Autosomal recessive hereditary spastic paraplegia clinical and genetic characteristics of a well-defined cohort

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1 DOI /s ORIGINAL ARTICLE Autosomal recessive hereditary spastic paraplegia clinical and genetic characteristics of a well-defined cohort G. Yoon & B. Baskin & M. Tarnopolsky & K. M. Boycott & M. T. Geraghty & E. Sell & S. Goobie & W. Meschino & B. Banwell & P. N. Ray Received: 18 March 2013 /Accepted: 21 May 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract We describe the clinical and genetic features of a well-characterized cohort of patients with autosomal recessive hereditary spastic paraplegia (ARHSP) in the province of Ontario. Patients with documented corticospinal tract abnormalities were screened by whole gene sequencing and multiplex ligation probe amplification for mutations in nine genes known to cause ARHSP. Of a cohort of 39 patients, a genetic diagnosis was established in 17 (44 %) and heterozygous mutations were detected in 8 (21 %). Mutations were most frequent in SPG7 (12 patients), followed by SPG11 (10 patients), PNPLA6 (SPG39, 2 patients), and ZFYVE26 (SPG15, 2 patients). Although there are associations between some clinical manifestations of ARHSP and specific genes, many patients are tested at an early stage of the disease when phenotype/genotype correlations are not obvious. Accurate molecular characterization of well-phenotyped cohorts of patients will be essential to establishing the natural history of these rare degenerative disorders to enable future clinical trials. Keywords Spastic paraplegia. Ataxia. Genetics Introduction Hereditary spastic paraplegias (HSPs) are a heterogeneous group of disorders and may be inherited as an autosomal dominant, autosomal recessive, or X-linked trait. At least 57 Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. G. Yoon Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, Canada G. Yoon : B. Banwell Division of Neurology, The Hospital for Sick Children, Toronto, Canada B. Baskin : P. N. Ray Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Canada M. Tarnopolsky Division of Neuromuscular and Neurometabolic Disorders, McMaster University, Hamilton, Canada K. M. Boycott Department of Genetics, Children s Hospital of Eastern Ontario, Ottawa, ON, Canada M. T. Geraghty : E. Sell Department of Pediatrics, Children s Hospital of Eastern Ontario, Ottawa, ON, Canada S. Goobie Medical Genetics Program of Southwestern Ontario, London Health Sciences Centre, London, Canada W. Meschino Genetics Program, North York General Hospital, Toronto, Canada B. Baskin Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden P. N. Ray Department of Molecular Genetics, The University of Toronto, Toronto, Canada G. Yoon (*) Divisions of Neurology and Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, ON M5G 1X8, Canada grace.yoon@utoronto.ca

2 different loci have been shown to be associated with HSP to date [1]. The HSPs comprise a genetically and clinically heterogeneous group of neurodegenerative disorders characterized by progressive spasticity and hyperreflexia of the lower limbs. HSP is estimated to affect 1 in 20,000 individuals in the European population, with variable frequencies in distinct populations from 1.3 to 9/100,000 individuals [2 4]. Clinically, HSPs can be divided into two main groups: uncomplicated (or pure) and complicated (complex). Pure HSPs are characterized by corticospinal tract findings (slowly progressive lower extremity spasticity, weakness, and extensor plantar response), variable neurogenic urinary disturbances, and mild reduction of lower extremity vibration sense and proprioception. Complex HSP syndromes are characterized by the presence of additional neurological or non-neurological features, including peripheral neuropathy, epilepsy, ataxia, optic atrophy, retinopathy, dementia, ichthyosis, cognitive impairment, deafness, or bulbar symptoms [1]. HSP may manifest at any age from infancy through late