New genetic causes for complex hereditary spastic paraplegia

Journal of the Neurological Sciences 379 (2017) 283–292

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Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns

New genetic causes for complex hereditary spastic paraplegia Paulo Victor Sgobbi de Souza, Thiago Bortholin, Renan Braido Dias, Marco Antônio Troccoli Chieia, Stênio Burlin, Fernando George Monteiro Naylor, Wladimir Bocca Vieira de Rezende Pinto ⁎, Acary Souza Bulle Oliveira Division of Neuromuscular Diseases, Department of Neurology and Neurosurgery, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 8 February 2017 Received in revised form 1 May 2017 Accepted 13 June 2017 Available online 15 June 2017 Keywords: Hereditary spastic paraplegia Spastic ataxia Neurogenetics Spasticity

a b s t r a c t Introduction: Hereditary Spastic Paraplegia (HSP) represents a complex and heterogeneous group of rare neurodegenerative disorders that share a common clinical feature of weakness and lower limb spasticity that can occur alone or in combination with a constellation of other neurological or systemic signs and symptoms. Although the core clinical feature of weakness and lower limb spasticity is virtually universal, the genetic heterogeneity is almost uncountable with more than 70 genetic forms described so far. We performed review of medical records from twenty-one patients from seventeen Brazilian families with complex phenotype of HSP. All cases have previously negative mutations in SPG11/KIAA1840 and SPG7 gene and were evaluated by whole-exome sequencing. An extensive description of systemic and neurological signs has been described. Results: Whole-exome sequencing was unremarkable in eight patients and established a definite genetic diagnosis in thirteen patients of twelve non-related families. Mutations were found in genes previously implicated in other neurodegenerative disorders such as Amyotrophic Lateral Sclerosis, Hereditary Neuropathy, Spastic Ataxias, Neurodegeneration with Brain Iron Accumulation, Glycogen Metabolism, Congenital Lipodystrophy and aminoacyl-tRNA synthetases disorders. Conclusions: We report thirteen new genetically-proven cases of complex HSP, expanding the clinical spectrum of presentations of HSP, providing new pathophysiological mechanisms and disclosing new potential therapeutic targets. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Hereditary Spastic Paraplegia (HSP) comprises a rare and heterogeneous wide group of inherited neurodegenerative disorders characterized clinically by the presence of lower limb spasticity and weakness resulting from a primary retrograde distal axonopathy of the longest descending motor fibers of the corticospinal tract and posterior columns caused by different molecular mechanisms that dysrupt membrane vesicular trafficking, organelle morphogenesis, axonal transport, lipid metabolism, mitochondrial function and different steps of myelination process [1–4]. The genetic basis of HSP is complex with more than 76 monogenic forms described so far that can be inherited in all patterns of Mendelian inheritance (autosomal dominant, autosomal recessive, X-linked) and non-Mendelian mitochondrial maternal transmission with large clinical and genetic overlapping with other neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS), Charcot-Marie-Tooth Disease (CMT), Spastic Ataxias (SPAX), Spinocerebellar Ataxias (SCA), ⁎ Corresponding author at: Department of Neurology and Neurosurgery, Federal University of São Paulo (UNIFESP), Estado de Israel Street, 899, 04022-002, Vila Clementino, São Paulo, SP, Brazil. E-mail address: [email protected] (W.B.V.R. Pinto).

http://dx.doi.org/10.1016/j.jns.2017.06.019 0022-510X/© 2017 Elsevier B.V. All rights reserved.

Hypomyelinating-Leukodystrophy Disorders (HLD) and Neurodegeneration with Brain Iron Accumulation (NBIA) [1,2]. The diagnostic of HSP is based on the presence of some findings such as: (I) clinical symptoms of bilateral lower limb spasticity and weakness that may be non-progressive or slowly progressive; (II) neurological examination showing involvement of corticospinal tract (spasticity, hyperreflexia, extensor plantar responses); (III) family history with similar neurological complaints with all patterns of Mendelian inheritance or maternal inheritante, even though sporadic cases can also occur frequently and incomplete genetic predominance be common and not the exception; (IV) definite molecular genetic testing showing a previously gene related to condition or a new mutation in genes not related to disease but involved in molecular mechanisms that can lead to corticospinal tract dysfunction [1,2,5,6]. The HSP can be classified according to the clinical phenotype, pattern of inheritance or pathophysiological molecular mechanism [1,2]. According to the clinical phenotype, the HSP are classified in pure or complex (complicated) forms; pure HSP are those that exhibit isolated pyramidal dysfunction (paraparesis or quadraparesis, spasticity, brisk tendon reflexes, and extensor plantar responses) in association with sphincter disturbances and deep sensory loss; and complex HSP are those in which spastic paraplegia phenotype is associated with other neurological signs such as cerebellar dysfunction (ataxia, dysarthria,

