Journal of the Neurological Sciences 347 (2014) 352–355
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Short communication
A complex form of hereditary spastic paraplegia in three siblings due to somatic mosaicism for a novel SPAST mutation in the mother Anna Aulitzky a, Katrin Friedrich b,c, Dieter Gläser d, Regina Gastl a, Christian Kubisch b,c, Albert C. Ludolph a,⁎, Alexander E. Volk b,c,⁎⁎ a
Department of Neurology, University Hospital Ulm, Oberer Eselsberg 45, 89081 Ulm, Germany Institute of Human Genetics, University of Ulm, Albert-Einstein Allee 11, 89081 Ulm, Germany Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany d genetikum, Laboratory of Cytogenetics and Molecular Genetics, Wegener Str. 15, 89231 Neu-Ulm, Germany b c
a r t i c l e
i n f o
Article history: Received 24 July 2014 Received in revised form 11 September 2014 Accepted 24 September 2014 Available online 2 October 2014 Keywords: Hereditary spastic paraplegia SPG4 SPAST Somatic mosaicism Genetic testing De novo mutation
a b s t r a c t Hereditary spastic paraplegias (HSPs) represent a clinically and genetically heterogeneous group of diseases. Major symptoms comprise progressive bilateral leg stiffness, spasticity at rest and diffuse muscle weakness. Complex forms are characterized by additional symptoms like dementia, cerebellar dysfunction or seizures. Autosomal dominant, autosomal recessive, X-linked recessive and possibly mitochondrial inheritance have been described in familial HSP. The most frequently mutated gene in familial cases of uncomplicated autosomal dominant HSP is SPAST, however de novo mutations in SPAST are rarely found. Here, we report on the clinical and genetic findings in a family with three children afflicted by complex HSP and their unaffected parents. Although autosomal dominant inheritance seemed unlikely in this family, genetic testing revealed a novel SPAST mutation, c.1837GNC (p.Asp613His), in a heterozygous state in all affected individuals and somatic mosaicism of this mutation in the unaffected mother. Our study thus expands the knowledge on SPAST-associated HSP and emphasizes that de novo mutations and somatic mosaicism should be taken into consideration in HSP families presenting with a family history not suggestive for an autosomal dominant inheritance pattern. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hereditary spastic paraplegias are a clinically and genetically heterogeneous group of neurodegenerative disorders. Pure and complex forms are described. In pure HSP, progressive spasticity and weakness of the lower limbs with increased reflexes and a positive Babinski's sign are the leading symptoms. Complex HSP in contrast presents with variable additional symptoms like dementia, seizures, optic atrophy, ataxia, polyneuropathy, dystonia, hypogonadism, and microcephaly [1,2]. Urinary incontinence is a frequently and fecal incontinence a less frequently observed accompanying symptom in pure and complex HSP [3,4]. HSP is genetically highly heterogeneous with nearly all possible monogenic inheritance patterns described (i.e. autosomal dominant, autosomal recessive, X-linked recessive and possibly also mitochondrial transmission). To date, around 60 HSP related genes have been identified [5,6]. Mutations in the SPAST gene (SPG4) account for up to 50% of ⁎ Corresponding author. Tel.: +49 731 177 1201; fax: +49 731 177 1202. ⁎⁎ Correspondence to: A.E. Volk, Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Tel.: +49 40 7410 52120; fax: +49 40 7410 55138. E-mail addresses: [email protected] (A.C. Ludolph), [email protected] (A.E. Volk).
http://dx.doi.org/10.1016/j.jns.2014.09.046 0022-510X/© 2014 Elsevier B.V. All rights reserved.
