Met117Ile [12] c.880G>A p.Glu294Lys [20] c.899C>T p.Thr300Met [18] c.1077delT p.Ile360Phefs*15 [6] c.1358G>A p.Arg453Gln [14] c.1556C>T p.Thr519Met [20] c.1565C>A p.Thr522Asn [6, 12, 19] c.1886C>T p.Thr629Met [6, 14] c.1912G>A p.Glu638Lys [18] TWNK (NM_021830.4) c.85C>T p.Arg29* This work c.793C>T p.Arg265Cys [14] c.968G>A p.Arg323Gln [12] c.1172G>A p.Arg391His [8] c.1196A>G p.Asn399Ser [12, 13] c.1321T>G p.Trp441Gly [8] c.1519G>A p.Val507Ile [8] c.1754A>G p.Asn585Ser [8] c.1802G>A p.Arg601Gln [13] c.1886C>T p.Ser629Phe This work ERAL1 (NM_005702.3) c.707A>T p.Asn236Ile [9] Conclusions syndrome caused by TWNK mutations will manifest neu- Genetic testing is a useful tool to confirm a tentative clin- rological signs in adulthood. Recessive disorders caused ical diagnosis of Perrault syndrome, although the genetic by TWNK mutations constitute a phenotypic continuum, cause remains unidentified in over half of the cases. Stud- so that establishing genotype–phenotype correlations is a ies on genotype–phenotype correlation from the hitherto challenging task that still needs identification and molec- reported cases indicate that all patients with Perrault ular and clinical characterization of novel cases. Domínguez‑Ruiz et al. J Transl Med (2019) 17:290 Page 9 of 10 Supplementary information Received: 5 March 2019 Accepted: 18 August 2019 Supplementary information accompanies this paper at https​://doi. org/10.1186/s1296​7-019-2041-x. Additional file 1: Table S1. Relevant mutations obtained by whole- exome sequencing of subject II: 3. References 1. Perrault M, Klotz B, Housset E. Two cases of Turner syndrome with deaf- mutism in two sisters. Bull Mem Soc Med Hop Paris. 1951;67:79–84. Abbreviations 2. Newman WG, Friedman TB, Conway GS, Demain LAM. Perrault Syndrome. Aa: amino acid; AdPEO: autosomal dominant progressive external ophthal‑ In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Stephens K, moplegia; CT: computed tomography; EEG: electroencephalography; HURyC: Amemiya A, editors. GeneReviews. Seattle (WA): University of Washing‑ Hospital Universitario Ramón y Cajal; IGTP: Institute Germans Trias i Pujol; ton, Seattle; 2018. https​://www.ncbi.nlm.nih.gov/books​/NBK24​2617. IOSCA: Infantile-Onset SpinoCerebellar Ataxia; MRI: Magnetic Resonance Accessed 6 Feb 2019. Imaging; mtDNA: mitochondrial DNA; PRLTS: Perrault syndrome; WES: whole 3. Fiumara A, Sorge G, Toscano A, Parano E, Pavone L, Opitz JM. Perrault exome sequencing. syndrome: evidence for progressive nervous system involvement. Am J Med Genet A. 2004;128A:246–9. Acknowledgements 4. Pierce SB, Walsh T, Chisholm KM, Lee MK, Thornton AM, Fiumara A, et al. We thank patients and relatives for their participation in the study. Mutations in the DBP-deficiency protein HSD17B4 cause ovarian dys‑ genesis, hearing loss, and ataxia of Perrault Syndrome. Am J Hum Genet. Authors’ contributions 2010;87:282–8. AGM, AIPA, AMP and MMG performed the clinical examination of the patients. 5. Pierce SB, Chisholm KM, Lynch ED, Lee MK, Walsh T, Opitz JM, et al. Muta‑ MDR, MV, MAMP and IdC carried out the genotyping for microsatellite mark‑ tions in mitochondrial histidyl tRNA synthetase HARS2 cause ovarian ers, haplotype analysis, Sanger DNA sequencing and segregation study, at dysgenesis and sensorineural hearing loss of Perrault syndrome. Proc Natl the HURyC. MCJ and AMD performed whole-exome sequencing, analysis of Acad Sci USA. 2011;108:6543–8. exome data and the genetic segregation study at the IGTP. IdC prepared the 6. Pierce SB, Gersak K, Michaelson-Cohen R, Walsh T, Lee MK, Malach D, et al. first draft of the manuscript with the collaboration of all coauthors. All authors Mutations in LARS2, encoding mitochondrial leucyl-tRNA synthetase, read and approved the final manuscript. lead to premature ovarian failure and hearing loss in Perrault syndrome. Am J Hum Genet. 