High diagnostic yield and novel variants in very late-onset spasticity

Abstract

Hereditary spastic paraplegias (HSPs) are a diverse group of genetic conditions with variable severity and onset age. From a neurogenetic clinic, we identified 14 patients with very late-onset HSP, with symptoms starting after the age of 35. In this cohort, sequencing of known genetic causes was performed using clinically available HSP sequencing panels. We identified 4 patients with mutations in SPG7 and 3 patients with SPAST mutations, representing 50% of the cohort and indicating a very high diagnostic yield. In the SPG7 group, we identified novel variants in two patients. We have also identified two novel mutations in the SPAST group. We present sequencing data from cDNA and RT-qPCR to support the pathogenicity of these variants, and provide observations regarding the poor genotype-phenotype correlation in these conditions that should be the subject of future study.

Keywords:

• SPG7
• paraplegin
• SPAST
• hereditary spastic paraplegia
• late-onset
• primary lateral sclerosis

Introduction

Hereditary spastic paraplegia (HSP) is a heterogeneous group of disorders that are characterised by degeneration of descending cortical spinal tracts, causing progressive spasticity starting in the legs, and often including bladder dysfunction (Harding, 1983). Some subtypes of HSP may have presentation in late adulthood, the most well-known of these being HSP type 4 (caused by dominant SPAST mutations, which encode the protein spastin) and HSP type 7 (caused by biallelic SPG7 mutations, which encode the protein paraplegin) (Chelban et al., 2017; Pfeffer et al., 2015). Both of these proteins are members of the AAA protein family (ATPase associated with a variety of cellular activities), although they have differing cellular localizations and functions: spastin is involved in microtubule metabolism in the cytoskeleton (Errico, Ballabio, & Rugarli, 2002), whereas paraplegin is localised to mitochondria where it appears to have an important effect on mitochondrial dynamics and maintenance, along with its binding partner AFG3L2 (Gorman et al., 2015; Pfeffer et al., 2014).

One difficulty in the diagnosis of late-onset HSP can be the distinction from primary lateral sclerosis (PLS) which has many similarities in presentation but eventually includes bulbar involvement (which is not usually a feature of HSP). Given that PLS is not a Mendelian genetic disorder, there is a possibility that some patients with late-onset spasticity do not undergo genetic investigations.

In this study, we present findings from investigation of 14 patients with very late-onset spasticity, including several patients who had initially been diagnosed with PLS. Our study emphasises that even in this heterogeneous population with late-onset disease, that diagnostic yield can be very high.

Methods

Inclusion criteria

Patients with a diagnosis of HSP or PLS with onset after the age of 35 years were included. All participants provided written informed consent for research (University of Calgary REB15-5763). All patients were assessed from a single clinic specialising in neurogenetic disorders, by one or more of the authors who are neurologists (AH, LWK, and/or GP).

Clinical data

Patient data were summarised following detailed chart review, including onset age, clinical history, neurologic exam findings, neuroimaging findings, and genetic test results (see Table 1). All patients had sequencing performed with commercially available sequencing panels (Invitae, CA); or Blueprints (Helsinki Finland) as part of their clinical care.

Table 1. Clinical and genetic data of 7 patients with SPG7 and SPG4 mutations; N/A = not available; ExAC = exome aggregation consortium.