autosomal dominant HSP. Intrafamilial variability is considerable in families with SPAST-associated HSP. In most cases, mutations in SPAST are associated with an uncomplicated form of HSP, but complex forms have also been described [7,8]. Most individuals with SPG4 have inherited a mutation from an affected parent although the exact proportion of de novo SPAST mutations is unknown [9]. There is a single report in which somatic mosaicism for a SPAST mutation in one individual with late onset HSP is associated with HSP in his daughter and his grandson [10]. We now report another family with complex HSP in two siblings and one half-sibling due to somatic mosaicism for a novel SPAST mutation in the unaffected mother. 2. Methods All patients were examined by an experienced neurologist at the University Hospital Ulm and underwent magnetic resonance imaging and neurophysiological studies. Blood samples for genetic testing of all affected individuals and the unaffected mother were collected. Informed written consent was obtained for all individuals. The study was approved by the Ethics Committee of the University of Ulm. DNA was extracted from peripheral blood lymphocytes using a column extraction kit (Qiagen). Genetic testing was performed by direct sequencing and multiplex ligation dependent probe amplification (MPLA kit SALSA MLPA kit P165 HSP/
A. Aulitzky et al. / Journal of the Neurological Sciences 347 (2014) 352–355
Spastin; MRC-Holland). Primer sequences are available upon request. The sequences were compared to GenBank entry NM_014946.3. The mutation identified was checked in public databases (dbSNP (http:// www.ncbi.nlm.nih.gov/snp/), 1000 Genomes browser (http:// browser.1000genomes.org/index.html), Exome Variant Server (http://evs.gs.washington.edu/EVS/)), the Human Gene Mutation Database (HGMD® professional) and the mutation prediction programs Polyphen-2 [11], MutationTaster [12] and SIFT [13]. Multiple sequence alignment was performed with Clustal Omega (http:// www.ebi.ac.uk/Tools/msa/clustalo/). 3. Results Three children with complex spastic paraplegia and their unaffected mother were investigated (Fig. 1). Spastic gait was the initial symptom in the eldest girl (II.1; Fig. 1) which was first recognized at the age of three years. Symptoms slowly progressed over time and at an age of 26 years she presented with characteristic HSP-associated symptoms such as increased muscle reflexes of the lower limbs, a positive Babinski's sign, contractures of the joints and a slight paresis of the extensors on the lower limbs. Furthermore, she displayed signs of cerebellar dysfunction like dysmetria and dysdiadochokinesis. A brain MRI showed thinning of the corpus callosum. Her maternal half-brother (II.2, Fig. 1) developed progressive walking disturbances and spastic gait by the age of four years. At the age of 19 years he presented with spastic gait, increased reflexes, contractures of the lower limbs and cerebellar dysfunction. A brain MRI was unremarkable. He had no paresis but suffered from urinary and fecal incontinence. He is mentally handicapped, however no formal IQ testing was available. In the youngest sibling (II.3, Fig. 1) motor and cognitive development was delayed and at the age of three years spastic gait became evident. At the age of 16 years, he presented increased reflexes, contractures of the lower limbs, Babinski's sign, and cerebellar involvement. A clinical overview of all three patients is given in Table 1. Most probably unrelated to HSP, individual II.2 also showed hyperlordosis and individual II.3 had neurodermatitis. Neither their common mother nor their respective fathers were reported to suffer from HSP or any other neurodegenerative disorder, and a clinical evaluation of the mother at the age of 44 years did not show any signs of HSP. The family history was unremarkable with respect to neurologic and neurodegenerative diseases in other family members (as e.g. in the parents of the mother or the respective fathers) and there were no additional siblings or children of the affected individuals. Although a hereditary origin of the disease seemed likely, the family history offered no clear clue about the exact mode of inheritance. Autosomal dominant inheritance did not seem to be very likely, as the mother of three affected children showed no symptoms. Likewise autosomal recessive inheritance looked rather unlikely as the three affected children did not have a common father and as both fathers are not related to each other or to the mother. Last but not least, an X-chromosomal recessive mode of inheritance was not absolutely convincing due to
I 1
2
3
II 1
2
3
Fig. 1. Pedigree of the family. Circles represent females, squares represent males. Filled symbols indicate affected individuals. II.1 is the half-sister of the two brothers II.2 und II.3.
