Journal of the Neurological Sciences 349 (2015) 105–109
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Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Analysis of the C19orf12 and WDR45 genes in patients with neurodegeneration with brain iron accumulation Anne Tschentscher a, Gabriele Dekomien a, Sophia Ross b, Kirsten Cremer c, Guido M. Kukuk d, Jörg T. Epplen a,e, Sabine Hoffjan a,⁎ a
Department of Human Genetics, Ruhr-University, Bochum, Germany Pediatric Neurology, Children's Hospital Erlangen, Germany Department of Human Genetics, University of Bonn, Germany d Department of Radiology, University of Bonn, Germany e Faculty of Health, University Witten/Herdecke, Witten, Germany b c
a r t i c l e
i n f o
Article history: Received 1 July 2014 Received in revised form 24 November 2014 Accepted 24 December 2014 Available online 3 January 2015 Keywords: NBIA C19orf12 WDR45 MPAN BPAN Mutation analysis
a b s t r a c t Background: Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of diseases presenting with movement disorders and brain iron deposits. In addition to NBIA subtypes caused by mutations in PANK2 and PLA2G6, mutations in the C19orf12 gene were recently described as the third frequent cause of NBIA (called mitochondrial membrane protein-associated neurodegeneration, MPAN). Additionally, the X-linked gene WDR45 was found causative for a special subtype named static encephalopathy in childhood with neurodegeneration in adulthood (also called BPAN); however, analysis of this gene in a broader spectrum of NBIA has not been reported yet. Methods: In a heterogeneous cohort of 69 patients with suspected NBIA that did not carry mutations in PANK2 and PLA2G6, the coding region of C19orf12 was evaluated by Sanger sequencing. The WDR45 gene was analyzed via high resolution melting and subsequent sequence analysis. Results: Previously described homozygous C19orf12 mutations were found in 3/69 NBIA patients (4.3%). Analysis of the WDR45 gene revealed a novel heterozygous missense mutation in one female NBIA patient showing psychomotor retardation with secondary decline. Conclusions: C19orf12 mutations were confirmed in our heterogeneous NBIA cohort, while WDR45 mutations appear to be restricted to the subtype showing encephalopathy in childhood with neurodegeneration in adulthood. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Neurodegeneration with brain iron accumulation (NBIA) is a clinically and genetically heterogeneous group of neurodegenerative diseases, typically comprising movement disorders and brain iron deposits [1]. The wide phenotypic spectrum of NBIA, as well as parallels to other disorders such as Parkinsonism or spastic paraplegia [1,2], can make diagnosis difficult and, in some cases, the underlying genetic mechanisms still remain unknown.
Abbreviations: NBIA, neurodegeneration with brain iron accumulation; MPAN, mitochondrial membrane protein-associated neurodegeneration; BPAN, beta-propeller protein-associated neurodegeneration; PKAN, panthothenate kinase-associated neurodegeneration; PLAN, Phospholipase A2-associated neurodegeneration; HRM, high resolution melting; SENDA, static encephalopathy in childhood with neurodegeneration in adulthood. ⁎ Corresponding author at: Department of Human Genetics, Ruhr-University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany. Tel.: +49 234 3223823; fax: + 49 234 3214196. E-mail address: [email protected] (S. Hoffjan).
http://dx.doi.org/10.1016/j.jns.2014.12.036 0022-510X/© 2014 Elsevier B.V. All rights reserved.
The first subtype of NBIA, pantothenate kinase-associated neurodegeneration (PKAN) caused by mutations in the PANK2 gene, was described in 2001 [3]. Since then, mutations in various genes, such as PLA2G6, FA2H, FTL, AP and ATP13A2, have been added to the growing list of clinically and genetically distinct entities associated with brain iron accumulation [4]. Most recently, mutations in the C19orf12 and WDR45 genes were identified as additional causes for NBIA. Mutations in C19orf12 were first described in a homogeneous cohort from Poland where most cases carried an 11bp deletion founder mutation [5]. In a subsequent larger multinational study, C19orf12 mutations were found associated with a wide spectrum of phenotypes, typically including motor neuronopathy, cognitive decline and neuropsychiatric impairment [6]. MRI scans showed T2-hypointensities in globus pallidus and substantia nigra without an eye of the tiger sign [5]. Due to localization of the gene product in mitochondria, the name mitochondrial membrane protein-associated neurodegeneration (MPAN) was proposed for this clinical entity [5]. MPAN was suggested to be the third frequent NBIA subtype after PKAN (PANK2 gene) and PLAN
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(PLA2G6); however the mutational frequencies varied substantially between studies. Mutations in the WDR45 gene, located on the X chromosome, were recently identified in patients with a very specific phenotype that is referred to as static encephalopathy in childhood with neurodegeneration in adulthood (SENDA). This new X-linked NBIA subtype, affecting a gene product involved in autophagy [7], has been named beta-propeller protein-associated neurodegeneration (BPAN) [8]. Most affected individuals are females and all reported mutations so far occurred de novo. However, a few males with WDR45 mutations and a SENDA phenotype, most of them probably showing somatic mosaicism, have also been described [8]. To our knowledge, the frequency of WDR45 mutations in patients with a broader clinical NBIA phenotype has not been investigated yet. In order to further evaluate the mutational and clinical spectra of these two newly described NBIA subtypes, we screened a cohort of PANK2 and PLA2G6 mutation-negative patients clinically presenting with NBIA (n = 69) for mutations in C19orf12 and WDR45. 2. Materials and methods 2.1. Patient cohort Patients were recruited from an internal registry of the Department of Human Genetics of the Ruhr-University Bochum, including DNA samples sent by clinicians for the diagnosis of NBIA during the years 2006–2013 which had been tested negative for mutations in the PANK2 or PLA2G6 genes. All samples were obtained with informed consent of probands or their legal representatives. The study was approved by the Ethics Committee of the Ruhr-University Bochum and carried out in accordance with the Declaration of Helsinki protocols. The NBIA cohort comprised 69 individuals of different ethnicities and ages, born between 1932 and 2010 (summarized in Table 1). The clinical course varied substantially between patients, with age of onset in childhood for 45% of individuals and onset in adulthood for 42%.
