Evoked potentials as biomarkers of hereditary spastic paraplegias: A case-control study.

INTRODUCTION: The Hereditary Spastic Paraplegias (HSP) are a group of genetic diseases that lead to slow deterioration of locomotion. Clinical scales seem to have low sensitivity in detecting disease progression, making the search for additional biomarkers a paramount task. This study aims to evaluate the role of evoked potentials (EPs) as disease biomarkers of HSPs.
METHODS: A single center cross-sectional case-control study was performed, in which 18 individuals with genetic diagnosis of HSP and 21 healthy controls were evaluated. Motor evoked potentials (MEP) obtained with transcranial magnetic stimulation and somatosensory evoked potentials (SSEP) were performed in lower (LL) and upper limbs (UL).
RESULTS: Central motor conduction time in lower limbs (CMCT-LL) was prolonged in HSP subjects, with marked reductions in MEP-LL amplitudes when compared to the control group (p<0.001 for both comparisons). CMCT-UL was 3.59ms (95% CI: 0.73 to 6.46; p = 0.015) prolonged and MEP-UL amplitudes were reduced (p = 0.008) in the HSP group. SSEP-LL latencies were prolonged in HSP subjects when compared to controls (p<0.001), with no statistically significant differences for upper limbs (p = 0.147). SSEP-UL and SSEP-LL latencies presented moderate to strong correlations with age at onset (Rho = 0.613, p = 0.012) and disease duration (Rho = 0.835, p<0.001), respectively. Similar results were obtained for the SPG4 subgroups of patients.
CONCLUSION: Motor and somatosensory evoked potentials can adequately differentiate HSP individuals from controls. MEP were severely affected in HSP subjects and SSEP-LL latencies were prolonged, with longer latencies being related to more severe disease. Future longitudinal studies should address if SSEP is a sensitive disease progression biomarker for HSP.

Introduction

The Hereditary Spastic Paraplegias (HSP) are a group of monogenic neurodegenerative diseases with great clinical and genetic heterogeneity, currently with 83 different loci1 [1]. HSPs are rare diseases with prevalence estimations ranging from 2 to 9.1 per 100,000 individuals [2, 3]. The main features of these conditions are related to the retrograde degeneration of the longest axons of the corticospinal tract and the posterior columns [4].

HSP are classified in pure and complex forms [5]. An isolated pyramidal syndrome that predominantly affects the lower limbs, accompanied or not by neurogenic bladder and impaired vibratory sensation, characterizes “pure” HSP. Additional involvement of other systems (cognitive impairment, ataxia, parkinsonism, visual or auditory disorders, peripheral neuropathy, etc.) defines "complex" forms. Symptoms progress slowly, starting from childhood to late adulthood [4, 6], and complex forms are generally associated to a more severe disease course [2].

Although the natural history of HSPs is largely unknown, the available studies point to a very slow progression [2, 7] suggesting that clinical scales based on neurological examination might not present enough sensitivity to change for detecting disease progression, making the search for additional biomarkers a paramount task. Abnormalities in motor and somatosensory evoked potentials were previously described in HSPs, pointing neurophysiological measurements of the integrity of central motor and sensory pathways as candidate biomarkers of these diseases. However, results on motor evoked potentials (MEP) and somatosensory evoked potentials (SSEP) were heterogenous across reports, with most studies evaluating small samples or presenting poor genetic and clinical characterization [8-10].

Therefore, the aim of the present study was to characterize the role of MEP and SSEP in lower (LL) and upper limbs (UL) as biomarkers of HSPs, and to advance in the understanding of the pathophysiology of these disorders; especially concerning the involvement of central sensory pathways.

Methods

A single center exploratory cross-sectional case-control study was performed, in which a convenience sample of 18 individuals (from 11 families) with genetic diagnosis of HSP (12 SPG4, 3 SPG5, 1 SPG7, 1 SPG11 and 1 cerebrotendinous xanthomatosis, CTX) and 21 healthy controls were evaluated. Participants were included in the study from October 2019 to February 2021. The study was approved by the Ethics in Research Committee of Hospital de Clínicas de Porto Alegre (GPPG-HCPA 2019–0081), Porto Alegre, Brazil. All participants were verbally informed about the conditions of the study, and signed a written consent form. In the case of children under 18 years of age, the parents signed the consent. The control group was composed of family members unrelated to the cases, such as spouses, and individuals from the community of Porto Alegre.

Eligibility for cases were previous molecular diagnosis of HSP and acceptance in participating in the study. The single subject with CTX presented a complex form of HSP, details on this case are available elsewhere [11]. Healthy subjects, unrelated, but with similar sex and ages to cases, without previous diagnosis of neurological or systemic diseases associated to motor or sensory abnormalities were recruited as the control group. Considering the exploratory design of the study, no single primary outcome was defined and sample size estimations were not performed.

Data regarding sex, age at last examination, age at onset (first motor sign), disease duration and history of peripheral neuropathy were collected from patients and relatives or retrieved from electronic medical records. Severity of disease was evaluated with the Spastic Paraplegia Rating Scale (SPRS, range: 0–52, crescent in severity) [12]. We also analyzed motor-SPRS (mSPRS), excluding items related to pain and sphincter control (range: 0–44).