adulthood, and the age of symptom onset, rate of symptom progression, and extent of disability are variable both within and between HSP families, which renders diagnosis extremely difficult. We describe the clinical and genetic features of 25 out of a cohort of 39 patients with spastic paraplegia who were screened by whole gene sequencing and multiplex ligation-dependent probe amplification (MLPA) for mutations in nine genes known to cause autosomal recessive hereditary spastic paraplegia (ARHSP), and suggest an approach to genetic testing for these disorders. Methods Patients were ascertained at subspecialty clinics (neurology and genetics) throughout the province of Ontario from June 2009 to January All patients with typical features of spastic paraplegia with documented corticospinal tract involvement (progressive lower extremity spasticity, weakness, and extensor plantar response), and family history suggestive of sporadic or autosomal recessive inheritance were included in the study. Patients with biochemically or genetically confirmed metabolic disease or structural brain lesions (tumors, vascular, or brain malformations) were excluded from the study. All patients had normal chromosomal microarray (Agilent Oligo Array-44 K), lactate, plasma amino acids, urine organic acids, carnitine and acylcarnitine levels, vitamin B12 and vitamin E levels, lipid profiles, peroxisomal metabolites (very long chain fatty acids, pipecolic, and phytanic acids), and lysosomal enzyme activities. Mutations of SPAST (SPG4) were ruled out in sporadic cases. This study was approved by the Research Ethics Board of the Hospital for Sick Children. Sequence analysis DNA from peripheral blood samples were analyzed in the genetic diagnostic laboratory at The Hospital for Sick Children. All exons and flanking intron sequences of SPG11 (SPG11, NM_ ), CYP7B1 (SPG5A, NM_ ), SPG7 (NM_ ), ZFYVE26 (SPG15, NM_ ), SPG20 (NM_ ), SPG21 (NM_ ), PNPLA6 (SPG39, NM_ ), CCT5 (NM_ ), and GJC2 (SPG44) were sequenced both in forward and reverse direction. Sanger sequencing was performed according to standard protocols using BigDye terminator v1.1 (Life Technologies) and sequencing products were separated on an ABI model 3730 Capillary Sequencer (Life Technologies) and analyzed using SeqPilot software (JSI Medical systems). Classification of sequence variants is based on the ACMG recommendations for interpretation and reporting of sequence variations [5]. Deletion analysis MLPA (MRC-Holland, Amsterdam, The Netherlands) was performed to determine copy number changes in SPG7 and SPG11 using probe mixtures P213 HSP-2 and P306 SPG11, respectively. The data obtained were analyzed using GeneMarker software (Softgenetics, State College, PA, USA) for MLPA analysis. Abnormal copy number ranges are based on clinically validated samples used in our diagnostic laboratory. Height ratios of were determined as deletions. Quantitative real-time PCR using SYBR green chemistry (Life Technologies, Carlsbad, CA, USA) was used to determine copy number changes for every exon of the PNPLA6 gene using standard protocols. Results Molecular analyses A total of 39 patients were enrolled in this study, and a genetic diagnosis was established in 17 patients (44 %). For these 17 patients, mutations were most frequent in SPG7 (nine patients), followed by SPG11 (six patients), PNPLA6 (SPG39, one patient), and ZFYVE26 (SPG15, one patient). The types of mutations detected included missense, nonsense, splicing, frame shift, and exonic deletions. Sequence variants that were previously unreported but of the type expected to cause disease were classified as pathogenic, i.e., predicted to result in the introduction of a stop codon, alter the sequence of a splice junction, particularly the invariant AG/GT nucleotides or delete one or more nucleotides to cause frame shift. A missense variant that previously had not been described as disease causing or a polymorphism was considered pathogenic when predicted to have a