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P.V.S. Souza et al. / Journal of the Neurological Sciences 379 (2017) 283–292

nystagmus, tremor), axonal or demyelinating neuropathy (dysautonomia, sensory disturbances), cognitive impairment (dementia, intellectual disability or mental retardation), extrapyramidal features (parkinsonism, dystonia, chorea), psychiatric disturbances, myopathic features, epilepsy or abnormalities on brain and spinal cord MRI (leukodystrophy, hypomyelination, thin corpus callosum, hydrocephalus, brain iron accumulation, spinal cord atrophy); or non-neurological signs such as ophthalmological abnormalities (optic atrophy, retinitis pigmentosa, macular degeneration), dysmorphic features (microcephaly, short stature, facial dysmorphisms), skin changes (ichthyosis, vitiligo) and orthopedic abnormalities (scoliosis, hip dislocation, foot deformities) [1,2,6]. Herein, we describe the clinical and genetic findings of thirteen patients of eleven non-related Brazilian families with complex phenotype of HSP due to novel genetic causes not related to HSP contributing to expand the clinical phenotype spectrum and genetic mechanisms of HSP. 2. Material and methods 2.1. Patients selection and evaluation Twenty-one patients of seventeen non-related Brazilian families with clinical and radiological findings consistent with complex HSP with previously negative mutations in SPG11/KIAA1840 and SPG7 gene were identified and assessed from the Division of Neuromuscular Diseases of Universidade Federal de São Paulo (UNIFESP) in São Paulo, Brazil. Medical records and neuroimaging studies were reviewed, and all available individuals were reexamined. 2.2. Standard protocol approvals, registrations, and patient consents All patients gave written informed consent for genetic studies and for this publication, and study procedures were approved by institutional ethics committees. 2.3. Blood tests All patients were submitted to an investigative work-up of blood tests to exclude acquired or metabolic causes of spastic paraparesis. Analysis of very long chain fatty acids, B12 vitamin and serum copper was performed. Serological tests for Human Immunodeficiency Virus (HIV-I/II), Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), Herpes Simplex Virus (HSV-I/II), Varicella-Zoster Virus (VZV), Human Tlymphotropic Virus (HTLV), Brucella, Treponema pallidum and Borrelia burgdorferi, and rheumatologic tests with anti-nuclear antibodies (ANA), extra-nuclear antibodies (ENA), anti-neutrophil cytoplasmic antibodies (ANCA) and antiphospholipid antibodies were performed. 2.4. Brain magnetic resonance imaging All patients performed brain and cervical spinal cord magnetic resonance imaging (MRI) at 1.5 T or 3 T using age-specific protocols. 2.5. Neurophysiological studies All patients were submitted to nerve conduction studies (NCS) and electromyography (EMG) to evaluate the presence of sensory-motor neuropathy, myopathic findings or motor neuron features. 2.6. Clinical scales All patients were submitted to Spastic Paraplegia Rating Scale (SPRS) and Scale of the Assessment and Rating of Ataxia (SARA) evaluation.

2.7. Muscle histochemistry Muscle biopsy was performed in some selected patients. Muscle biopsies were performed using an open technique in the deltoid muscle. Muscle samples stored at a temperature of −80 °C were cut into fragments with a depth of 6-8 μm and mounted on slides, which were subsequently stained with hematoxylin and eosin, modified Gömöri trichrome, periodic acid-Schiff, Oil-red O, and submitted to reactions with the enzymes reduced nicotinamide adenine tetrazolium reductase, cytochrome C oxidase (COX), succinate dehydrogenase, and ATPase in two different pH values: 9.4 and 4.6.

2.8. Whole-exome sequencing Whole-exome sequencing (WES) was performed in all patients with the complex phenotype of HSP with Illumina's Nextera Rapid Capture according to the manufacturer's protocol and subsequent sequencing on the Illumina HiSeq platform where sequence alignment and variant call coverage of target bases with more than 10 reads was 96.8% and the pathogenicity of the mutations was assessed in pathogenicity prediction programs such as SIFT, PolyPhen-2 and Mutation Taster as well as in studies reported in the medical literature.