Table 1 Clinical data for the NBIA cohort. NBIA (n = 69) Sex Age of manifestation Childhood Adolescence Adulthood n/a Major symptoms Ocular manifestations Dystonia Dysarthria Spasticity Gait disturbance Parkinsonism Ataxia Choreatic movements Developmental delay Cognitive decline Dementia Psychiatric disorders Seizures n/a MRI imaging Eye of the tiger sign T2 signal hypointensities of basal ganglia Other MRI abnormalities Without findings n/a Ethnicity European
Turkish Argentinian n/a Family history Affected relative(s) Inconspicuous Consanguineous n/a
34 males/35 females 31 (45%) 8 (11.6%) 29 (42%) 1 (1.4%) 4 (5.8%) 19 (27.5%) 12 (17.4%) 14 (20.3%) 11 (15.9%) 4 (5.8%) 5 (7.2%) 6 (8.7%) 10 (14.5%) 6 (8.7%) 5 (7.2%) 6 (8.7%) 7 (10.1%) 12 (17.4%) 7 (10.1%) 11 (15.9%) 13 (18.8%) 1 (1.4%) 37 (53.6%) German 53.6% French 5.8% other European 7.2% 14.5% 1.4% 17.4% 9 (13%) 27 (39.1%) 6 (8.7%) 27 (39.1%)
2.2. Mutational analysis 2.2.1. C19orf12 DNA was extracted from peripheral lymphocytes according to standard methods [9]. Mutation analysis of the C19orf12 gene in the NBIA cohort was performed by PCR amplification of all three exons followed by direct sequencing according to the method of Sanger. Primers were used as previously described by Hogarth et al. [6]; primer sequences are listed in Supplementary Table 1. The reference sequence corresponds to the longest transcript variant (NM_001031726.3) as shown in Dogu et al. [10]. 2.2.2. WDR45 Mutational analysis of the WDR45 gene in the NBIA cohort was conducted via high resolution melting (HRM) analysis followed by direct sequencing of atypical curves as described before [11]. Primers were designed with the LightScanner® Primer design software, and the primer sequences are listed in Supplementary Table 1. The reference sequence used was NM_007075.3. 3. Results 3.1. C19orf12 In three patients with suspected NBIA we identified homozygous mutations in C19orf12 that had been previously described as pathogenic (Table 2). Two patients (MPAN-1 and MPAN-2) showed the homozygous mutation c.C32T (p.Thr11Met) which only affects the longer transcript variant of C19orf12 and has been reported repeatedly [5,10,12, 13]. Both affected subjects were from consanguineous Turkish families,
but, to our knowledge, were not related to each other. Both presented in early adulthood with similar symptoms including dystonia, spasticity, progressive dementia and psychiatric disorders. Bilateral signal hypointensities in basal ganglia were also marked in both patients. DNA was only available from the parents of MPAN-1 who were heterozygous carriers of the Thr11Met mutation. One sister of MPAN-1 was similarly affected and equally found homozygous for Thr11Met. Since the age of 30, she had shown a rapidly progressing Parkinson-like phenotype with gait instability, frequent falls, speech difficulties, tremor and rigor, dystonia as well as dementia. Even though she was initially successfully treated with Levodopa and Amantadine, within two years she was requiring a high level of care and died from pneumonia at the age of 32 years. At least one sibling of MPAN-2 had also been diagnosed with a neurodegenerative disorder, but more detailed clinical information was not available for this family. MPAN-3 showed the homozygous recurrent mutation p.Gly69Arg [2,5,6,14]. She presented at age 10 with gait impairment and repeated falls due to spasticity of the lower limbs. Later on she developed optic atrophy and psychiatric symptoms such as impulse control difficulties and depression. MRI scans showed bilateral signal alteration in globus pallidus and crus cerebri. A variant frequently found in combination with the Gly69Arg missense mutation, c.424A NG, p.Lys142Glu (rs146170087) [6], was also found in our patient, albeit in heterozygous state. The non-consanguineous parents of MPAN-3 were confirmed to be heterozygous carriers of Gly69Arg, and the father additionally carried the Lys142Glu variant.