2013;92:614–20. Funding 7. Jenkinson EM, Rehman AU, Walsh T, Clayton-Smith J, Lee K, Morell RJ, This research was supported by grants from the Instituto de Salud Carlos III et al. Perrault syndrome is caused by recessive mutations in CLPP, encod‑ (ISCIII), Madrid, Spain (PI14/01162 and PI17/00572; National Plan for Scientific ing a mitochondrial ATP-dependent chambered protease. Am J Hum and Technical Research and Innovation 2013–2016, with cofounding from the Genet. 2013;92:605–13. European Regional Development Fund (ERDF), “A way to make Europe”) (to 8. Morino H, Pierce SB, Matsuda Y, Walsh T, Ohsawa R, Newby M, et al. IdC); S2017/BMD-3721-RAREGENOMICS-CM from the Consejería de Educación Mutations in Twinkle primase-helicase cause Perrault syndrome with e Investigación de la Comunidad de Madrid (to MAMP); and CP08/00027, neurologic features. Neurology. 2014;83:2054–61. CPII14/00029, FIS PI14/00136, and FIS PI17/00534 (to AM-D). 9. Chatzispyrou IA, Alders M, Guerrero-Castillo S, Zapata Perez R, Haagmans MA, Mouchiroud L, et al. A homozygous missense mutation in ERAL1, Availability of data and materials encoding a mitochondrial rRNA chaperone, causes Perrault syndrome. The datasets used and/or analysed during the current study are available from Hum Mol Genet. 2017;26:2541–50. the corresponding author on reasonable request. However, the Whole Exome 10. Lieber DS, Hershman SG, Slate NG, Calvo SE, Sims KB, Schmahmann JD, Sequencing datasets generated and/or analysed during the current study are et al. Next generation sequencing with copy number variant detection not publicly available because of the European Union General Data Protection expands the phenotypic spectrum of HSD17B4-deficiency. BMC Med Regulation (GDPR). Genet. 2014;15:30. 11. Chen K, Yang K, Luo SS, Chen C, Wang Y, Wang YX, et al. A homozygous Ethics approval and consent to participate missense variant in HSD17B4 identified in a consanguineous Chinese This study was conducted according to the ethical principles for medical Han family with type II Perrault syndrome. BMC Med Genet. 2017;18:91. research involving human subjects according to the Declaration of Helsinki. 12. Demain LA, Urquhart JE, O’Sullivan J, Williams SG, Bhaskar SS, Jenkinson Written informed consents were obtained for all individuals included in the EM, et al. Expanding the genotypic spectrum of Perrault syndrome. Clin study, which was approved by the Ethical Committees of the Hospital Univer‑ Genet. 2017;91:302–12. sitario Ramón y Cajal (Madrid) and the University Hospital Germans Trias i Pujol 13. Ołdak M, Oziębło D, Pollak A, Stępniak I, Lazniewski M, Lechowicz U, (HUGTiP) in Badalona. et al. Novel neuro-audiological findings and further evidence for TWNK involvement in Perrault syndrome. J Transl Med. 2017;15:25. Consent for publication 14. Lerat J, Jonard L, Loundon N, Christin-Maitre S, Lacombe D, Goizet C, Consent for publication was obtained from the three affected subjects of et al. An application of NGS for molecular investigations in Perrault family SH19. syndrome: study of 14 families and review of the literature. Hum Mutat. 2016;37:1354–62. Competing interests 15. Ahmed S, Jelani M, Alrayes N, Mohamoud HS, Almramhi MM, Anshasi W, The authors declare that they have no competing interests. et al. 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