Electrophysiological procedures

MEPs were measured to the first dorsal interosseus and tibialis anterior muscles after muscle activation. MEPs were obtained by single pulse transcranial magnetic stimulation with the Neuro-MS Paired Monophasic Transcranial Magnetic Stimulator (Neurosoft, Russia) device, in which an eight-shaped magnetic stimulating coil was placed over the motor cortex (total motor conduction time, TMCT) of the dominant hemisphere (C3 or C4, based on 10–20 EEG system), orientating the coil at 45% degrees from C3-C4 positions to nasion, over the seventh cervical vertebra for UL and over the fifth lumbar vertebra for LL (peripheral motor conduction time, PMCT). The pulse intensity started at the motor threshold value and increased up to about 20% of this threshold, single pulses were delivered with a frequency of 1 Hz. Ten MEPs were recorded and their amplitudes and latencies were averaged. The recording sensitivity was 100μV and 5ms per division and the filter for lower and higher frequencies was 5Hz and 10kHz, being analyzed during 100ms. Central motor conduction time (CMCT) was obtained with the direct method, by subtracting TMCT from PMCTs. When CMCT was absent, a ceiling value of 100ms was imputed. MEP amplitudes were measured from baseline to peak.

SSEPs were obtained using Neuropack M1 MEB-9200 (Nihon Kohden, Japan), in which the stimulus was generated through electrical pulses of 0.2ms applied 3 times/sec with intensities ranging from 2 to 20mV applied in medial malleolus and wrists, over the median and posterior tibial nerves respectively. On average 200 to 250 potentials were performed and superimposed to check for the reproducibility of the stimulus. Central recording electrodes were placed on the scalp over the primary sensitive area (Fz, Cz, C3, C4) with peripheral check points at the Erb point for the UL and popliteal fossa for the LL. The recording sensitivity was 2μV and 5ms per division and the filter for lower and higher frequencies was 10-2500Hz, being analyzed during 100ms. The N20 peak latencies was considered for the UL and the N50 peak latencies was considered for the LL. All neurophysiological evaluations were performed by the same evaluator (SFB), in order to reduce measurement bias. Examples of MEP and SSEP recording are presented in .

Statistical analysis

Statistical tests were selected according to the distribution of data given by Shapiro-Wilk test and histograms. Age, age at onset, disease duration, SPRS scores and CMCT-UL presented normal distributions and were presented as means and standard deviations. The other continuous variables in the study exhibited a non-parametric distribution and were shown as median and interquartile ranges. Comparisons between cases and controls for continuous variables were performed by Mann-Whitney U-test for CMCT and for non-parametric variables and by two-tailed unpaired Student’s t-test for parametric variables and by Fisher’s Exact Test for categorical variables. CMCT and the SSEP latencies were considered prolonged in a given subject when the values exceeded 2 standard deviations above the mean value for the control group of the study. Correlations were performed with Spearman correlation test for CMCT and for non-parametric variables. Statistical significance was defined as p<0.05.

Results

The main demographic characteristics of the sample are summarized in and the main motor and somatosensory evoked potentials findings are described in . Among all the 18 individuals with HSP, only the subject with CTX had complains about decreased pain sensation in the LL. All datasets can be found in S3 Table.

Motor evoked potentials

MEP amplitudes in UL and LL were decreased in HSP subjects when compared to healthy controls (p = 0.008, ; p<0.0001, ; respectively). Similar results were found in the SPG4 subgroup for amplitudes of MEP-LL (p = 0.001, ), but not for amplitudes of MEP-UL, which presented a trend for reduced amplitudes when compared to healthy controls (p = 0.053, ). No statistically significant correlations of MEP amplitudes for the overall HSPs or SPG4 subgroup were found with disease severity variables ( and respectively)

Evoked potentials abnormalities in hereditary spastic paraplegias.

CMCT: Central Motor Conduction Time; HSP: Hereditary spastic paraplegia; LL: lower limbs; MEP: motor evoked potential; msec: milliseconds; mV: millivolt; SSEP: Somatosensory Evoked Potential; UL: upper limbs; μV: microvolt. *p<0.05; **p<0.01; ***p<0.001.

Evoked potentials abnormalities in SPG4.

CMCT: Central Motor Conduction Time; HSP: Hereditary spastic paraplegia; LL: lower limbs; MEP: motor evoked potential; msec: milliseconds; mV: millivolt; SSEP: Somatosensory Evoked Potential; UL: upper limbs; μV: microvolt. ***p<0.001.

CMCT in lower limbs (LL) was strikingly different when compared to healthy controls (p<0.001, ). CMCT-UL was 3.59ms (95% CI: 0.73 to 6.46; p = 0.015, ) longer in HSP subjects when compared to healthy controls. Similar results were found for the SPG4 subgroup for CMCT-LL (p<0.001, ), but not for CMCT-UL (p = 0.813, ) when compared to healthy controls. No statistically significant correlations of CMTCs for the overall HSPs or SPG4 subgroup were found with other disease severity variables ( and respectively).