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Table 2 Mutations in the C19orf12 and WDR45 genes identified in the present study. Patient
Gene
Mutation
Main clinical symptoms
Mutation described in
MPAN-1 Male,*74
C19orf12, Ex2
c.32CNT p.Thr11Met (hom)
Deschauer et al. (2012) [15] Dogu et al. (2013) [10] Schulte et al. (2013) [12] Dezfouli et al. (2013) [13]
MPAN-2 Female, *84
C19orf12, Ex2
c.32CNT p.Thr11Met (hom)
MPAN-3 Female, *01
C19orf12, Ex3
BPAN-1 Female, *83
WDR45, Ex9
c.205GNA p.Gly69Arg (hom) c.424ANG p.Lys142Glu (het) c.626CNA p.Ala209Asp (het)
• Onset at ~25 years, progressive gait disturbance, dystonia, spasticity, dementia, hallucinations • T2-signal hypointensity in globus pallidus and substantia nigra, progressive cerebral atrophy • Signs of proximal denervation in EMG • Patient from Turkish consanguineous parents, sister similarly affected (age of onset: 28 years) • Psychiatric problems, rigidity, dementia and gait disturbance since the age of 20 • T2-signal hypointensity in globus pallidus • Patient from a Turkish consanguineous family with another affected sibling • Onset at 10 years, bilateral spasticity, MRI signal abnormalities in globus pallidus, optic atrophy, psychiatric symptoms • German patient, non-consanguineous family, no other affected family members • Severe global developmental delay in early infancy, expressive speech disorder, generalized seizures, hypertonia • Secondary worsening with progressive gait disturbance at 27 years of age • Hypointensities of globus pallidus in T2-weighed MRI • German patient with two healthy brothers, mother does not carry the mutation
present study
3.2. WDR45 Mutational analysis of WDR45 via HRM analysis showed a deviating curve that was confirmed to be, to our best knowledge, an unknown sequence alteration in only one patient with a diagnosis of NBIA. This patient (BPAN-1) harbored a heterozygous missense mutation resulting in a substitution of alanine by asparagine at position 209 (c.626C N A, p.Ala209Asp; see Fig. 1), that was uniformly predicted as damaging by standard prediction tools (MutationTaster, PolyPhen2, SIFT). The affected amino acid is highly conserved throughout species. Clinically, the female patient of German origin showed severe global developmental delay from early childhood on with expressive language impairment, seizures and a high muscle tone. From 27 years of age onwards, symptoms worsened with progressive gait impairment and frequent falls. MRI imaging showed T2 hypointensities in globus pallidus and substantia nigra, consistent with iron deposits, as well as moderately dilated ventricles (Fig. 2). The Ala209Asp mutation was not detected in the subject's mother; her father could not be analyzed because he was already deceased. He had not shown any neurological symptoms. The patient has two healthy brothers, and the mother had had two early miscarriages.
Hartig et al. (2011) [5] Goldmann et al. (2013) [14] Hogarth et al. (2013) [6]
comparatively low frequency may be due to the large phenotypic spectrum of patients referred for evaluation in the internal registry used for the present study.
4. Discussion Mutations in the C19orf12 and WDR45 genes have recently been identified as causative for the two NBIA subtypes MPAN and BPAN, respectively. In order to assess the phenotypic and genotypic spectrum of MPAN, some studies had already been conducted following its first description in 2011 [5]. Little is known, however, about the impact of WDR45 mutations in patients that do not fulfill the strict criteria of SENDA. In the present study we aimed at evaluating the mutational and phenotypic spectrum for the C19orf12 and WDR45 genes. It has been suggested that MPAN overall accounts for 6–10% of NBIA cases [16]. However, the reported mutational frequencies differed substantially between studies. The original report in a homogeneous Polish population found C19orf12 mutations in 13/23 (N 50%) of patients without PANK2 mutations; however, because of a founder mutation, this frequency is probably by far overestimated [5]. In a cohort evaluated by Hogarth et al. [6], 23/161 (14.3%) of PANK2- and PLA2G6negative NBIA patients had alterations in C19orf12, but in an Italian cohort, only 3/46 (6.5%) could be confirmed as suffering from MPAN [17]. Comparable to this latter report, in the present study we identified homozygous pathogenic mutations in C19orf12 in 3/69 patients (4.3%) formerly tested negative for mutations in PANK2 and PLA2G6. This
Fig. 1. Pedigree, genomic sequence and amino acid alignment for patient BPAN-1, showing a novel heterozygous missense mutation in the WDR45 gene. a) Pedigree of patient BPAN1 b) sequence of BPAN-1 and her mother, along with the reference sequence c) amino acid alignment corresponding to the shown sequence with amino acid position, human, rhesus, mouse, dog, X. Tropicalis and zebrafish alignments.