353
Table 1 Clinical details. Patient ID Age at examination Sex Initial symptom Age at onset Spastic upper limb Spastic lower limb Contractures upper limbs Contractures lower limb Reflexes upper limbs Reflexes lower limbs Babinski's sign Paresis upper limbs Paresis lower limbs Pallesthesia upper limbs Pallesthesia lower limbs Cerebellar signs Mental retardation Incontinence
II.1 26 female Spastic gait 4 − + − + ++ +++ + − + 8/8 7/8 + − −
II.2 19 male Spastic gait 4 − + − + ++ +++ + − − 8/8 8/8 + + Urinary and fecal
II.3 16 male Spastic gait 3 − + − − ++ +++ + − − 8/8 8/8 + + −
rather severe disease in the affected girl. In principle, the pedigree might suggest maternal inheritance due to a mitochondrial mutation. However convincing evidence about a causative role of mutations in the mitochondrial genome in early-onset complicated HSP is missing even if a single publication reported a HSP-like late-onset disease with a mitochondrial mutation in a single family [14]. In a more pragmatic approach, we therefore ought to first test the most common disease genes for HSP regardless of the underlying inheritance pattern. Genetic testing of the SPAST gene was unremarkable in the MLPA analysis, however sequencing revealed a substitution of guanine to cytosine at cDNA position 1837 (c.1837GNC; Fig. 2A) in a heterozygous state in patient II.2. On protein level this variation is predicted to lead to a substitution of a negatively charged asparagine by histidine (p.Asp613His). This substitution had not been described in patients with HSP before according to the Human Gene Mutation Database. The mutation was not listed in ~12,500 control alleles sequenced by the NHLBI Exome Sequencing Project and was not annotated in either dbSNP or the 1000 Genomes Project database. The affected amino acid is highly conserved among species (Fig. 2B) and predicted to be deleterious by different prediction programs (Polyphen-2, MutationTaster, SIFT; Fig. 2C). Even more, the mutation segregates with the phenotype and was also present in the other affected individuals (II.1, II.3; Fig. 2A) in a heterozygous state. Interestingly, genetic testing of the mother reproducibly showed a low-grade somatic mosaicism for the mutation c.1837GNC at least in blood cells (Fig. 2A), which after all explained the unusual family history. 4. Discussion Direct sequencing of the SPAST gene revealed the novel mutation c.1837GNC in a heterozygous state in three half-siblings with a complex form of HSP. Genetic testing of this specific mutation in the healthy mother proved somatic mosaicism in her as the altered allele could also be detected in DNA derived from blood cells. The mutation is most probably pathogenic as it (i) segregates with the phenotype, (ii) has not been listed in public databases precluding that it represents a more common polymorphism, (iii) is highly conserved among species and (iv) is predicted to be pathogenic by different prediction programs. Furthermore, a substitution of this asparagine to a nonpolar alanine (p.Asp613Ala) has previously been described in another patient with HSP [15] pointing to a crucial role of this amino acid for the function of spastin. In contrast to the patients described here, the patient with p.Asp613Ala showed a pure HSP and a later age of onset (36 years). Our patients had early-onset HSP accompanied by cerebellar disturbances and mental retardation.
354
A. Aulitzky et al. / Journal of the Neurological Sciences 347 (2014) 352–355
D
F
G
D
T
T
V
A
*
control D
F
G
D/H
T
T
V
B D. rerio X. laevis chicken human mouse
*
C
G/C
I.2 G/C
II.1 G/C
II.2 G/C
II.3
Fig. 2. A: Electropherograms showing the mutation c.1837GNC of the SPAST gene (arrow) in individuals II.1, II.2, II.3 in a heterozygous state. The altered allele carrying a cytosine could also, although to a lesser amount be detected in DNA derived from blood cells in the mother (I.2). B: Multiple sequence alignment of SPAST orthologs surrounding the D613 position in the human protein. C: Results of different prediction programs for the mutations p.Asp613His and p.Asp613Ala.