3 damaging effect on the protein using four in silico tools (SIFT, Polyphen, Align GVGD, MutationTaster). Sequence changes were predicted to cause splice site alterations when determined to affect splicing by either destroying an existing splice site or creating a new one by four in silico tools (SpliceSiteFinder-like, MaxEntScan, NNSPLICE, Human Splicing Finder). Prediction of pathogenicity of novel missense mutations and splice site alterations are presented as Electronic supplementary material. Of note, exonic deletions were found in seven patients (41 %) in our cohort of patients with confirmed genetic diagnoses of ARHSP. These rearrangements were present in the compound heterozygous state in two patients with SPG11 and one patient with SPG7, two siblings were homozygous for deletion of exon 6 of the SPG7 gene, and one patient was compound heterozygous for deletion of exons of the PNPLA6 gene, in addition to a truncating mutation (Table 1). Eight of 39 patients had only one confirmed mutation. These included three patients with mutations in SPG11, three patients with mutations in SPG7, one patient with a mutation in ZFYVE26, and one patient with a mutation in PNPLA6. All three SPG7 patients were heterozygous for the c.1529c>t (p.ala510val) mutation (Table 2). No patient in our cohort was found to have a mutation in CYP7B1 (SPG5A), SPG20, SPG21, CCT5, orgjc2 (SPG44). One patient with clinical features suggestive of SPG11 was found to be heterozygous for a previously reported c.3664_3665inst (p.lys1222ilefsx15) mutation [6] in SPG11. A second mutation was not identified by sequencing or the additional use of MLPA for detection of exonic deletions. As her clinical and neuroimaging features were typical of SPG11, further effort was made to detect a second mutation in SPG11. Subsequent mrna analysis using RT- PCR and sequencing of muscle tissue from the right quadriceps revealed a deletion of exon 27 in the SPG11 transcript (r.4667_4774del). This would result in deletion of 36 amino acids (p.tyr1556_val1592delinsphe) in the SPG11 protein. Further molecular investigation revealed that exon 27 in the patient s genomic DNA was inverted and therefore resulted in exon skipping in the SPG11 transcript. This also explains why a deletion of exon 27 could not be detected with MLPA analysis. Clinical features of 17 patients with confirmed ARHSP Seven of the 17 patients (41 %) had a family history suggestive of autosomal recessive inheritance; the remaining 10 patients (59 %) had no family history of neurological symptoms. Age at onset of symptoms varied according to genotype with the ZFYVE26 (SPG15) mutation carriers having the earliest mean age at symptom onset (3 years), followed by PNPLA6 (SPG39, 6 years) and SPG11 (12.5 years). Patients with SPG7 presented in adulthood with a mean age at onset of 31 years. The predominant presenting symptom was gait disturbance (15/17 patients, 88 %). Motor delay was reported in 2/17 patients (12 %) and the majority (14/17, 82 %) reported significant difficulties with ambulation and falling by adulthood. Learning disability, requiring a modified education program, was present in 7/17 (41 %) patients, 6 of whom were determined to have SPG11. It was also a feature in the patient with SPG39. As expected, all patients demonstrated abnormalities in corticospinal tract function (spasticity, hyperreflexia, extensor plantar response) and 13/17 (76 %) also had lower extremity weakness. Muscle atrophy was present in 5/17 (29 %) patients (two each with SPG11 and SPG7, and one patient with SPG15). Neuropathy was present in 6/17 patients, four with SPG11 and two with SPG7. Cognitive regression was present in four patients, three with SPG11, and one with SPG7. abnormalities (dysarthria, scanning speech) were present in 10/17 (59 %) patients, and one patient with SPG11 had swallowing difficulties. Of note, speech abnormalities were most common in patients with SPG7 (six patients), followed by SPG11 (four patients) and in one patient with SPG15. All patients