3. Results 3.1. Genetic findings A definitive pathogenic genetic mutation was identified in 13/21 (62%) of complex HSP patients with previously negative tests for mutations in the SPG11/KIAA1840 and SPG7 genes. We identified mutations in 12 genes not related to the phenotype of HSP but related to other neurodegenerative conditions with corticospinal tract dysfunction such as ALS (CHCHD10, FIG4 and VCP), Spastic Ataxias (VAMP1 and MTPAP), NBIA (PLA2G6), Congenital Lipodystrophy (CAV1), Hereditary Neuropathy (DNMT1 and GAN1), Glycogen Metabolism (GBE1) and mitochondrial aminoacyl-tRNA synthetase dysfunction (NARS2 and SARS2). Most variants had been previously reported as pathogenic with new mutations in the SARS2 and VCP genes predicted as pathogenic by in silico analysis. Variants of unknown significance were not reported. The thirteen patients belong to twelve non-related Brazilian families, with positive family history in 11/13 (84%) cases and 7/13 (53%) families with an autosomal dominant pattern of inheritance. The pathogenic mutations identified are summarized in Table 1.

3.2. Clinical findings In this Brazilian cohort of thirteen patients with complex HSP the main neurological complaint that characterized the complex phenotype was neuropathy (9/13) followed by cognitive impairment (8/13), cerebellar ataxia (7/13) and extrapyramidal features (6/13). The most common non-neurological signs were orthopedic abnormalities (7/13), endocrinopathies (7/13) and sleep disorders (7/13). The mean age at onset was 20 years, ranging from age 3 to 45 years. Neurological and systemic features are summarized in Tables 2 and 3.

3.3. Neuroimaging findings In this cohort of patients with complex hereditary spastic paraplegia the main neuroimaging abnormalities (Fig. 1) were cerebellar atrophy (9/13), thin corpus callosum (5/13), cervical spinal cord atrophy (3/ 13) and white matter abnormalities (3/13). The radiological, neurophysiological and muscle biopsy findings are summarized in Table 4.


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Table 1 Summary of neurogenetic variants identified in all HSP patients. Patient Family history

Consanguinity Gene involved

Prior association with HSP

Variant type

Allelic variantsa

Seggregation within family

1 2

Y Y

N Y

CHCHD10 PLA2G6

N N

He CH

Y Parents He

3

Y

Y

PLA2G6

N

CH

4 5 6 7 8 9 10

N Y Y Y Y Y N

Y Y N N Y N N

NARS2 SARS2 VAMP1 MTPAP GAN VCP GBE1

N Y Y N N Y N

Ho Ho He He Ho He CH

11 12 13

Y Y Y

N N N

DNMT1 CAV1 FIG4

Y N N

He He He

c.176CNT (p.Ser59Leu) c.1634A NG (p.Lys545Arg); c.2370TNG (p.Tyr790a) c.1634A NG (p.Lys545Arg); c.2370TNG (p.Tyr790a) c.822CNG (p.Gln274His) c.1343A NT (p.His448Leu) c.342T NG (p.Ser114Arg) c.1432A NG (p.Asn478Asp) c.1429C NT (p.Arg477Thr) c.625T NG (p.Cys209Gly) c.986ªNC (p.Tyr329Cys) c.1544ªNG (pArg515His) c.2053GNA (p.Ala685Tre) 1 bp del-A (p.Ile134fsThr137) c.1207C N T (p.Gln403a)

Parents He Parents He Parents He Y Parents He Parents He Y N/A Y Y Y

Italics represented in table are representing each of the involved genes which were diagnosed for each of the referred patients. CH: compound heterozygous; He: Heterozygous; Ho: Homozygous; N; No; N/A: non-available data; Y: Yes. a No pathogenic variants have been priorly linked to HSP.

(ALS-FTD). Neurological examination revealed spastic paraparesis with brisk tendon reflexes, dysmetry in the upper limbs with action tremor and global ophthalmoparesis with bilateral ptosis. NCS was normal and EMG did not show signs of motor neuron involvement or myopathic features. Muscle biopsy exhibited ragged-red fibers with subsarcolemmal mitochondrial proliferation and COX-deficient fibers. Genetic testing identified a heterozygous missense mutation in CHCHD10 gene (c.176CNT; p.Ser59Leu).