In respect of the clinical phenotype, it is interesting that the mutation p.Asp613Ala was – in contrast to the mutation p.Asp613His identified in our patients – predicted to be tolerated by SIFT which may point to a possible genotype–phenotype correlation (Fig. 2C). The family history of the patient described before was unremarkable. If this may suggest a de novo mutation is speculative but may indicate a hot spot for mutations in SPAST and should be kept in mind for further studies. In our family the novel mutation c.1837GNC occurred de novo in the mother. The mutation is present in the mother's germline as all three affected children inherited the mutation. As the mutation could also be detected in blood the mutation event in the mother must have occurred at an early embryonic stage. Whether the mutation is also present in neuroectodermal tissue and especially in the central nervous system is unclear. Therefore, a prediction on a possible clinical phenotype in the mother is not possible and a later onset of disease symptoms in the mother cannot be excluded. On the first view, the pedigree might have suggested a mitochondrial mode of inheritance. However, genetic testing helped to unravel the causative nuclear genetic alteration in the family and indeed identified a novel, most probably causative mutation in SPAST. In conclusion, de novo mutation events and somatic mosaicism may be taken into consideration in families with HSP
presenting with a family history counterintuitive for autosomal dominant inheritance. Disclosure statement The authors declare that they do not have any actual or potential conflicts of interest. Acknowledgments We are indebted to the family for the participation in this study. The project was supported by the BMBF-funded German Network for motor neuron diseases (01GM1103A; MND-NET; ACL). References [1] Harding AE. Classification of the hereditary ataxias and paraplegias. Lancet May 21 1983;1(8334):1151–5. [2] Blackstone C. Cellular pathways of hereditary spastic paraplegia. Annu Rev Neurosci 2012;35:25–47. [3] Braschinsky M, Zopp I, Kals M, Haldre S, Gross-Paju K. Bladder dysfunction in hereditary spastic paraplegia: what to expect? J Neurol Neurosurg Psychiatry Mar 2010; 81(3):263–6. [4] Jennum P, Neerup Jensen L, Fenger K, Nielsen JE, Fuglsang-Frederiksen A, Nielsen JE. Motor evoked potentials from the external anal sphincter in patients with
A. Aulitzky et al. / Journal of the Neurological Sciences 347 (2014) 352–355
[5] [6]
[7] [8]
[9]
[10]
autosomal dominant pure spastic paraplegia linked to chromosome 2p. J Neurol Neurosurg Psychiatry Oct 2001;71(4):561–2. Noreau A, Dion PA, Rouleau GA. Molecular aspects of hereditary spastic paraplegia. Exp Cell Res Jul 1 2014;325(1):18–26. Novarino G, Fenstermaker AG, Zaki MS, Hofree M, Silhavy JL, Heiberg AD, et al. Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders. Science Jan 31 2014;343(6170):506–11. Fink JK. Hereditary spastic paraplegia: clinico-pathologic features and emerging molecular mechanisms. Acta Neuropathol Sep 2013;126(3):307–28. Finsterer J, Loscher W, Quasthoff S, Wanschitz J, Auer-Grumbach M, Stevanin G. Hereditary spastic paraplegias with autosomal dominant, recessive, X-linked, or maternal trait of inheritance. J Neurol Sci Jul 15 2012;318(1–2):1–18. Depienne C, Tallaksen C, Lephay JY, Bricka B, Poea-Guyon S, Fontaine B, et al. Spastin mutations are frequent in sporadic spastic paraparesis and their spectrum is different from that observed in familial cases. J Med Genet Mar 2006;43(3):259–65. Depienne C, Fedirko E, Faucheux JM, Forlani S, Bricka B, Goizet C, et al. A de novo SPAST mutation leading to somatic mosaicism is associated with a later age at onset in HSP. Neurogenetics Aug 2007;8(3):231–3.
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[11] Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods Apr 2010;7(4):248–9. [12] Schwarz JM, Rodelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods Aug 2010;7(8): 575–6. [13] Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res May 2001;11(5):863–74. [14] Verny C, Guegen N, Desquiret V, Chevrollier A, Prundean A, Dubas F, et al. Hereditary spastic paraplegia-like disorder due to a mitochondrial ATP6 gene point mutation. Mitochondrion Jan 2011;11(1):70–5. [15] Alvarez V, Sanchez-Ferrero E, Beetz C, Diaz M, Alonso B, Corao AI, et al. Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia. BMC Neurol Oct 8 2010;10 [89-2377-10-89].