Patient Number	HSP Subtype	Onset age	Ethnicity	Clinical presentation	Family History	Physical exam findings	MRI findings	Mutation 1	Mutation 2	Zygosity
Patient 1	SPG7	49	Korean	Progressive gait difficulties and arm weakness	None	Multidirectional Ophthalmoplegia, Spasticity in upper and lower extremities, Decrease vibration and proprioception, wide-based gait	Normal brain and spinal cord	c. 244C > T, p.Gln82*, ExAC absent		Homozygous
Patient 2	SPG7	36	FrenchCanadian	Bladder urgency, gait difficulties, Diplopia, sensory symptoms, swallowing difficulties	None	Spasticity in the lower extremities, ataxia, decreased vibration sensation	Mild cerebellar atrophy Normal spine	c.988-1G > A, disrupts splice acceptor, activates 2 cryptic splice sites. ExAC 0.00002		Homozygous
Patient 3	SPG7	63	Scottish	progressive gait difficulties and arm weakness	None	Spasticity in the lower extremities and upper extremities (Upper < lower)	Spine MRI was normal Brain MRI chronic small vessel disease	c.1045G > A, p.Gly349Ser, rs141659620, ExAC 0.0008	Unidentified***	Compound heterozygous (presumed)
Patient 4	SPG7	37	Mexican	Lower extremities weakness and numbness	None	Left beating nystagmus Spasticity in lower extremities Reduced vibration	Normal brain and Spinal cord	c.850_851delinsC, p.Phe284Profs*. ExAC absent	c.2014G > A (p.Gly672Arg), rs369503365, ExAC 0.000008	Compound heterozygous
Patient 5	SPG4	53	British	Leg weakness Bladder urgency	Affected daughter	Lower extremities weakness increased tony and hyper reflexia	Spinal cord normal	c.1573C > T (p.Gln525*). ExAC absent.	Heterozygous	Heterozygous
Patient 6	SPG4	38	British	Lower extremities weakness	None	Spasticity and hyper reflexia in the lower extremities, bilateral feet drop	Atrophy of brain Small corpus callosum posterior periventricular signal abnormalities Normal spine	c.156T > G (p.Tyr52*). ExAC absent.		Heterozygous
Patient 7	SPG4	56**	Italian	Asymptomatic	Affected son	Mild spasticity on exam, normal strength, brisk reflexes on lower extremities	N/A	c.1004 + 1G > C (altered splicing). ExAC absent.		Heterozygous

*Stop codon.

** This patient was clinically asymptomatic at the time of diagnosis but had subclinical signs on examination.

*** Refer to article text and Figure 1(E), indicating degradation of the transcript from this allele, due to a presently unknown genetic lesion.

cDNA sequencing and RT-qPCR

Whole blood was collected into Paxgene RNA tubes. RNA was extracted using standard protocols. cDNA was synthesised using Superscript III (Invitrogen, Carlsbad, CA, USA) with random hexamers. Sequencing for patients 2, 3, and 7 was performed using custom primers, PCR performed using Qiagen Taq polymerase, PCR clean-up with ExoI-SAP, followed by Sanger sequencing using Bigdye on a ABI3130XL sequencer. For RT-qPCR, custom primers for SPG7 and AFG3L2 were optimised using Perfecta SYBR Green (QuantaBio, Beverly, MA, USA) on a StepOne Plus qPCR system (Applied Biosystems, Foster city, CA, USA). Transcript levels were normalised against GAPDH and B2M. Control comparisons for RT-qPCR were from healthy control spouses who are not known to have any neurological disease, and provided written informed consent. Data were analysed in aggregate from three separate replicates performed by two different operators (MA, KM) and statistical significance measured using a t-test. All primer sequences are available upon request. Additional date available in supplement material.

Results

Clinical features

From the 14 included patients, seven had variants identified in SPG7 or SPAST. The clinical features are summarised in Table 1. The mean age at presentation was 46 years (range 36–63). Among SPG7 patients, the most common clinical feature upon presentation was gait difficulties (100%). Cerebellar signs in the form of ataxia were found in three patients (75%). Two (50%) patients had ocular findings; one had external ophthalmoplegia and the other had left beating nystagmus. All four patients underwent brain imaging and only one patient was found to have cerebellar atrophy (25%). Patients with SPAST mutations were found to have lower extremities spasticity, weakness and hyperreflexia on the exam (N = 3). One patient did not have symptoms but had physical findings of HSP and a positive family history.

SPG7 variants

Patient 1 had a homozygous c.244C > T mutation causing p.Gln82*. This mutation is a novel variant, in silico prediction tools suggest it is pathogenic, and stop-gain mutations are reported as pathogenic in numerous other cases of SPG7-related disease.

Patient 2 had a homozygous splice site mutation in c.988-1G > A which produces two cryptic splice sites, resulting in a deletion of 2 and 21 nucleotides, respectively (Figure 1(A)). This mutation had been previously reported in the literature to be pathogenic in the French Canadian population (Choquet et al., 2016).