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A
B
Fig. 2. MRI scan of patient BPAN-1 carrying a novel heterozygous missense mutation in WDR45 (c.626CNA, p.Ala209Asp) at the age of 30 years. A AxialT2-weighted MR images show an abnormal hypointense signal of the globus pallidus typical for iron accumulation (arrows). The “eye of the tiger” sign is absent. B AxialT2-weighted gradient echo MR images of the same patient reveal a signal drop in the globus pallidus that is characteristic for pathologic iron accumulation (arrows).
The Thr11Met mutation has been described in a number of publications [5,10,12,13,15]. Both patients carrying this mutation in homozygous state in the present investigation (MPAN-1 and MPAN-2) were from consanguineous Turkish families. Clinical presentation of the two patients, with disease onset in early adulthood, severe cognitive decline and psychiatric problems, was similar to that of other cases homozygous for this mutation in an Iranian [13] and a Turkish [10] cohort, as well as to a single Polish case [5]. Taking into account data from our two patients as well as the patients described in the literature, it appears that the Thr11Met mutation may be associated with a later age of onset (in early adulthood) which might be due to the fact that this mutation only affects the longer splice variant [5,12]. On the other hand, disease progression seemed rather rapid for this mutation. The recurrent mutation p.Gly69Arg [5,6,14] is supposed to disrupt the subcellular distribution of C19orf12 [2]. To our knowledge, only one other patient equally carrying this mutation in homozygous state has been described in the literature that showed a disease course comparable to our patient MPAN-3 (age of onset in the first decade, bilateral spasticity and optic atrophy — i.e. ID2 in Ref. [5]). In our patient, we also found another missense variant (c.424AN G, p.Lys142Glu) in heterozygous state that was suggested to be an innocuous SNP co-segregating with the Gly69Arg mutation [6]. Our data confirm the notion that this missense variant is probably not pathogenic but argue against complete co-segregation of this sequence variant with the Gly69Arg mutation, since it was only found on the paternal, but not the maternal allele. Mutations in WDR45 were so far identified in only a small fraction of NBIA cases with a highly uniform phenotype consisting of childhood encephalopathy followed by neurodegeneration in adulthood (a syndrome formerly named SENDA) [8,16]. However, it was suggested that the phenotypic spectrum might broaden when more patients with different disease courses would be investigated [18]. We therefore screened our entire heterogeneous NBIA cohort also for WDR45 mutations and detected a heterozygous missense mutation in only one patient (BPAN-1) whose phenotype very much corresponds to that of the cases described for SENDA in the literature. The female German patient carrying this mutation initially presented with severe global developmental delay that was suspected to be caused by oxygen deficiency during delivery. At the age of 27, her condition began to deteriorate with progressive gait disturbance and frequent falls, and MRI scans at that time were suggestive of NBIA. The novel heterozygous missense mutation (c.626C N A p.Ala209Asp) in exon 9 of the WDR45 gene identified in this patient was uniformly predicted as damaging by
standard prediction tools and affects an amino acid that is highly conserved throughout species. We therefore strongly suspect it to be pathogenic; however, functional studies are needed to further evaluate the pathogenic relevance of this mutation. The mutation was not identified in the patient's mother, but since the father was not available for testing, we can only assume that it had occurred de novo in our patient. We are aware of the fact that mutation scanning with the HRM method, as used for the analysis of WDR45 in the present study, may not be as sensitive as complete sequence analysis. Yet, this method has been successfully used for a number of different diagnostic settings [19] and a recent meta-analysis for detection of BRAF mutations identified an overall sensitivity and specificity of 0.99 for the HRM method [20], so that we feel confident that we have not missed mutations. In conclusion, we confirmed c19orf12 mutations at a noticeable but comparatively low frequency in a clinically heterogeneous NBIA cohort. WDR45 mutations, on the other hand, appear infrequent in NBIA patients that do not show the typical SENDA phenotype. Since the BPAN phenotype in childhood can include hand stereotypes, seizures, disordered sleep and spasticity, features strongly reminiscent of Rett syndrome, it was suggested that WDR45 should be evaluated in patients with a Rett-like phenotype that lack mutations in known Rett genes [18]. This theory is supported by the recent report of a patient that showed a typical clinical Rett phenotype and carried a novel splice mutation in WDR45 [21]. Therefore, investigating additional Rett cohorts may further broaden the phenotypic spectrum of WDR45 mutations in the near future. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jns.2014.12.036. Financial disclosures of all authors Anne Tschentscher was supported by a dissertation grant of the “Heinrich und Alma Vogelsang Stiftung”. The authors declare that they have no conflict of interest. Acknowledgements Anne Tschentscher was supported by a dissertation grant of the “Heinrich und Alma Vogelsang Stiftung”. We thank the patients and their families for participating in the study.