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Short communication
A complex form of hereditary spastic paraplegia in three siblings due to somatic mosaicism for a novel SPAST mutation in the mother Anna Aulitzky a, Katrin Friedrich b,c, Dieter Gläser d, Regina Gastl a, Christian Kubisch b,c, Albert C. Ludolph a,⁎, Alexander E. Volk b,c,⁎⁎ a
Department of Neurology, University Hospital Ulm, Oberer Eselsberg 45, 89081 Ulm, Germany Institute of Human Genetics, University of Ulm, Albert-Einstein Allee 11, 89081 Ulm, Germany Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany d genetikum, Laboratory of Cytogenetics and Molecular Genetics, Wegener Str. 15, 89231 Neu-Ulm, Germany b c
a r t i c l e
i n f o
Article history: Received 24 July 2014 Received in revised form 11 September 2014 Accepted 24 September 2014 Available online 2 October 2014 Keywords: Hereditary spastic paraplegia SPG4 SPAST Somatic mosaicism Genetic testing De novo mutation
a b s t r a c t Hereditary spastic paraplegias (HSPs) represent a clinically and genetically heterogeneous group of diseases. Major symptoms comprise progressive bilateral leg stiffness, spasticity at rest and diffuse muscle weakness. Complex forms are characterized by additional symptoms like dementia, cerebellar dysfunction or seizures. Autosomal dominant, autosomal recessive, X-linked recessive and possibly mitochondrial inheritance have been described in familial HSP. The most frequently mutated gene in familial cases of uncomplicated autosomal dominant HSP is SPAST, however de novo mutations in SPAST are rarely found. Here, we report on the clinical and genetic findings in a family with three children afflicted by complex HSP and their unaffected parents. Although autosomal dominant inheritance seemed unlikely in this family, genetic testing revealed a novel SPAST mutation, c.1837GNC (p.Asp613His), in a heterozygous state in all affected individuals and somatic mosaicism of this mutation in the unaffected mother. Our study thus expands the knowledge on SPAST-associated HSP and emphasizes that de novo mutations and somatic mosaicism should be taken into consideration in HSP families presenting with a family history not suggestive for an autosomal dominant inheritance pattern. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hereditary spastic paraplegias are a clinically and genetically heterogeneous group of neurodegenerative disorders. Pure and complex forms are described. In pure HSP, progressive spasticity and weakness of the lower limbs with increased reflexes and a positive Babinski's sign are the leading symptoms. Complex HSP in contrast presents with variable additional symptoms like dementia, seizures, optic atrophy, ataxia, polyneuropathy, dystonia, hypogonadism, and microcephaly [1,2]. Urinary incontinence is a frequently and fecal incontinence a less frequently observed accompanying symptom in pure and complex HSP [3,4]. HSP is genetically highly heterogeneous with nearly all possible monogenic inheritance patterns described (i.e. autosomal dominant, autosomal recessive, X-linked recessive and possibly also mitochondrial transmission). To date, around 60 HSP related genes have been identified [5,6]. Mutations in the SPAST gene (SPG4) account for up to 50% of ⁎ Corresponding author. Tel.: +49 731 177 1201; fax: +49 731 177 1202. ⁎⁎ Correspondence to: A.E. Volk, Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Tel.: +49 40 7410 52120; fax: +49 40 7410 55138. E-mail addresses: [email protected] (A.C. Ludolph), [email protected] (A.E. Volk).
http://dx.doi.org/10.1016/j.jns.2014.09.046 0022-510X/© 2014 Elsevier B.V. All rights reserved.
autosomal dominant HSP. Intrafamilial variability is considerable in families with SPAST-associated HSP. In most cases, mutations in SPAST are associated with an uncomplicated form of HSP, but complex forms have also been described [7,8]. Most individuals with SPG4 have inherited a mutation from an affected parent although the exact proportion of de novo SPAST mutations is unknown [9]. There is a single report in which somatic mosaicism for a SPAST mutation in one individual with late onset HSP is associated with HSP in his daughter and his grandson [10]. We now report another family with complex HSP in two siblings and one half-sibling due to somatic mosaicism for a novel SPAST mutation in the unaffected mother. 2. Methods All patients were examined by an experienced neurologist at the University Hospital Ulm and underwent magnetic resonance imaging and neurophysiological studies. Blood samples for genetic testing of all affected individuals and the unaffected mother were collected. Informed written consent was obtained for all individuals. The study was approved by the Ethics Committee of the University of Ulm. DNA was extracted from peripheral blood lymphocytes using a column extraction kit (Qiagen). Genetic testing was performed by direct sequencing and multiplex ligation dependent probe amplification (MPLA kit SALSA MLPA kit P165 HSP/
A. Aulitzky et al. / Journal of the Neurological Sciences 347 (2014) 352–355