Figure 1. (A) Sanger sequencing of cDNA from a control (upper chromatograph) and Patient 2 (lower chromatograph) indicating overlapping sequences (blue arrow) that confirm a 2 and 21 nucleotide deletion. (B) Agarose gel electrophoresis of cDNA from Patient 7, indicating two PCR products (red arrow) revealing the normal-sized product and the smaller product with the 134 base pair deletion of exon 6 (the two lanes to right are normal controls with only a single normal-sized PCR product). (C) Sanger sequencing of the above PCR product indicates the presence of a heterozygous deletion of exon 6, with joining of exons 5 and 7. (D) RT-qPCR of SPG7 transcript levels demonstrate significantly reduced transcript levels in Patients 1, 2, and 3 (p < .05). (E) Sanger sequencing at the position of the c.1045G > A mutation from a control (upper chromatograph) and Patient 3 (lower chromatograph) demonstrating the mutation appears to be homozygous (although it is heterozygous in genomic DNA), indicating that cDNA from the opposite allele is not transcribed or is degraded.

Patient 3 had a heterozygous mutation in c.1045G > A (p.Gly349Ser). This variant was previously reported as pathogenic (rs141569620) (van Gassen et al., 2012), and has been reported in the single heterozygous state in a patient with a similar phenotype (Krüger et al., 2016). We attempted to identify any cryptic splicing abnormalities by sequencing the SPG7 cDNA in this patient with primers binding to the splice junctions at exons 1–2 and 14–15. This indicated a normal-sized product with normal exon order. However, the c.1045G > A mutation appeared to be homozygous in cDNA (Figure 1(E)) indicating that the cDNA from the opposing allele is either not transcribed or is degraded.

Patient 4 had a frameshift mutation (c.850_851 delTTinsC), previously reported in patients from one Moroccan family (Elleuch et al., 2006). The second mutation, c.2014G > A (p.Gly672Arg), was previously described as a variant of unknown clinical significance from a single patient in a Dutch cohort (Brugman et al., 2008; van Gassen et al., 2012).

SPAST mutations

All patients with SPAST mutations had a typical phenotype with slowly progressive uncomplicated paraparesis starting in the legs. Patient 5 had a c.1573C > T (p.Gln525*) mutation that was previously described (Loureiro et al., 2009). Patient 6 had a novel mutation, c.156T > G (p.Tyr52*), which is considered to be likely pathogenic based on the fact that early termination mutants are a known cause of disease. Patient 7 had a c.1004 + 1G > C novel mutation, which causes skipping of exon 6 due to disruption of the 5′ splice site (Figure 1(B,C)).

RT-qPCR data

RT-qPCR was performed to indicate the presence of reduced SPG7 transcript levels in patients compared with controls (Figure 1(D)). The results indicate a reduction in SPG7 transcript levels in patients compared to controls, which was statistically significant for Patients 1, 2, and 3 (p < .05).

Discussion

Diagnostic yield of SPG7 and SPAST sequencing was very high in this ethnically diverse cohort, and emphasises the utility of such testing as part of routine care for patients with very late-onset HSP, or diagnoses of PLS that have not developed bulbar involvement. Most of the prior studies done in HSP7 were conducted in ethnically-homogenous populations such as Dutch (Brugman et al., 2008), British (Pfeffer et al., 2015), French Canadian (Choquet et al., 2016) and Spanish cohorts (Sánchez‐Ferrero et al., 2013, p. 7). In this series with very late onset spasticity, half of the patients had variants identified in either SPG7 or SPAST.

Prior series have considered the diagnosis of patients with very late onset spasticity. Variable penetrance in HSP is well-known, and may in part be related to very late onset in subsets of patients: in a study of familial HSP categorised the condition as ‘type 1’ or ‘type 2’ based on age of onset below or above 35 years, in which these very late onset cases made up 9 of 52 cases in the cohort (Harding, 1981). As knowledge of the genetic causes of these conditions has improved, more recent series indicate that onset age later than age 35 is fairly common. One large series of individuals with SPAST mutations indicated that at the time of their assessment, 14 of 238 patients were not yet clinically affected, and 45 of 238 patients were subclinically affected (Fonknechten et al., 2000). Recently, another large series demonstrated that 52% of cases had an age of onset after 35 years and that the peak age of onset was around the age of 40 years (Schüle et al., 2016). The broad distribution of presentation age was mainly accounted for by cases due to SPAST and SPG7 mutations, which was also demonstrated in another recent large series (Chrestian et al., 2017). We are not aware of studies that specifically attempted to define the diagnostic yield of genetic testing in patients with very-late onset spasticity. As such, our findings are valuable by identifying a high diagnostic yield in this patient population.