Somatosensory evoked potentials (SSEP)

Cortical SSEP latencies in lower limbs (p<0.001, ), but not in upper limbs (p = 0.147, ), were prolonged in HSP subjects when compared to healthy controls. SEP-LL presented a direct correlation with age (Rho = 0.628, p = 0.012) and disease duration (Rho = 0.835, p<0.001, ), and moderate, but non-significant correlations with SPRS (Rho = 0.483, p = 0.068) and motor SPRS (Rho = 0.502, p = 0.056, ) in the overall HSP group. SEP-UL presented a direct correlation with age (Rho = 0.698, p = 0.003) and age at onset (Rho = 0.613, p = 0.012, ) in the overall HSP group. Similar results were obtained for the SPG4 subgroups of patients, with prolonged cortical SEP latencies in lower limbs (p = 0.001, ), but not in upper limbs (p = 0.593, ). In the SPG4 subgroup, SEP-LL presented a direct correlation with disease duration (Rho = 0.811, p = 0.002, ) and SEP-UL presented a direct correlation with age (Rho = 0.834, p = 0.001) and age at onset (Rho = 0.720, p = 0.008, ). Neither SEP-UL nor SEP-LL correlate significantly with age in the control group (Rho = 0.250, p = 0.274; Rho = 0.260, p = 0.255, respectively). No statistically significant correlations of SEP latencies for the overall HSPs or SPG4 subgroup were found with other disease severity variables ( and respectively).

Correlations of somatosensory evoked potential with disease severity variables.

LL: lower limbs; MEP: motor evoked potential; SSEP: Somatosensory Evoked Potential; SPRS: Spastic Paraplegia Rating Scale; msec: milliseconds; mSPRS: Motor Spastic Paraplegia Rating Scale; UL: upper limbs.

Discussion

In the present study a detailed neurophysiological characterization of the integrity of central motor and sensory pathways in individuals with HSPs was performed. Our results indicated that motor and somatosensory evoked potentials can distinguish HSP subjects from healthy controls. MEPs were more severely affected in HSP subjects and SSEP-LL latencies were prolonged, with longer latencies being related to more severe disease.

Motor evoked potential presented decreased amplitudes in both the UL and the LL in the overall HSP group, but for SPG4, only MEP-LL amplitudes were reduced. Several studies have described decreased or absent MEP-LL amplitudes in HSPs [8–10, 13, 14]; however, for MEP-UL amplitudes the results from different authors were more heterogeneous, with some studies finding similar results to healthy controls [10, 13]. No significant correlations between MEP-LL and MEP-UL amplitudes with disease severity variables were found in this work, and most previous studies did not report evaluating these correlations [8–10, 13, 14].

Central motor conduction times in the lower limbs were altered in the majority HSP individuals, being absent in 9/18 (50%). In a recent systematic review of MEPs in HSPs [9], 78% of studies found abnormalities in the CMCT-LL. A study involving three centers in Germany that evaluated 128 patients with HSP, 54 of them with confirmed genetic diagnosis, reported prolongation of the CMCT-LL in 37% of the cases and absence in 36%. In the subgroup of 35 patients with SPG4, 48% presented prolonged CMCT-LL [8]. Another multicenter study carried out in Italy performed neurophysiological characterization of 49 subjects with confirmed genetic diagnosis of HSP, describing that CMCT-LL was prolonged or absent in all cases, except for one individual with SPG4 with mild phenotype. However, the correlations of CMCT findings with disease severity variables and the number of subjects who performed each of the evaluations were not clearly described for this measurement [15]. Another Italian study that evaluated 12 patients with SPG4 with an average disease duration of 20 years described prolonged CMCT-LL obtained by both the direct and indirect methods [10].

Central motor conduction times in the upper limbs were prolonged in the overall HSP group, but without differences between SPG4 and controls. Previous studies showed variable results for CMCT-UL, being abnormal in 59% of the studies reported in a recent systematic review [9]. In the multicenter studies reported above, 45% of Italian [15] and 32% (28% prolonged and 4% absent) of German patients presented prolonged CMTC-UL; however, CMCT-UL was normal in all subjects with SPG4 evaluated in the German study [8]. Eight studies reported normal CMCT-UL in HSPs, 4 of which in cohorts that included only patients with SPG4 [9, 10, 16–18].

We did not find any significant correlations between CMCTs and age at onset, disease duration and SPRS, which is similar to that found in most previous studies [9, 10, 19]. The exception was the study by Karle et al; which described a weak correlation between CMCT-LL with total SPRS and its spasticity sub-score. Another interesting data from this study was the genotype neurophysiological-phenotype correlation, in which subjects with missense variants had lower CMCT-LL latencies than subjects with truncating variants and in-frame deletions in SPAST [8]. Interestingly, such findings have been replicated in advanced neuroimaging studies in which SPG4 patients with missense variants had less severe corticospinal tract diffusivity abnormalities than patients with truncating variants in SPAST [20].