Spastin; MRC-Holland). Primer sequences are available upon request. The sequences were compared to GenBank entry NM_014946.3. The mutation identified was checked in public databases (dbSNP (http:// www.ncbi.nlm.nih.gov/snp/), 1000 Genomes browser (http:// browser.1000genomes.org/index.html), Exome Variant Server (http://evs.gs.washington.edu/EVS/)), the Human Gene Mutation Database (HGMD® professional) and the mutation prediction programs Polyphen-2 [11], MutationTaster [12] and SIFT [13]. Multiple sequence alignment was performed with Clustal Omega (http:// www.ebi.ac.uk/Tools/msa/clustalo/). 3. Results Three children with complex spastic paraplegia and their unaffected mother were investigated (Fig. 1). Spastic gait was the initial symptom in the eldest girl (II.1; Fig. 1) which was first recognized at the age of three years. Symptoms slowly progressed over time and at an age of 26 years she presented with characteristic HSP-associated symptoms such as increased muscle reflexes of the lower limbs, a positive Babinski's sign, contractures of the joints and a slight paresis of the extensors on the lower limbs. Furthermore, she displayed signs of cerebellar dysfunction like dysmetria and dysdiadochokinesis. A brain MRI showed thinning of the corpus callosum. Her maternal half-brother (II.2, Fig. 1) developed progressive walking disturbances and spastic gait by the age of four years. At the age of 19 years he presented with spastic gait, increased reflexes, contractures of the lower limbs and cerebellar dysfunction. A brain MRI was unremarkable. He had no paresis but suffered from urinary and fecal incontinence. He is mentally handicapped, however no formal IQ testing was available. In the youngest sibling (II.3, Fig. 1) motor and cognitive development was delayed and at the age of three years spastic gait became evident. At the age of 16 years, he presented increased reflexes, contractures of the lower limbs, Babinski's sign, and cerebellar involvement. A clinical overview of all three patients is given in Table 1. Most probably unrelated to HSP, individual II.2 also showed hyperlordosis and individual II.3 had neurodermatitis. Neither their common mother nor their respective fathers were reported to suffer from HSP or any other neurodegenerative disorder, and a clinical evaluation of the mother at the age of 44 years did not show any signs of HSP. The family history was unremarkable with respect to neurologic and neurodegenerative diseases in other family members (as e.g. in the parents of the mother or the respective fathers) and there were no additional siblings or children of the affected individuals. Although a hereditary origin of the disease seemed likely, the family history offered no clear clue about the exact mode of inheritance. Autosomal dominant inheritance did not seem to be very likely, as the mother of three affected children showed no symptoms. Likewise autosomal recessive inheritance looked rather unlikely as the three affected children did not have a common father and as both fathers are not related to each other or to the mother. Last but not least, an X-chromosomal recessive mode of inheritance was not absolutely convincing due to
I 1
2
3
II 1
2
3
Fig. 1. Pedigree of the family. Circles represent females, squares represent males. Filled symbols indicate affected individuals. II.1 is the half-sister of the two brothers II.2 und II.3.
353
Table 1 Clinical details. Patient ID Age at examination Sex Initial symptom Age at onset Spastic upper limb Spastic lower limb Contractures upper limbs Contractures lower limb Reflexes upper limbs Reflexes lower limbs Babinski's sign Paresis upper limbs Paresis lower limbs Pallesthesia upper limbs Pallesthesia lower limbs Cerebellar signs Mental retardation Incontinence
II.1 26 female Spastic gait 4 − + − + ++ +++ + − + 8/8 7/8 + − −
II.2 19 male Spastic gait 4 − + − + ++ +++ + − − 8/8 8/8 + + Urinary and fecal
II.3 16 male Spastic gait 3 − + − − ++ +++ + − − 8/8 8/8 + + −
rather severe disease in the affected girl. In principle, the pedigree might suggest maternal inheritance due to a mitochondrial mutation. However convincing evidence about a causative role of mutations in the mitochondrial genome in early-onset complicated HSP is missing even if a single publication reported a HSP-like late-onset disease with a mitochondrial mutation in a single family [14]. In a more pragmatic approach, we therefore ought to first test the most common disease genes for HSP regardless of the underlying inheritance pattern. Genetic testing of the SPAST gene was unremarkable in the MLPA analysis, however sequencing revealed a substitution of guanine to cytosine at cDNA position 1837 (c.1837GNC; Fig. 2A) in a heterozygous state in patient II.2. On protein level this variation is predicted to lead to a substitution of a negatively charged asparagine by histidine (p.Asp613His). This substitution had not been described in patients with HSP before according to the Human Gene Mutation Database. The mutation was not listed in ~12,500 control alleles sequenced by the NHLBI Exome Sequencing Project and was not annotated in either dbSNP or the 1000 Genomes Project database. The affected amino acid is highly conserved among species (Fig. 2B) and predicted to be deleterious by different prediction programs (Polyphen-2, MutationTaster, SIFT; Fig. 2C). Even more, the mutation segregates with the phenotype and was also present in the other affected individuals (II.1, II.3; Fig. 2A) in a heterozygous state. Interestingly, genetic testing of the mother reproducibly showed a low-grade somatic mosaicism for the mutation c.1837GNC at least in blood cells (Fig. 2A), which after all explained the unusual family history. 4. Discussion Direct sequencing of the SPAST gene revealed the novel mutation c.1837GNC in a heterozygous state in three half-siblings with a complex form of HSP. Genetic testing of this specific mutation in the healthy mother proved somatic mosaicism in her as the altered allele could also be detected in DNA derived from blood cells. The mutation is most probably pathogenic as it (i) segregates with the phenotype, (ii) has not been listed in public databases precluding that it represents a more common polymorphism, (iii) is highly conserved among species and (iv) is predicted to be pathogenic by different prediction programs. Furthermore, a substitution of this asparagine to a nonpolar alanine (p.Asp613Ala) has previously been described in another patient with HSP [15] pointing to a crucial role of this amino acid for the function of spastin. In contrast to the patients described here, the patient with p.Asp613Ala showed a pure HSP and a later age of onset (36 years). Our patients had early-onset HSP accompanied by cerebellar disturbances and mental retardation.