3.4. Case reports 3.4.1. Family 1 The proband patient (Patient 1) from Family 1 was a 28-year-old man who presented with a 3-year history of gait impairment, slurred speech and diplopia. Family history (Fig. 2) showed two brothers, the mother and the maternal grandfather with similar symptoms and two uncles with Amyotrophic Lateral Sclerosis-Frontotemporal Dementia

Table 2 Summary of neurological clinical features present in all described patients. Patients

1

2

3

4

5

6

7

8

9

10

11

12

13

Age (y) Onset (y) Gender Spasticity Weakness Amyotrophy Brisk TR Babinski sign Deep sensory loss Pes cavus Dysarthria Dysphagia Nystagmus Ocular movements Eyelid Ptosis Visual loss Constipation Neurogenic bladder Dysautonomia Ataxia Tremor Dystonia Chorea Parkinsonism Facial weakness Epilepsy Headache Vertigo Hearing loss Cognitive impairment Psychiatric disturbance SARA SPRS

28 25 M 2+

23 5 M 3+ P X X X X X X X X OA

25 3 F 2+ Q X X X X X

29 21 M 1+ Q X X X X

19 12 M 2+ P X X X

48 40 M 2+ P

26 20 M 2+ P X X X

13 7 F 3+ P X X X X X X X X

48 38 F 2+ P X X X

55 45 F 2+ Q

21 11 F 1+ P X X X

16 12 M 1+ P

50 32 M 3+ P X X X X X X X

X

X X X

X X X X X

OP X

X X

X X X

X X X X

12 23

X X OA

X X X 39 48

X X X X

X X X

X X

X X 22 31

X X

X X

X X X X X 6 13

X X X X

X

X SVGP X X X X X X

X SVGP

X X

X X

X

X X

X

18 24

X X

X

5 16

X X

X

X

X OD

X

X

X X X

X X X

X X

X X X

X X

X X

X

X X

X 16 20

X

X

X X

X

X X X X

7 35

X 8 32

X X 8 37

X X X 12 30

X X X 20 18

X X 27 44

F: female; ICARS: International Cooperative Ataxia Rating Scale score; M: male; OA: Oculomotor apraxia; OD: ocular dysmetria; OP: Ophthalmoparesis; P: paraparesis; Q: quadriparesis; SARA: Scale for the Assessment and Rating of Ataxia score; SVGP: Supranuclear Vertical Gaze Palsy; TR: tendon reflexes; X: present; y: years; 1+: mild; 2+: moderate; 3+: severe.

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Table 3 Summary of systemic (non-neurological) features present in all HSP patients. Patients Funduscopic Orthopedic Endocrine Cardiopathy Pneumopathy Nephropathy Hematological Immunological Genitourinary Dermatological Sleep disturbances

1 S H MVP

2

3

4

AO S, PCV

OA S, PCV

AO S H

5

6

DM

FSG

NP

7

8

9

10

OA S, TE H, DM

12

AO

PR

H, DM

H, DM

13

S H

As T Ig

11

PH NP T

T Ig

CVID

Ig

VP Kinky hair OSAS

RSBD

OSAS

RSBD

RSBD

RSBD

Scleroderma Narcolepsy

Acanthosis nigricans

As: Asthma; CVID: Common Variable Immunodeficiency; DM: diabetes mellitus; FSG: focal segmental glomerulosclerosis; ICARS: International Cooperative Ataxia Rating Scale score; H: hypothyroidism; Ig: IgA deficiency; MVP: mitral valve prolapse; NP: Nephrolithiasis; OA: Optic atrophy; OSAS: Obstructive Sleep Apena Syndrome; PCV: pes calcaneovarus; PH: pulmonary hypertension; PR: pigmentary retinopathy; RSBD: REM Sleep Behavior Disorder; S: scoliosis; SARA: Scale for the Assessment and Rating of Ataxia score; T: thrombocytopenia; TE: talipes equinovarus; VP: vaginal prolapse; X: present.