Therefore, all these data indicate important changes in CMCT in patients with HSPs, particularly when evaluated in the lower limbs. The absence of MEP in lower limbs in a significant proportion of HSP subjects indicates that this measurement has a low threshold for a ceiling effect, which may prevent the detection of relevant correlations with variables related to disease severity. In the case of SPG4, CMCT-LL results are similar to the overall HSPs, but differ for CMCT-UL, which present similar latencies to controls in most studies. Most series that evaluated the CMCT-LL, including ours, had an average disease duration close to 20 years, with moderate to severe disease according to SPRS. Due to the ceiling effect that was observed for CMCT-LL, it is unlikely that this variable will be a good biomarker of disease progression for HSP subjects with similar severity and disease durations to those reported so far; however, it will be essential that future longitudinal studies assess CMCT-LL at early disease stages, looking for an early-stage disease progression biomarker. The normal or slightly altered results of the CMCT-UL, on the other hand, suggest that future longitudinal studies should assess the progression of latencies and amplitudes of MEP in the upper limbs, seeking for an eventual role of this measure as a disease progression biomarker.

With regard to somatosensory evoked potentials, the present study found important changes only in lower limbs, in which the cortical SSEP-LL latencies were prolonged in the overall HSP and SPG4 subgroup. SSEPs are less explored in the literature, and available data are more controversial. In the German study by Karle and collaborators SSEP-UL latencies were normal in 91% of cases, whereas SSEP-LL latencies were altered in 34% of cases, being prolonged in 27% and absent in 7% [8]. In the small Italian series that evaluated only SPG4 patients, SSEP-UL latencies were normal, whereas SSEP-LL latencies were absent in 25% of cases. However, because absent potentials data were censored, reported SSEP-LL latencies were similar to the control group [10]. In the multicenter Italian study by Martinuzzi and collaborators, cortical SSEP-LL latencies were obtained from 44 subjects with HSP, being prolonged in 30/44 (68%). SSEP-UL latencies were evaluated only in the 30 subjects with abnormal SSEP-LL, being changed in 21/30 (70%), including 9/22 (40%) of patients with SPG4 and in most patients with SPG5, SPG7 and SPG15 [15]. A congress abstract reported cortical SSEP latencies in 28 subjects with SPG4, describing changes in SSEP-UL and SEP-LL latencies in 25% and 38% of cases, respectively. The authors also reported severe temporal dispersion with decreased SSEP-LL amplitude in 61% of patients [21].

In the present study, SSEP-LL latencies strongly correlated with disease duration and moderately with age and showed trends to moderate correlations with SPRS and its motor sub score, with similar results for correlation with disease duration in the SPG4 subgroup. Despite not differing from controls, SSEP-UL latencies showed a moderate direct correlation with age at onset and with the age of subjects with HSP and with SPG4. As we did not find any correlation between age and SSEP-LL and SSEP-UL latencies in healthy controls, the correlations identified with age in HSP likely represent a confounding factor due to its high intercorrelation with age at onset and disease duration. Due to the exploratory nature of this study and its small sample size, it was not possible to perform more robust statistical analyzes to correct for this bias. The study by Karle et al. reported a correlation of SSEP-LL latencies with clinical sensory deficit [8]; however, correlations with other clinical variables were not previously described [8, 10, 15, 21].

Prolonged cortical SSEP-LL latencies in HSP are consistent with the clinical findings of vibratory sensation impairment even in pure forms of HSPs and with the results of neuroimaging studies, which indicate that the neurological changes also affect sensory pathways. Advanced neuroimaging studies using voxel-based morphometry show widespread damage to white matter in all forms of HSP and also damage to gray matter in complex forms [22]. In addition to microstructural changes in the corticospinal tract, changes in posterior brain subcortical regions were also reported in SPG4 subjects [20] as well as reduction in the cervical and dorsal spinal cord area [18, 22, 23]. Of note the reduction of spinal cord area was not accompanied by its flattening, which was interpreted as similar findings to pathological reports of SPG4 individuals, in which lateral and posterior columns involvement were the major macroscopic finding, suggesting both corticospinal tracts and posterior column involvement in this subtype [23]. Post-mortem neuropathological studies have also confirmed the involvement of the gracilis and cuneirform fascicles, with maximum involvement in the region of the dorsal spine [4]. Additionally, considering that dorsal root ganglion cells, which are pseudounipolar neurons, present the longest axons in humans [24] and that HSPs are one of the groups of dying-back axonopathies of long tracts, it was expected that sensory pathways abnormalities detected by SSEP would be found and would be relevant for these diseases.

Our results suggest that there are changes in SSEP-LL latencies in patients with HSP and that these latencies are correlated with longer disease durations. Future studies with larger sample sizes will be able to better detail the possible correlation with disease severity measured by SPRS. Thus, SSEP-LL latencies can be considered as disease biomarkers of HSPs and it will be essential that future longitudinal studies evaluate SSEP-LL and SSEP-UL latencies in HSPs, seeking to evaluate its role as a disease progression biomarker.

Study limitations

The major study limitation was its small sample size and its exploratory design. Although several statistically significant differences were found for different EP measurements; we likely did not have enough power to detect smaller differences between groups and weaker to moderate correlations of MEPs and SSEPs latencies with other disease severity variables. Another study limitation is the lack of nerve conduction studies (NCS). CMCTs were obtained by the direct method, which is not influenced by motor NCS. Abnormalities in sensory NCS might have affected SSEPs; however, since only 2/18 individuals in the study presented peripheral neuropathy, both with complex HSP, and the SSEPs results of the overall HSP and the SPG4 subjects (all with pure HSP and with no evidence of peripheral neuropathy) were similar, it is unlikely that the lack of correction for sensory NCS have influenced the study results in a significant manner.