with confirmed SPG11 or SPG15 had thinning of the corpus callosum on brain imaging (Table 1). Cerebellar signs were present in seven patients, with cerebellar atrophy noted in one patient who was homozygous for a c.1529c>t (p.ala510val) mutation in SPG7, one patient who was compound heterozygous for a c.1996g>c (p.gly666arg) point mutation and deletion of exons of SPG7, and one patient with SPG39 who was compound heterozygous for two novel mutations of the PNPLA6 gene, c.1672_1674delinsta (p.r558x) and deletion of exons In contrast to previously reported patients with SPG39 [7], our one patient did not have amyotrophy but had multiple medical issues including short stature, hypothyroidism, and hypogonadotrophic hypopituitarism requiring hormone supplementation, speech delay, learning disability, cerebellar signs, and mild dysmorphic features, in addition to progressive lower extremity spasticity and weakness. Initial brain imaging at age 12 years was normal; however, a follow-up study at age 15 years revealed significant atrophy of the anterior cerebellar vermis (Fig. 1). Clinical features of eight patients with heterozygous mutations in ARHSP genes The sole patient heterozygous for the c.2944_2947dup (p.arg983fs) mutation in PNPLA6 appears to have a pure spastic paraplegia phenotype thus far, but is only eight years of age. Two patients heterozygous for mutations in SPG11 and the one patient heterozygous for a mutation in ZFYVE26

4 Table 1 Clinical and genetic characteristics of 17 patients with genetically confirmed autosomal recessive hereditary spastic paraplegia Gene Mutation allele 1 Reported/ novel Mutation allele 2 Reported/ novel Current age Age of onset Presenting symptom Gender Ethnicity Motor delay delay 4 PNPLA6 c.1672_1674delinsta (p.arg558x) 1 SPG11 c.442+1g>c (r.spl) Reported [19] c.6898_6899delct (p.leualafsx39) 5 SPG11 c.5093 T>A (p.leu1698x) 6 SPG11 c.6197_6198delag (p.gln2066argfsx5) 8 SPG11 c.3664_3665inst (p.lys1222ilefsx15) Novel del exons Novel 17 6 Toe walking, spastic gait Reported [20] Spastic gait, poor coordination M English + F Italian + Novel del exons Reported [21] Spastic gait M Mennonite Novel del exon 20 Novel Spastic gait M Afghani Reported [21] r.4667_4774del Novel 17 6 Gait abnormality, poor coordination F English/Dutch 15 SPG11 c g>c (r.spl?) Novel c g>c (r.spl?) Novel Cognitive decline F Iranian Novel Motor delay M French Canadian SPG11 c.6082c>t (p.gln2028x) Novel c.6598a>t (p.lys2200x) 25 SPG15 c.273+8g>a (r.spl?) Novel c.273+8g>a (r.spl?) Novel 18 3 Spastic gait M Filipino 2 SPG7 c.415c>t(p.arg139x) Reported [22] c.1053dupc Reported [23] Poor balance M English (p.gly352argfsx44) 11 SPG7 c.1529c>t(p.ala510val) Reported [24] c.1529c>t(p.ala510val) Reported [24] Gait abnormality M Caucasian 13 SPG7 c.1529c>t(p.ala510val) Reported [24] c T>C (r.spl?) Novel Poor balance M Caucasian 24 SPG7 c.1045g>a (p.gly349ser) Reported [24] c.1996g>c (p.gly666arg) Novel Gait disturbance M French Canadian + 27 SPG7 c.759-1g>t (r.spl?) Novel c _1457del (r.spl?) Novel Gait disturbance M Caucasian 30 SPG7 c.1529c>t (p.ala510val) Reported [24] c.4delg (p.ala2fsx64) Novel Gait abnormality M French Canadian 31 SPG7 del exon 6 Novel del exon 6 Novel Spastic gait F East Indian 44 SPG7 del exon 6 Novel del exon 6 Novel Spastic gait M East Indian 45 SPG7 c.1996g>c (p.gly666arg) Novel del exons Novel Gait abnormality M Lebanese/French Canadian Learning disability Bladder dysfunction Falls Swallowing difficulty abnormalities Muscle atrophy/ amyotrophy Cerebellar signs Dementia Neuropathy Other features MRI findings Hypopituitarism Cerebellar atrophy Thin corpus callosum Thin corpus callosum 6 + Thin corpus callosum Cognitive decline, Thin corpus callosum dystonia, bradykinesia Cognitive decline Thin corpus callosum Severe autism Thin corpus callosum Mild muscle atrophy Thin corpus callosum Severe scanning speech