3.4.2. Family 2 The proband patient (Patient 2) from Family 2 was a 23-year-old man who presented with a 18-year-history of gait instability and progressive visual loss. Past medical history revealed a normal pregnancy and neonatal period with normal developmental milestones, with

cognitive impairment since 7-years-old. Family history (Fig. 2) demonstrated consanguineous parents and a sister with similar neurological complaints. Neurological examination showed spastic paraplegia with global brisk tendon reflexes, bilateral Babinski sign, cervical and right upper limb dystonia, global ophthalmoparesis and optic atrophy. The

Fig. 1. Neuroimaging findings in HSP patients. (A) Sagittal T1-weighted brain MR image disclosing thin corpus callosum, mild cortical atrophy and mild cerebellar atrophy (Patient 1, Family 1). (B) Sagittal, (D) axial T1-weighted images and (C) axial FLAIR brain MR images disclosing thin corpus callosum, moderate olivopontocerebellar atrophy, cortical atrophy and mild periventricular and profound white matter changes (Patient 2, Family 2). (E) Sagittal T1-weighted, (F,G) axial T2-weighted brain MR images disclosing thin corpus callosum, diffuse leukoencephalopathy involving periventricular, profound and subcortical areas with relative sparing of the basal ganglia (Patient 7, Family 6). (H) Sagittal T1-weighted and (I,J) axial T2-weighted brain MR images disclosing marked cerebellar atrophy and thin splenium of the corpus callosum (Patient 8, Family 7). (K) Sagittal T1-weighted, (M,N) axial FLAIR sequence and (L) coronal T2-weighted brain MR images disclosing marked diffuse thin corpus callosum, cerebellar atrophy, frontotemporal cortical atrophy and compensatory enlargement of the cerebral lateral ventricles (Patient 9, Family 8). (O) Coronal brain MR images disclosing marked bilateral hyperintensity in corticospinal tract pathway in T2weighted images (Patient 13, Family 12).


287

Table 4 Neurorradiological, neurophysiological and muscle biopsy findings of all patients. Patients

1

2

TCC White matter abnormality CA CoA Abnormal brain iron accumulation Hydrocephalus CSCA NCS EMG Muscle biopsy

X

X

X

X X

3

4

5

6

7

8

X X

X X

X

X

9 X X X X

10

11

X

RRF

ASN CD RRF

ASN CD RRF

X

X

X X

X ASN CD NA

13

X

X

ASN CD NA

12 X

ASN CD NA

X X ASN CD NA

X ASN CD NA

SN

SN

RRF

NA

ASN: axonal sensorimotor neuropathy; CA: cerebellar atrophy; CoA: cortical atrophy; CSCA: cervical spinal cord atrophy; CD: chronic denervation; EMG: electromyography; NA: neurogenic amyotrophy; NCS: nerve conduction studies; RRF: ragged-red fibers; SN: sensory neuropathy; TCC: thin corpus callosum; X: present.

sister of proband patient (Patient 3) was a 25-year-old woman with 22year-old history with gait abnormalities and urinary incontinence. Past medical history revealed a normal pregnancy and neonatal period, with delayed motor and cognitive milestones and bilateral visual loss. Neurological examination showed spastic quadraplegia with global brisk tendon reflexes, cervical dystonia, optic atrophy, global ophtalmoparesis, bilateral pes cavus, peroneal muscular atrophy and scoliosis. Genetic analysis revealed compound heterozygous missense mutations in the PLA2G6 gene (c.1634A NG [p.Lys545Arg]; c.2370T N G [p.Tyr790*]) in the two patients. 3.4.3. Family 03 The proband patient (Patient 4) was a 29-year-old man that presented with 21-year history of frequent falls and urinary incontinence. Past medical history (Fig. 2) disclosed chronic kidney disease, cognitive impairment and epilepsy since adolescence with unknown etiology. Neurological examination showed spastic quadriplegia with brisk tendon reflexes and extensor plantar responses, scoliosis and severe hypoesthesia in the lower limbs. Genetic studies exhibited homozygous missense mutations in the NARS2 gene (c.822GNC [p.Gln274His]). 3.4.4. Family 04 The proband patient (Patient 5) was a 19-year-old man with a 7year history of weakness and rigidity in the lower limbs. Family history