Conclusion

Motor and somatosensory evoked potentials can distinguish HSP subjects from controls. MEP were severely affected and SSEP-LL latencies were prolonged, with longer latencies being related to more severe disease, indicating that SSEP-LL are candidate disease biomarkers for HSP. Future longitudinal studies should address if CMCT in earlier disease stages and SSEP latencies are sensitive disease progression biomarker for HSPs that could be used as surrogate outcome measures for future clinical trials.

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(PDF)

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Examples of motor and somatosensory evoked potentials.

A) Example of a motor evoked potential (MEP); B) Example of a Somatosensory Evoked Potential (SSEP).

(TIF)

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Correlations of evoked potentials with clinical findings in the overall HSP group.

CMCT: Central Motor Conduction Time; HSP: Hereditary spastic paraplegias; LL: lower limbs; MEP: motor evoked potential; ms: milliseconds; mV: millivolt; SSEP: Somatosensory Evoked Potential; UL: upper limbs; μV: microvolt.

(DOCX)

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Correlations of evoked potentials with clinical findings in the SPG4 subgroup.

CMCT: Central Motor Conduction Time; HSP: Hereditary spastic paraplegias; MEP: motor evoked potential; ms: milliseconds; mV: millivolt; SSEP: Somatosensory Evoked Potential; UL: upper limbs; μV: microvolt.

(DOCX)

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Full dataset with clinical, genetic and neurophysiological information.

AA, Amino acid; ACMG, American College of Medical Genetics and Genomics; CMCT: Central Motor Conduction Time; LL: lower limbs; SPRS, Spastic Paraplegia Rating Scale; SSEP: Somatosensory Evoked Potential; UL: upper limbs.

(XLSX)

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10 Sep 2021

PONE-D-21-19134Evoked potentials as biomarkers of hereditary spastic paraplegiasPLOS ONE

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Reviewer #1: This is a nice study looking at evoked potentials in HSP. More data is certainly needed in this area, given this is still one of the most promising biomarkers for this condition. The paper is very well written and the methodology is complete.

However, a few comments could be made about the study which may improve it further:

- The title could perhaps better represent the contents of the study, from reading the title 'Evoked potentials as biomarkers in hereditary spastic paraplegia' it is not clear whether it is a review, systematic review or original research. The authors should consider an alternative title that is more informative such as ‘A single centre cross-sectional case-control study of evoked potentials as biomarkers of hereditary spastic paraplegia’.

- For the cerebrotendinous xanthomatosis patient to be included, the authors should clarify that this patient had a predominant HSP phenotype, since the phenotypic spectrum of this condition is variable.

- The genetic findings should be included, perhaps in a supplementary table (this would address the following point '3. Have the authors made all data underlying the findings in their manuscript fully available?').

- I thought the discussion could have been more concise and focused on the findings of the study.

- It is already known that CMCT is absent or prolonged in HSP, so the authors should make it clear what the unique contribution of there study is.

Reviewer #2: In their original article „Evoked potentials as biomarkers of hereditary spastic paraplegias”, Brighente et al. present the results of a cross sectional evoked potential study in 18 HSP patients (among these 12 SPG4) and a control cohort. As these measures are easy to perform electrophysiological biomarkers, they would be of high interest for the HSP field, and longitudinal data are still scarce. The present study, however, is limited by the low patient number, a missing disease mimic cohort (e.g., multiple sclerosis), the cross sectional design, missing p-value correction for multiple testing, and the insufficient consideration of existing studies.

Previous studies are described “poor” although some of them contained more patients with more detailed characterization than in the current work (e.g., PMID24107482 and PMID27077743). It is also claimed that “previous studies did not report evaluating these correlations [8,9,10,11,12]” although it was indeed performed in ref. [8].

Substitution of an absent TMS LL response with a value of CMCT 100ms appears arbitrary and must at least be critically discussed. With this calculation, it is not surprising that the difference for CMCT-LL is highly significant. The same is true for the SEP-LL latency.

The correlation analyses for age at onset and disease duration show significant correlations. A basic influence of age at examination itself could also explain these results. Therefore, authors should also calculate correlations to age at examination (both for the HSP and for the control cohort).

As shown in Suppl. tables 1 and 2, a high number of correlation analyses was performed and the authors highlighted the significant findings in the main text and figure 3. However, with multiple correlation analyses, p values need to be corrected for multiple testing (e.g., FDR method).

Additional issues:

Text word count on the title page is missing.

When stating “discriminatory validity” within the Abstract and in the discussion/ conclusions, authors should also perform ROC analysis and indicate AUC values. Also, the authors mention “prolonged”, but do not state reference/ cut-off values and how these were defined.

It is not reported where controls were recruited (from hospital staff, or unrelated family members, or community?).