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Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Analysis of the C19orf12 and WDR45 genes in patients with neurodegeneration with brain iron accumulation Anne Tschentscher a, Gabriele Dekomien a, Sophia Ross b, Kirsten Cremer c, Guido M. Kukuk d, Jörg T. Epplen a,e, Sabine Hoffjan a,⁎ a
Department of Human Genetics, Ruhr-University, Bochum, Germany Pediatric Neurology, Children's Hospital Erlangen, Germany Department of Human Genetics, University of Bonn, Germany d Department of Radiology, University of Bonn, Germany e Faculty of Health, University Witten/Herdecke, Witten, Germany b c
a r t i c l e
i n f o
Article history: Received 1 July 2014 Received in revised form 24 November 2014 Accepted 24 December 2014 Available online 3 January 2015 Keywords: NBIA C19orf12 WDR45 MPAN BPAN Mutation analysis
a b s t r a c t Background: Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of diseases presenting with movement disorders and brain iron deposits. In addition to NBIA subtypes caused by mutations in PANK2 and PLA2G6, mutations in the C19orf12 gene were recently described as the third frequent cause of NBIA (called mitochondrial membrane protein-associated neurodegeneration, MPAN). Additionally, the X-linked gene WDR45 was found causative for a special subtype named static encephalopathy in childhood with neurodegeneration in adulthood (also called BPAN); however, analysis of this gene in a broader spectrum of NBIA has not been reported yet. Methods: In a heterogeneous cohort of 69 patients with suspected NBIA that did not carry mutations in PANK2 and PLA2G6, the coding region of C19orf12 was evaluated by Sanger sequencing. The WDR45 gene was analyzed via high resolution melting and subsequent sequence analysis. Results: Previously described homozygous C19orf12 mutations were found in 3/69 NBIA patients (4.3%). Analysis of the WDR45 gene revealed a novel heterozygous missense mutation in one female NBIA patient showing psychomotor retardation with secondary decline. Conclusions: C19orf12 mutations were confirmed in our heterogeneous NBIA cohort, while WDR45 mutations appear to be restricted to the subtype showing encephalopathy in childhood with neurodegeneration in adulthood. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Neurodegeneration with brain iron accumulation (NBIA) is a clinically and genetically heterogeneous group of neurodegenerative diseases, typically comprising movement disorders and brain iron deposits [1]. The wide phenotypic spectrum of NBIA, as well as parallels to other disorders such as Parkinsonism or spastic paraplegia [1,2], can make diagnosis difficult and, in some cases, the underlying genetic mechanisms still remain unknown.
Abbreviations: NBIA, neurodegeneration with brain iron accumulation; MPAN, mitochondrial membrane protein-associated neurodegeneration; BPAN, beta-propeller protein-associated neurodegeneration; PKAN, panthothenate kinase-associated neurodegeneration; PLAN, Phospholipase A2-associated neurodegeneration; HRM, high resolution melting; SENDA, static encephalopathy in childhood with neurodegeneration in adulthood. ⁎ Corresponding author at: Department of Human Genetics, Ruhr-University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany. Tel.: +49 234 3223823; fax: + 49 234 3214196. E-mail address: [email protected] (S. Hoffjan).
http://dx.doi.org/10.1016/j.jns.2014.12.036 0022-510X/© 2014 Elsevier B.V. All rights reserved.
The first subtype of NBIA, pantothenate kinase-associated neurodegeneration (PKAN) caused by mutations in the PANK2 gene, was described in 2001 [3]. Since then, mutations in various genes, such as PLA2G6, FA2H, FTL, AP and ATP13A2, have been added to the growing list of clinically and genetically distinct entities associated with brain iron accumulation [4]. Most recently, mutations in the C19orf12 and WDR45 genes were identified as additional causes for NBIA. Mutations in C19orf12 were first described in a homogeneous cohort from Poland where most cases carried an 11bp deletion founder mutation [5]. In a subsequent larger multinational study, C19orf12 mutations were found associated with a wide spectrum of phenotypes, typically including motor neuronopathy, cognitive decline and neuropsychiatric impairment [6]. MRI scans showed T2-hypointensities in globus pallidus and substantia nigra without an eye of the tiger sign [5]. Due to localization of the gene product in mitochondria, the name mitochondrial membrane protein-associated neurodegeneration (MPAN) was proposed for this clinical entity [5]. MPAN was suggested to be the third frequent NBIA subtype after PKAN (PANK2 gene) and PLAN
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(PLA2G6); however the mutational frequencies varied substantially between studies. Mutations in the WDR45 gene, located on the X chromosome, were recently identified in patients with a very specific phenotype that is referred to as static encephalopathy in childhood with neurodegeneration in adulthood (SENDA). This new X-linked NBIA subtype, affecting a gene product involved in autophagy [7], has been named beta-propeller protein-associated neurodegeneration (BPAN) [8]. Most affected individuals are females and all reported mutations so far occurred de novo. However, a few males with WDR45 mutations and a SENDA phenotype, most of them probably showing somatic mosaicism, have also been described [8]. To our knowledge, the frequency of WDR45 mutations in patients with a broader clinical NBIA phenotype has not been investigated yet. In order to further evaluate the mutational and clinical spectra of these two newly described NBIA subtypes, we screened a cohort of PANK2 and PLA2G6 mutation-negative patients clinically presenting with NBIA (n = 69) for mutations in C19orf12 and WDR45. 2. Materials and methods 2.1. Patient cohort Patients were recruited from an internal registry of the Department of Human Genetics of the Ruhr-University Bochum, including DNA samples sent by clinicians for the diagnosis of NBIA during the years 2006–2013 which had been tested negative for mutations in the PANK2 or PLA2G6 genes. All samples were obtained with informed consent of probands or their legal representatives. The study was approved by the Ethics Committee of the Ruhr-University Bochum and carried out in accordance with the Declaration of Helsinki protocols. The NBIA cohort comprised 69 individuals of different ethnicities and ages, born between 1932 and 2010 (summarized in Table 1). The clinical course varied substantially between patients, with age of onset in childhood for 45% of individuals and onset in adulthood for 42%.