5 Table 1 (continued) Dementia Neuropathy Other features MRI findings Cerebellar signs Muscle atrophy/ amyotrophy abnormalities Falls Swallowing difficulty Bladder dysfunction Learning disability Cerebellar atrophy Scanning speech 31 Cognitive deficits Cognitive deficits Cerebellar atrophy (SPG15) all had thinning of the corpus callosum on MRI. The third patient with presumed SPG11 had a definite mutation predicted to cause disease, c.6091c>t (p.arg2031x), but a second mutation has yet to be identified. She did not have a thin corpus callosum on brain imaging, but presented with photosensitivity, dermatomyositis, and alopecia in addition to corticospinal tract signs. All three SPG7 patients were heterozygous for the c.1529c>t (p.ala510val) mutation and had clinical features that were indistinguishable from those with two mutations in the SPG7 gene. The clinical features of the nine patients with only one mutation are presented in Table 2. Discussion Whole gene sequencing combined with MLPA of nine genes known to cause autosomal recessive hereditary spastic paraplegia (ARHSP) enabled the identification of mutations in both alleles for 17/39 (44 %) patients and in one allele for 8/39 (21 %) patients in whom a clinical diagnosis of ARHSP was suspected. The majority of mutations were private, which supports whole gene sequencing as the preferred initial diagnostic option for this group of disorders. No patient in our cohort was found to have a mutation in CYP7B1 (SPG5A), SPG20, SPG21, CCT5, or GJC2 (SPG44). This is likely due to the relatively small size of our cohort and the relative rarity of mutations in these genes in patients with ARHSP. Mutations in CYP7B1 have been found to account for approximately 9 % of ARHSP in previous studies [8, 9] and genetic testing of larger cohorts of patients will be required to establish accurate prevalence data for these rarer forms of ARHSP. A significant proportion of patients in our cohort with confirmed ARHSP (7/17, 41 %) were found to have exonic deletions in either the compound heterozygous or homozygous state in one of the ARHSP genes. These mutations cannot be detected using whole gene sequencing methods and require the use of additional techniques such as MLPA, highlighting the importance of such a combined approach to significantly increase the sensitivity of mutation detection in ARHSP. One patient with a heterozygous mutation in SPG11 did not have a second mutation detected by MLPA, and mrna studies on muscle tissue were required to identify the second mutation. While not routinely available for clinical diagnostic purposes, further studies utilizing these techniques may lead to identification of more patients with ARHSP caused by complex genomic rearrangements. Our data suggests that at least some mutations in ARHSP are undetectable using clinically available approaches. Eight patients with a clinical diagnosis of ARHSP were found to be heterozygous for a mutation in one of the ARHSP genes, with the second mutation undetected thus far. Potential reasons for this include promoter mutations,

6 Table 2 Clinical and genetic characteristics of nine patients with a clinical diagnosis of hereditary spastic paraplegia but only one mutation in a known ARHSP gene Gene Mutation allele 1 Reported/ novel Mutation allele 2 Current age Age of onset Presenting symptom Gender Ethnicity Motor delay delay 16 PNPLA6 c.2944_2947dup (p.arg983argfsx86) Novel Not detected 8 6 Gait abnormality M Caucasian + 7 SPG11 c.6091c>t(p.arg2031x) Reported [25] Not detected 12 4 Gait abnormality, stiffness F Pakistani + 9 SPG11 c.3037a>g (r.(spl?)) Novel Not detected Stiffness M Portuguese 10 SPG11 c.3037a>g (r.(spl?)) Novel Not detected 25 2 Gait abnormality, falls F Caucasian 29 SPG15 c.5215c>t (p.arg1739x) Novel Not detected 32 6 Motor delay F Caucasian + 12 SPG7 c.1529c>t (p.ala510val) Reported [24] Not detected Gait abnormality F Caucasian 22 SPG7 c.1529c>t (p.ala510val) Reported [24] Not detected Gait disturbance M Caucasian 28 SPG7 c.1529c>t (p.ala510val) Reported [24] Not detected Gait disturbance M Caucasian Learning disability Bladder dysfunction Falls Swallowing difficulty abnormalities Muscle atrophy/ amyotrophy Cerebellar signs Dementia Neuropathy Other features MRI findings Photosensitivity, dermatomyositis, alopecia 9 + Thin corpus callosum Congenital sensorineural hearing loss Thin corpus callosum Mild LD Thin corpus callosum, brain atrophy, periventricular white matter changes Tremor Diffuse WM changes on MRI Severe motor neuropathy