Single data points (i.e. one point per patient/ control) should be shown in the figures.

typos: p.15 “then”, p.20 “suspicion”, p.25 “earlies”

**********

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Reviewer #1: Yes: Kishore Raj Kumar

Reviewer #2: No

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12 Oct 2021

RESPONSE TO REVIEWERS

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

Response: The text was checked according to the template suggested by the journal.

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Response: Additional information about the consent form and control group was attached in the body of the article.

“All participants were verbally informed about the conditions of the study, and signed a written consent form. In the case of children under 18 years of age, the parents signed the document. The control group was composed of family members unrelated to the cases, such as spouses, and individuals from the community of Porto Alegre.”

3.Thank you for stating the following in the Acknowledgments Section of your manuscript:

“We are grateful to patients for participating in this study. The study was funded by Fundo de Incentivo à Pesquisa e Eventos-Hospital de Clínicas de Porto Alegre (FIPE-HCPA) (Grant Number: 2019-0081).

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Response: The funding information was withdrawal from this section and provided only in the online submission form.

4 – In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

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Response: In order to attend PLOS Data policy we have now added Supplemental Table 3, an Excel file in which anonymized raw data for clinical, genetic and neurophysiological variants is provided.

Point 5

COMENTS

Reviewer 1

Reviewer #1:

This is a nice study looking at evoked potentials in HSP. More data is certainly needed in this area, given this is still one of the most promising biomarkers for this condition. The paper is very well written and the methodology is complete.

However, a few comments could be made about the study which may improve it further

Response: We thank the reviewer comment

- The title could perhaps better represent the contents of the study, from reading the title 'Evoked potentials as biomarkers in hereditary spastic paraplegia' it is not clear whether it is a review, systematic review or original research. The authors should consider an alternative title that is more informative such as ‘A single centre cross-sectional case-control study of evoked potentials as biomarkers of hereditary spastic paraplegia’.

Response: We agree with the reviewer point. The title was changed to:

“Evoked potentials as biomarkers of hereditary spastic paraplegias: a case-control study”

- For the cerebrotendinous xanthomatosis patient to be included, the authors should clarify that this patient had a predominant HSP phenotype, since the phenotypic spectrum of this condition is variable.

Response: We have added in the eligibility criteria section that the single subject with CTX presented a complex form of HSP and that details on this case are available elsewhere (Burguez et al, 2017).

- The genetic findings should be included, perhaps in a supplementary table (this would address the following point '3. Have the authors made all data underlying the findings in their manuscript fully available?').

Response: We have added Supplemental Table 3, an Excel file in which anonymized raw data for clinical, genetic and neurophysiological variants is provided.

- I thought the discussion could have been more concise and focused on the findings of the study.

Response: We have reviewed the discussion section and performed cuts in the text in order to reduce its length.

- It is already known that CMCT is absent or prolonged in HSP, so the authors should make it clear what the unique contribution of there study is.

Response: We agree that it is already known that CMCT is absent or very

prolonged in HSP; however, some previous studies did not have a complete genetic characterization and there is a lack of data regarding the correlation of CMCT with other clinical severity outcome. So, we consider that the confirmation of CMCT prolongation in HSP in our population was an interesting finding. Also, the lack of correlation of CMCT with other disease severity variables may indicate that this biomarker in patients with mean disease duration of around 15 years is too much compromised, probably presenting a ceiling effect that hamper significant correlations with other disease severity biomarkers like clinical scales and disease duration. It will be interesting to evaluate CMCT in HSP patients in the first years of clinical presentation, indicating that future studies with CMCT in HSP are needed.

Nevertheless, the most relevant finding in this sample was the data found on sensory latencies. The sensory system is generally not considered a core system involved in the pathophysiology of HSP and we have found differences from controls and significant correlations with disease duration and age at onset, and a trend for a moderate direct correlation with SPRS severity. Perhaps the fact that the sensory system remains more preserved than the motor makes it a better disease biomarker during intermediate or late stages of the disease.

We have added in the conclusion that SSEP-LL are candidate disease biomarkers for HSP, trying to emphasize this major result.

Reviewer 2

In their original article “Evoked potentials as biomarkers of hereditary spastic paraplegias”, Brighente et al. present the results of a cross sectional evoked potential study in 18 HSP patients (among these 12 SPG4) and a control cohort. As these measures are easy to perform electrophysiological biomarkers, they would be of high interest for the HSP field, and longitudinal data are still scarce.

Response: We thank the reviewer comment and we agree his/her points.

The present study, however, is limited by the low patient number, a missing disease mimic cohort (e.g., multiple sclerosis), the cross sectional design, missing p-value correction for multiple testing, and the insufficient consideration of existing studies.

Response: We thank the reviewer comment. We will address the comments point by point bellow

Low patient number: We agree with this point, which was stated in the study limitations sections on discussion; however, considering the disease rarity and the number of subjects in previous studies with HSP and neurophysiological characterization we consider that our study provides significant addition to scientific literature. We have recruited all subjects from a reference center in a state of 10 million inhabitants in Southern Brazil with genetic confirmation of HSP diagnosis. Some patients didn´t agree to participate due to the pandemic of COVID-19. For sure, future multicenter studies will be needed to achieve larger samples sizes and robust conclusions; however, these future studies should focus on the clues given by studies like ours, as studying CMCT in early stages of HSP and to assess longitudinally SSEP to see if it is also a disease progression biomarker.