Table 1 Clinical data for the NBIA cohort. NBIA (n = 69) Sex Age of manifestation Childhood Adolescence Adulthood n/a Major symptoms Ocular manifestations Dystonia Dysarthria Spasticity Gait disturbance Parkinsonism Ataxia Choreatic movements Developmental delay Cognitive decline Dementia Psychiatric disorders Seizures n/a MRI imaging Eye of the tiger sign T2 signal hypointensities of basal ganglia Other MRI abnormalities Without findings n/a Ethnicity European
Turkish Argentinian n/a Family history Affected relative(s) Inconspicuous Consanguineous n/a
34 males/35 females 31 (45%) 8 (11.6%) 29 (42%) 1 (1.4%) 4 (5.8%) 19 (27.5%) 12 (17.4%) 14 (20.3%) 11 (15.9%) 4 (5.8%) 5 (7.2%) 6 (8.7%) 10 (14.5%) 6 (8.7%) 5 (7.2%) 6 (8.7%) 7 (10.1%) 12 (17.4%) 7 (10.1%) 11 (15.9%) 13 (18.8%) 1 (1.4%) 37 (53.6%) German 53.6% French 5.8% other European 7.2% 14.5% 1.4% 17.4% 9 (13%) 27 (39.1%) 6 (8.7%) 27 (39.1%)
2.2. Mutational analysis 2.2.1. C19orf12 DNA was extracted from peripheral lymphocytes according to standard methods [9]. Mutation analysis of the C19orf12 gene in the NBIA cohort was performed by PCR amplification of all three exons followed by direct sequencing according to the method of Sanger. Primers were used as previously described by Hogarth et al. [6]; primer sequences are listed in Supplementary Table 1. The reference sequence corresponds to the longest transcript variant (NM_001031726.3) as shown in Dogu et al. [10]. 2.2.2. WDR45 Mutational analysis of the WDR45 gene in the NBIA cohort was conducted via high resolution melting (HRM) analysis followed by direct sequencing of atypical curves as described before [11]. Primers were designed with the LightScanner® Primer design software, and the primer sequences are listed in Supplementary Table 1. The reference sequence used was NM_007075.3. 3. Results 3.1. C19orf12 In three patients with suspected NBIA we identified homozygous mutations in C19orf12 that had been previously described as pathogenic (Table 2). Two patients (MPAN-1 and MPAN-2) showed the homozygous mutation c.C32T (p.Thr11Met) which only affects the longer transcript variant of C19orf12 and has been reported repeatedly [5,10,12, 13]. Both affected subjects were from consanguineous Turkish families,
but, to our knowledge, were not related to each other. Both presented in early adulthood with similar symptoms including dystonia, spasticity, progressive dementia and psychiatric disorders. Bilateral signal hypointensities in basal ganglia were also marked in both patients. DNA was only available from the parents of MPAN-1 who were heterozygous carriers of the Thr11Met mutation. One sister of MPAN-1 was similarly affected and equally found homozygous for Thr11Met. Since the age of 30, she had shown a rapidly progressing Parkinson-like phenotype with gait instability, frequent falls, speech difficulties, tremor and rigor, dystonia as well as dementia. Even though she was initially successfully treated with Levodopa and Amantadine, within two years she was requiring a high level of care and died from pneumonia at the age of 32 years. At least one sibling of MPAN-2 had also been diagnosed with a neurodegenerative disorder, but more detailed clinical information was not available for this family. MPAN-3 showed the homozygous recurrent mutation p.Gly69Arg [2,5,6,14]. She presented at age 10 with gait impairment and repeated falls due to spasticity of the lower limbs. Later on she developed optic atrophy and psychiatric symptoms such as impulse control difficulties and depression. MRI scans showed bilateral signal alteration in globus pallidus and crus cerebri. A variant frequently found in combination with the Gly69Arg missense mutation, c.424A NG, p.Lys142Glu (rs146170087) [6], was also found in our patient, albeit in heterozygous state. The non-consanguineous parents of MPAN-3 were confirmed to be heterozygous carriers of Gly69Arg, and the father additionally carried the Lys142Glu variant.