7 Fig. 1 Initial normal brain MRI (a) of a patient with SPG39. Cerebellar atrophy (b) is present on a follow-up MRI 3 years after the initial study deep intronic mutations, or other mechanisms not detected using current diagnostic methods, as highlighted above. Another possibility could be digenic inheritance in these patients, with mutations in genes that have thus far not been identified. However, digenic inheritance has not been reported in ARHSP to date. There is also the possibility that these patients are true carriers and that the changes identified are those of the population frequency. This is most likely not the case since the frequency of these changes should be found at a similar level in the normal population. One potential exception is the three SPG7 patients in our cohort who were heterozygous for the c.1529c>t (p.ala510val) mutation. Our results support those of a recent study [10] where a significant number of patients with clinical features in keeping with SPG7 were found to carry the c.1529c>t (p.ala510val) mutation in the heterozygous state. The authors of this study suggested that some mutations in SPG7 may act in a dominant manner, with variable penetrance, Fig. 2 Proposed diagnostic approach for the autosomal recessive hereditary spastic paraplegias Family history of autosomal recessive HSP ARHSP Diagnostic Pathway Patient with lower extremity weakness, spasticity and extensor plantar response Clinical Testing Clinical assessment (MRI brain and Spinal Cord, EMG/NCV, Vitamin B12, E metabolic/leukodystrophy investigations) AR HSP suspected AR HSP suspected Genetic assessment of proband Thin corpus callosum on brain MRI No thinning of the corpus callosum Mutation analysis of relatives SPG11 and SPG15 Childhood onset Adult onset SPG5A and SPG39 SPG7

8 with c.1529c>t (p.ala510val) being a recurrent example. Further prospective studies of large cohorts of patients with SPG7, in addition to functional studies, will be required to fully understand the pathogenicity of these rare variants. Although there are published data suggesting an association between some clinical manifestations of ARHSP and specific genes [11 18], many patients are tested at an early stage of the disease when phenotype/genotype correlations are not obvious. The distinction between pure and complex forms of HSP is not always clear, as exemplified by our patient with complex phenotype and SPG39, and in the future it may be most practical to categorize the HSP syndromes by their constellation of features and avoid the arbitrary terms complex or pure. The integration of clinical, electrophysiological, and neuroimaging studies is necessary for efficient, cost-effective diagnosis of this complex group of disorders, and we propose an approach to genetic diagnosis of ARHSP, outlined in Fig. 2. We recognize that in the near future, the cost and accessibility of next-generation sequencing approaches will become such that prioritization of genes for mutation detection will no longer be necessary, but genotype phenotype correlation will become even more important to enable accurate variant interpretation in multiple genes. Long-term, prospective studies of wellcharacterized cohorts of patients will be essential to establishing phenotype/genotype correlations that may be used to guide genetic testing. In addition, prospective studies of clinically and molecularly characterized patients will be essential to establishing the natural history of these rare disorders to enable future clinical trials for these rare degenerative disorders. 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