A missing disease mimic cohort (e.g., multiple sclerosis): Considering that we were not searching for a diagnostic test, as HSP are genetic conditions and the final genetic diagnosis is achieved by the gold standard (genetic testing) we did not consider that a disease mimic group would be necessary for the study aims. We changed the term discriminatory validity to discriminate case and healthy controls to avoid any misunderstanding regarding this point. Importantly, a given disease biomarker does not need to be specific of HSP. The main intention here was to assess if the biomarker was different from healthy controls, which meant that it is associated with the disease state and to assess if patients with greater severities indicated by different variables (eg: disease duration, SPRS, age at onset) presented worse results than patients with milder disease, trying to search for disease severity biomarkers.

Cross sectional design: Indeed, this design does not allow us to asses the properties of MEP and SSEP as disease progression biomarkers. Only longitudinal studies will be able to do so. We are following these subjects and we intend to report the longitudinal findings after 24 months in a future publication.

Missing p-value correction for multiple testing: As disclosed by the reviewer the study has a small sample and it is also exploratory, so we didn´t perform correction for multiple testing. We will discuss this issue in more details bellow.

insufficient consideration of existing studies: we have performed a systematized review of the literature for this project before it began and we have cited the main studies that were found. Of note, the systematic review by Siow et al summarized many of the studies regarding MEP and we have cited this comprehensive review.

Previous studies are described “poor” although some of them contained more patients with more detailed characterization than in the current work (e.g., PMID24107482 and PMID27077743). It is also claimed that “previous studies did not report evaluating these correlations [8,9,10,11,12]” although it was indeed performed in ref. [8].

Response: Some of the previous works have larger number of patients, like the ones cited by the reviewer; however, even with a larger sample, the study PMID27077743 had broader objectives and the neurophysiological characterization lacks a lot of details. On the other hand, the study by Karle et al is very well described, being a multicenter German study with a larger number of subjects, 128. As we have stated in the discussion section, the study by Karle et al, along with the others cited papers did not presented data on the amplitudes of MEP, indeed they have focused on CMCT findings. In the discussion section, when we are describing CMCT results we have detailed the findings of the study by Karle et al and we highlight such parts bellow:

“A study involving three centers in Germany that evaluated 128 patients with HSP-suspicion, 54 of them with confirmed genetic diagnosis, reported prolongation of the CMCT-LL in 37% of the cases and absence in 36% . In the subgroup of 35 patients with SPG4, 48% presented prolonged CMCT-LL [8].

…The exception was the study by Karle et al., which described a weak correlation between CMCT-LL with total SPRS and its spasticity sub-score. Another interesting data from this study was the genotype neurophysiological-phenotype correlation, in which subjects with missense variants had lower CMCT-LL latencies than subjects with truncating variants and in-frame deletions in SPAST [8].”

Substitution of an absent TMS LL response with a value of CMCT 100ms appears arbitrary and must at least be critically discussed. With this calculation, it is not surprising that the difference for CMCT-LL is highly significant. The same is true for the SEP-LL latency.

Response: The use of the ceiling value of 100ms for CMCT in absent TMS LL response was indeed arbitrary and intended to avoid the exclusion of cases with the greatest severity on this biomarker. However, the chosen value had no impact on the obtained p-values, since for both between groups comparisons and for correlations involving CMCT and SSEP we have used ranked non-parametric tests (Mann-Whitney U-test and Spearman) in which the rank, but the not the raw value is used to calculate p-values and Rho. We have added the statistical method for evaluating CMCT more clearly in the statistical analysis, to avoid misinterpretations. We have provided data always as median and interquartile ranges, except for CMCT-UL which did not have a ceiling effect and in which the data was normally distributed.

The correlation analyses for age at onset and disease duration show significant correlations. A basic influence of age at examination itself could also explain these results. Therefore, authors should also calculate correlations to age at examination (both for the HSP and for the control cohort).

Response: We consider that the raised issues were already addressed during the paper. In the results section we have informed that “SEP-LL presented a direct correlation with age (Rho=0.628, p=0.012)… in the overall HSP group” …” In the SPG4 subgroup…SEP-UL presented a direct correlation with age (Rho=0.834, p=0.001). We have also detailed that “Neither SEP-UL nor SEP-LL correlate significantly with age in the control group (Rho=0.250, p=0.274; Rho=0.260, p=0.255, respectively).”.

In the discussion section we have described that: “Despite not differing from controls, SSEP-UL latencies showed a moderate direct correlation with age at onset and with the age of subjects with HSP and with SPG4. As we did not find any correlation between age and SSEP-LL and SSEP-UL latencies in healthy controls, the correlations identified with age in HSP likely represent a confounding factor due to its high intercorrelation with age at onset and disease duration. Due to the exploratory nature of this study and its small sample size, it was not possible to perform more robust statistical analyzes to correct for this bias.”

As shown in Suppl. tables 1 and 2, a high number of correlation analyses was performed and the authors highlighted the significant findings in the main text and figure 3. However, with multiple correlation analyses, p values need to be corrected for multiple testing (e.g., FDR method).