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Table 2 Mutations in the C19orf12 and WDR45 genes identified in the present study. Patient
Gene
Mutation
Main clinical symptoms
Mutation described in
MPAN-1 Male,*74
C19orf12, Ex2
c.32CNT p.Thr11Met (hom)
Deschauer et al. (2012) [15] Dogu et al. (2013) [10] Schulte et al. (2013) [12] Dezfouli et al. (2013) [13]
MPAN-2 Female, *84
C19orf12, Ex2
c.32CNT p.Thr11Met (hom)
MPAN-3 Female, *01
C19orf12, Ex3
BPAN-1 Female, *83
WDR45, Ex9
c.205GNA p.Gly69Arg (hom) c.424ANG p.Lys142Glu (het) c.626CNA p.Ala209Asp (het)
• Onset at ~25 years, progressive gait disturbance, dystonia, spasticity, dementia, hallucinations • T2-signal hypointensity in globus pallidus and substantia nigra, progressive cerebral atrophy • Signs of proximal denervation in EMG • Patient from Turkish consanguineous parents, sister similarly affected (age of onset: 28 years) • Psychiatric problems, rigidity, dementia and gait disturbance since the age of 20 • T2-signal hypointensity in globus pallidus • Patient from a Turkish consanguineous family with another affected sibling • Onset at 10 years, bilateral spasticity, MRI signal abnormalities in globus pallidus, optic atrophy, psychiatric symptoms • German patient, non-consanguineous family, no other affected family members • Severe global developmental delay in early infancy, expressive speech disorder, generalized seizures, hypertonia • Secondary worsening with progressive gait disturbance at 27 years of age • Hypointensities of globus pallidus in T2-weighed MRI • German patient with two healthy brothers, mother does not carry the mutation
present study
3.2. WDR45 Mutational analysis of WDR45 via HRM analysis showed a deviating curve that was confirmed to be, to our best knowledge, an unknown sequence alteration in only one patient with a diagnosis of NBIA. This patient (BPAN-1) harbored a heterozygous missense mutation resulting in a substitution of alanine by asparagine at position 209 (c.626C N A, p.Ala209Asp; see Fig. 1), that was uniformly predicted as damaging by standard prediction tools (MutationTaster, PolyPhen2, SIFT). The affected amino acid is highly conserved throughout species. Clinically, the female patient of German origin showed severe global developmental delay from early childhood on with expressive language impairment, seizures and a high muscle tone. From 27 years of age onwards, symptoms worsened with progressive gait impairment and frequent falls. MRI imaging showed T2 hypointensities in globus pallidus and substantia nigra, consistent with iron deposits, as well as moderately dilated ventricles (Fig. 2). The Ala209Asp mutation was not detected in the subject's mother; her father could not be analyzed because he was already deceased. He had not shown any neurological symptoms. The patient has two healthy brothers, and the mother had had two early miscarriages.
Hartig et al. (2011) [5] Goldmann et al. (2013) [14] Hogarth et al. (2013) [6]
comparatively low frequency may be due to the large phenotypic spectrum of patients referred for evaluation in the internal registry used for the present study.
4. Discussion Mutations in the C19orf12 and WDR45 genes have recently been identified as causative for the two NBIA subtypes MPAN and BPAN, respectively. In order to assess the phenotypic and genotypic spectrum of MPAN, some studies had already been conducted following its first description in 2011 [5]. Little is known, however, about the impact of WDR45 mutations in patients that do not fulfill the strict criteria of SENDA. In the present study we aimed at evaluating the mutational and phenotypic spectrum for the C19orf12 and WDR45 genes. It has been suggested that MPAN overall accounts for 6–10% of NBIA cases [16]. However, the reported mutational frequencies differed substantially between studies. The original report in a homogeneous Polish population found C19orf12 mutations in 13/23 (N 50%) of patients without PANK2 mutations; however, because of a founder mutation, this frequency is probably by far overestimated [5]. In a cohort evaluated by Hogarth et al. [6], 23/161 (14.3%) of PANK2- and PLA2G6negative NBIA patients had alterations in C19orf12, but in an Italian cohort, only 3/46 (6.5%) could be confirmed as suffering from MPAN [17]. Comparable to this latter report, in the present study we identified homozygous pathogenic mutations in C19orf12 in 3/69 patients (4.3%) formerly tested negative for mutations in PANK2 and PLA2G6. This
Fig. 1. Pedigree, genomic sequence and amino acid alignment for patient BPAN-1, showing a novel heterozygous missense mutation in the WDR45 gene. a) Pedigree of patient BPAN1 b) sequence of BPAN-1 and her mother, along with the reference sequence c) amino acid alignment corresponding to the shown sequence with amino acid position, human, rhesus, mouse, dog, X. Tropicalis and zebrafish alignments.