354
A. Aulitzky et al. / Journal of the Neurological Sciences 347 (2014) 352–355
D
F
G
D
T
T
V
A
*
control D
F
G
D/H
T
T
V
B D. rerio X. laevis chicken human mouse
*
C
G/C
I.2 G/C
II.1 G/C
II.2 G/C
II.3
Fig. 2. A: Electropherograms showing the mutation c.1837GNC of the SPAST gene (arrow) in individuals II.1, II.2, II.3 in a heterozygous state. The altered allele carrying a cytosine could also, although to a lesser amount be detected in DNA derived from blood cells in the mother (I.2). B: Multiple sequence alignment of SPAST orthologs surrounding the D613 position in the human protein. C: Results of different prediction programs for the mutations p.Asp613His and p.Asp613Ala.
In respect of the clinical phenotype, it is interesting that the mutation p.Asp613Ala was – in contrast to the mutation p.Asp613His identified in our patients – predicted to be tolerated by SIFT which may point to a possible genotype–phenotype correlation (Fig. 2C). The family history of the patient described before was unremarkable. If this may suggest a de novo mutation is speculative but may indicate a hot spot for mutations in SPAST and should be kept in mind for further studies. In our family the novel mutation c.1837GNC occurred de novo in the mother. The mutation is present in the mother's germline as all three affected children inherited the mutation. As the mutation could also be detected in blood the mutation event in the mother must have occurred at an early embryonic stage. Whether the mutation is also present in neuroectodermal tissue and especially in the central nervous system is unclear. Therefore, a prediction on a possible clinical phenotype in the mother is not possible and a later onset of disease symptoms in the mother cannot be excluded. On the first view, the pedigree might have suggested a mitochondrial mode of inheritance. However, genetic testing helped to unravel the causative nuclear genetic alteration in the family and indeed identified a novel, most probably causative mutation in SPAST. In conclusion, de novo mutation events and somatic mosaicism may be taken into consideration in families with HSP
presenting with a family history counterintuitive for autosomal dominant inheritance. Disclosure statement The authors declare that they do not have any actual or potential conflicts of interest. Acknowledgments We are indebted to the family for the participation in this study. The project was supported by the BMBF-funded German Network for motor neuron diseases (01GM1103A; MND-NET; ACL). References [1] Harding AE. Classification of the hereditary ataxias and paraplegias. Lancet May 21 1983;1(8334):1151–5. [2] Blackstone C. Cellular pathways of hereditary spastic paraplegia. Annu Rev Neurosci 2012;35:25–47. [3] Braschinsky M, Zopp I, Kals M, Haldre S, Gross-Paju K. Bladder dysfunction in hereditary spastic paraplegia: what to expect? J Neurol Neurosurg Psychiatry Mar 2010; 81(3):263–6. [4] Jennum P, Neerup Jensen L, Fenger K, Nielsen JE, Fuglsang-Frederiksen A, Nielsen JE. Motor evoked potentials from the external anal sphincter in patients with
A. Aulitzky et al. / Journal of the Neurological Sciences 347 (2014) 352–355
[5] [6]
[7] [8]
[9]
[10]
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