Response: The study sample size is small and it has an exploratory nature, therefore we did not perform corrections for multiple comparisons. The exploratory design is depicted in the methods section and also in the limitations section.

After the reviewer comment, we have performed the FDR method and only the SSEP-LL correlation with disease duration remained significant at a Benjamini-Hochberg Adjusted P value of 0.024. Considering that the results related to SPRS and age at onset (moderate correlations) should be confirmed in future multicenter studies, with greater study power, we decided to keep our original statistical design and we have reported the results without corrections. We reinforce the exploratory nature of the results to support our decision. Being too restrictive in small sample size studies will likely increase Type II error, which is not the objective of an exploratory study like ours.

Additional issues:

Text word count on the title page is missing.

Response: We added word for title and abstract. Word count for the manuscript is not required according to the journal policies. We would add it anyway; however, because Tables should appear inside the text of the main file, as well as legends, we did not add this information.

When stating “discriminatory validity” within the Abstract and in the discussion/ conclusions, authors should also perform ROC analysis and indicate AUC values.

Response: We changed the term “discriminatory validity” to “the given variable discriminate case and healthy controls” to avoid any misunderstanding regarding this point. Considering that we didn´t aim to evaluate the diagnostic properties of MEP e SSPE, ROC curves were not calculated.

Also, the authors mention “prolonged”, but do not state reference/ cut-off values and how these were defined.

Response: We considered that normality values for neurophysiological variables taken from books or guidelines would be less reliable than a comparison with the local population, so we decided to evaluate a healthy control group. In the present version we have avoided the term prolonged, but we also have described that: “CMCT and the SSEP latencies were considered prolonged in a given subject when the values exceeded 2 standard deviations above the mean value for the control group of the study” in the methods section.

It is not reported where controls were recruited (from hospital staff, or unrelated family members, or community?).

Response: The control group was composed of family members unrelated to the cases, such as spouses, and individuals from the community of Porto Alegre. We have added this in the methods section.

Single data points (i.e. one point per patient/ control) should be shown in the figures.

Response: Done. Figures were updated accordingly.

typos: p.15 “then”, p.20 “suspicion”, p.25 “earlies”

Response: Corrected. Thanks.

Submitted filename: RESPONSE TO REVIEWERS.pdf

Click here for additional data file.

19 Oct 2021

Evoked potentials as biomarkers of hereditary spastic paraplegias: a case-control study

PONE-D-21-19134R1

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25 Oct 2021

PONE-D-21-19134R1

Evoked potentials as biomarkers of hereditary spastic paraplegias: a case-control study

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Table 1

Main demographics characteristics of the study sample.

	Healthy Controls	HSP	SPG4	p-value
	n = 21	n = 18	n = 12
Female sex	12/21 (57.1%)	7/18 (38%)	5/12 (41%)	0.3411
Age (years)	35.2 (10.4)	39.7 (18.7)	38.17 (6.25)	ns2
Height (meters)	1.66 (0.1)	1.64 (0.17)	1.61 (0.18)	ns2
Age at Onset (years)	-	23.06 (15.9)	22.83 (5,47)	-
Disease duration (years)	-	16.6 (9.07)	15.33 (2.74)	-
SPRS	-	19.9 (10.65)	17.25 (3.02)	-
SPRS motor	-	16.8 (8.9)	15.17 (2.74)	-

Data are shown as mean and standard deviation. HSP: Hereditary spastic paraplegias; ns, not statistically significant; SPRS: Spastic Paraplegia Rating Scale; mSPRS: Motor Spastic Paraplegia Rating Scale.

1Fisher’s Exact Test.

2 two-tailed unpaired Student’s t-test comparing HSP and controls and SPG4 subgroup and controls.

Table 2

Main motor and somatosensory evoked potentials findings.

	Healthy Controls	HSP overall	p-value1	SPG4	p-value2
	n = 21	n = 18		n = 12
CMCT–UL (ms) 1	9.76 (0.87)	13.36 (6.42)	0.015	9.14 (3.69)	0.81
CMCT–LL (ms)	18.11 (4.67)	100 (63.51)	<0.001	69.0 (37.21)	<0.001
MEP amplitude-UL (μV)	318.4 (464.71)	86.64 (235.03)	0.008	90.54 (585.95)	0.53
MEP amplitude-LL (mV)	100.6 (86.1)	0 (0)	<0.001	0 (47.92)	<0.001
SEP UL (ms)	18.8 (2.52)	20.17 (7.09)	0.147	19.60 (6.31)	0.59
SEP LL (ms)	45 (4.10)	72.1 (27.2)	<0.001	72.1 (36.0)	<0.001

Data are shown as median and interquartile range except for 1CMCT–UL. which is shown as mean and standard deviation. CMCT: Central Motor Conduction Time; HSP: Hereditary spastic paraplegias; LL: lower limbs; MEP: motor evoked potential; ms: milliseconds; mV: millivolt; SSEP: Somatosensory Evoked Potential; UL: upper limbs; μV: microvolt.

1 comparisons between the overall HSP and the control group

2 comparisons between the SPG4 subgroup and the control group