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A
B
Fig. 2. MRI scan of patient BPAN-1 carrying a novel heterozygous missense mutation in WDR45 (c.626CNA, p.Ala209Asp) at the age of 30 years. A AxialT2-weighted MR images show an abnormal hypointense signal of the globus pallidus typical for iron accumulation (arrows). The “eye of the tiger” sign is absent. B AxialT2-weighted gradient echo MR images of the same patient reveal a signal drop in the globus pallidus that is characteristic for pathologic iron accumulation (arrows).
The Thr11Met mutation has been described in a number of publications [5,10,12,13,15]. Both patients carrying this mutation in homozygous state in the present investigation (MPAN-1 and MPAN-2) were from consanguineous Turkish families. Clinical presentation of the two patients, with disease onset in early adulthood, severe cognitive decline and psychiatric problems, was similar to that of other cases homozygous for this mutation in an Iranian [13] and a Turkish [10] cohort, as well as to a single Polish case [5]. Taking into account data from our two patients as well as the patients described in the literature, it appears that the Thr11Met mutation may be associated with a later age of onset (in early adulthood) which might be due to the fact that this mutation only affects the longer splice variant [5,12]. On the other hand, disease progression seemed rather rapid for this mutation. The recurrent mutation p.Gly69Arg [5,6,14] is supposed to disrupt the subcellular distribution of C19orf12 [2]. To our knowledge, only one other patient equally carrying this mutation in homozygous state has been described in the literature that showed a disease course comparable to our patient MPAN-3 (age of onset in the first decade, bilateral spasticity and optic atrophy — i.e. ID2 in Ref. [5]). In our patient, we also found another missense variant (c.424AN G, p.Lys142Glu) in heterozygous state that was suggested to be an innocuous SNP co-segregating with the Gly69Arg mutation [6]. Our data confirm the notion that this missense variant is probably not pathogenic but argue against complete co-segregation of this sequence variant with the Gly69Arg mutation, since it was only found on the paternal, but not the maternal allele. Mutations in WDR45 were so far identified in only a small fraction of NBIA cases with a highly uniform phenotype consisting of childhood encephalopathy followed by neurodegeneration in adulthood (a syndrome formerly named SENDA) [8,16]. However, it was suggested that the phenotypic spectrum might broaden when more patients with different disease courses would be investigated [18]. We therefore screened our entire heterogeneous NBIA cohort also for WDR45 mutations and detected a heterozygous missense mutation in only one patient (BPAN-1) whose phenotype very much corresponds to that of the cases described for SENDA in the literature. The female German patient carrying this mutation initially presented with severe global developmental delay that was suspected to be caused by oxygen deficiency during delivery. At the age of 27, her condition began to deteriorate with progressive gait disturbance and frequent falls, and MRI scans at that time were suggestive of NBIA. The novel heterozygous missense mutation (c.626C N A p.Ala209Asp) in exon 9 of the WDR45 gene identified in this patient was uniformly predicted as damaging by
standard prediction tools and affects an amino acid that is highly conserved throughout species. We therefore strongly suspect it to be pathogenic; however, functional studies are needed to further evaluate the pathogenic relevance of this mutation. The mutation was not identified in the patient's mother, but since the father was not available for testing, we can only assume that it had occurred de novo in our patient. We are aware of the fact that mutation scanning with the HRM method, as used for the analysis of WDR45 in the present study, may not be as sensitive as complete sequence analysis. Yet, this method has been successfully used for a number of different diagnostic settings [19] and a recent meta-analysis for detection of BRAF mutations identified an overall sensitivity and specificity of 0.99 for the HRM method [20], so that we feel confident that we have not missed mutations. In conclusion, we confirmed c19orf12 mutations at a noticeable but comparatively low frequency in a clinically heterogeneous NBIA cohort. WDR45 mutations, on the other hand, appear infrequent in NBIA patients that do not show the typical SENDA phenotype. Since the BPAN phenotype in childhood can include hand stereotypes, seizures, disordered sleep and spasticity, features strongly reminiscent of Rett syndrome, it was suggested that WDR45 should be evaluated in patients with a Rett-like phenotype that lack mutations in known Rett genes [18]. This theory is supported by the recent report of a patient that showed a typical clinical Rett phenotype and carried a novel splice mutation in WDR45 [21]. Therefore, investigating additional Rett cohorts may further broaden the phenotypic spectrum of WDR45 mutations in the near future. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jns.2014.12.036. Financial disclosures of all authors Anne Tschentscher was supported by a dissertation grant of the “Heinrich und Alma Vogelsang Stiftung”. The authors declare that they have no conflict of interest. Acknowledgements Anne Tschentscher was supported by a dissertation grant of the “Heinrich und Alma Vogelsang Stiftung”. We thank the patients and their families for participating in the study.
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