This study reports novel variants in Patients 1, 6, and 7. This study also supports the pathogenicity of other variants that have only been previously reported in single patients/families: in Patient 4, the c.2014G > A mutation is reported from a single patient in a Dutch cohort (Brugman et al., 2008; van Gassen et al., 2012) and this variant does not contain an entry in ClinVar (accessed 3 April 2018). The c.850_851delTTinsC mutation in Patient 4 is reported from a separate single family (Elleuch et al., 2006). The appearance of these variants in new unrelated patients supports the pathogenicity of these variants, as does the reduced SPG7 transcript levels.

Patient 3 presented with symptoms suggestive of motor neuron disease with fasciculations, atrophy, and spasticity. He was found to harbour a single heterozygous SPG7 mutation in c.1045G > A which was previously reported in another patient with ALS (Krüger et al., 2016). We have reason to believe that a second genetic lesion is affecting the opposing SPG7 allele, given that sequencing of cDNA was only able to identify the allele containing the c.1045G > A variant (Figure 1(E)), suggesting that the other transcript is either not transcribed or is degraded. This is the probable explanation for the reduction of SPG7 transcript levels in this patient (Figure 1(D)). We attempted to identify any cryptic splicing abnormalities but the sizing of the cDNA from exons 2–14 yielded a normal result. Identifying the second genetic lesion in this patient will be a matter for future study.

It is increasingly known that some patients with HSP7 appear to have dominantly inherited disease (Pfeffer et al., 2014; Sánchez‐Ferrero et al., 2013), with a phenotype similar to patients having recessive SPG7 mutations. The mechanisms by which single heterozygous SPG7 mutations can sometimes cause disease is not currently understood but may relate to other gene modifier effects that are not yet identified. It is also possible that the unidentified second genetic lesion in Patient 3 may also be present in other single heterozygous HSP7 patients. Although the nature of this second lesion is currently unknown, sequencing of cDNA in other single heterozygous cases could provide indirect evidence for a second genetic lesion in these cases, as in Patient 3 from this report.

In the case of Patient 4, it would have been helpful to be able to prove that the mutations were on opposite alleles, although unfortunately, family members were not available for sequencing, and attempts to demonstrate this using studies on the patient’s cDNA were not successful (data not shown). The identified SPG7 variants remain the presumed cause of the patient’s condition given the typical HSP7 phenotype in this patient, and the presence of two heterozygous variants that have both previously been reported in patients with spasticity.

Overall these findings support an additional conclusion regarding the extremely poor genotype-phenotype correlation in these disorders. For example, Patient 1 can be said to have the most severe genetic lesion of our cohort, having a homozygous stop-gain mutation after only 82 amino acids. However, despite this, the patient had a very late onset, at age 49, and remained independently functioning at the time of last assessment at age 57. His phenotype actually has similar severity to single heterozygous SPG7 mutation patients that have been previously described (Pfeffer et al., 2014). The very poor genotype-phenotype correlation may be due to the interaction of additional genetic factors that may explain the observation of patients with HSP7 phenotypes associated with single heterozygous mutations (Pfeffer et al., 2015; Sánchez‐Ferrero et al., 2013). There is precedent for the interaction of different Mendelian disease genes causing unique phenotypes (Balci et al., 2017; Posey et al., 2017), and detailed study of such situations can produce novel mechanistic insights (Lee et al., 2018; Niu et al., 2018). Given the extensive unexplained heterogeneity in HSP, particularly in SPG7- and SPAST-related disease, these conditions would appear to be the best candidates for study, to identify novel modifier mechanisms. On account of the differing cellular functions of spastin and paraplegin, it appears plausible that the mechanisms of phenotypic variability are likely to be distinct for each of these conditions.

Acknowledgements

Special thanks to Dr. Chris White and Dr. William Murphy who had referred patients who were included in this study.

Disclosure statement

No potential conflict of interest was reported by the authors.