(Fig. 2) disclosed an old sister with similar symptoms that died of respiratory impairment in the first decade of life. Neurological examination showed spastic paraplegia, bilateral Babinski sign, dysmetria in the upper limbs, pes cavus and hammertoes. Genetic analysis showed a new homozygous missense mutation in the SARS2 gene (c.1343A N T [p.His448Leu]). 3.4.5. Family 05 A 48-year-old man (Patient 6) with a 8-year-history of gait imbalance and slurred speech. Past medical history revealed urinary incontinence, constipation and erectile dysfunction. Familial history (Fig. 2) showed two sisters, the father, two aunts, two uncles and paternal grandmother with similar symptoms. Neurological examination showed mild spastic paraparesis, global brisk tendon reflexes, dysmetria in the upper limbs, cervical dystonia, slow saccades and supranuclear gaze palsy. Genetic studies revealed heterozygous missense mutation in the VAMP1 gene (c.342TN G; p.Ser114Arg). 3.4.6. Family 06 The proband patient (Patient 7) was a 26-year-old man who presented with 20-year-history of gait impairment and tremor in the upper limbs. Past medical history revealed delayed neuropsicomotor millestones. Family history (Fig. 2) showed consanguineous parents and a cousin with similar symptoms. Neurological examination showed

Fig. 2. Pedigree charts from Patients 1 (A), 2 (B), 3 (C), 4 (D), 5 (E) and 6 (F).

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spastic paraparesis, global brisk tendon reflexes, bilateral Babinski sign, oromandibular and cervical dystonia, optic atrophy, supranuclear vertical gaze palsy. Genetic investigation revealed homozygous missense mutation in the MTPAP gene (c.1432ANG; p.Asn478Asp). 3.4.7. Family 07 The proband patient (Patient 8) was a 13-year-old woman with a 6year-history of gait imbalance with urinary incontinence. Past medical history revealed delayed neuropsychomotor development and cognitive impairment. Familial history (Fig. 3) showed consanguineous parents and two cousins with similar neurological pictures. Neurological examination showed spastic paraparesis, hyperreflexia, ankle clonus, scoliosis and pes cavus. Genetic tests exhibited a homozygous missense mutation in the GAN gene (c.1429CNT; p.Arg477Thr). 3.4.8. Family 08 A 48-year-old woman (Patient 9) presented with a 10-year-history of gait imbalance and rigidity of the lower limbs. Past medical disclosed progressive dysphagia, facial weakness and psychiatric abnormalities with persecutory delusions. Family history (Fig. 3) disclosed two sisters with Paget disease of bone, two brother with similar symptoms and the father with ALS-FTD. Neurological examination showed moderate spastic paraparesis, brisk tendon reflexes with extensor plantar responses, facial weakness, cervical dystonia, pes cavus and distal limb muscle atrophy of the lower limbs. Genetic studies revealed heterozygous missense mutation in the VCP gene (c.625T NG; p.Cys209Gly) in the patient and her two brothers with similar neurological picture. 3.4.9. Family 09 The proband patient (Patient 10) was a 55-year-old woman with a 10-year-history of weakness in the lower limbs and urinary incontinence. Past medical history showed psychiatric abnormalities with visual and auditory hallucinations, persecutory delusions and two suicide attempts. Family history (Fig. 3) was unremarkable. Neurologic examination showed moderate spastic quadraparesis, brisk tendon reflexes, bilateral Babinski signs, pes cavus and orthosthatic hypotension.

Genetic analysis revealed heterozygous missense mutation in the GBE1 gene (c.986AN C [p.Tyr329Ser]; c.1544ANG [p.Arg515His]). 3.4.10. Family 10 A 21-year-old woman (Patient 11) with 10-year-history of gait impairment and muscle cramps. Past medical history disclosed delayed neurodevelopmental millestones with cognitive impairment, common variable immunodeficiency and deafness. Family history (Fig. 3) showed four cousins with hereditary neuropathy with unknown genetic etiology and an uncle with similar symptoms. Neurological examination showed spastic paraparesis, trunk ataxia, pes cavus and distal muscular atrophy in the lower limbs. Genetic tests showed heterozygous missense mutation in the DNMT1 gene (c.2053GNA; p.Ala685Tre). 3.4.11. Family 11 The proband patient (Patient 12) was a 16-year-old boy with a 4year-history of gait impairment and slurred speech. Past medical history disclosed congenital cataracts and pigmentary retinopathy. Family history (Fig. 3) showed a sister with similar neurological picture, two aunts with partial lipodystrophy with multiple endocrinopathies. Neurological examination showed mild spastic paraparesis, spastic-ataxic gait, hyperreflexia with extensor plantar responses and aquileu clonus. Genetic tests confirmed heterozygous mutation in the CAV1 gene (1 bp del-A [p.Ile134fsThr137]) in the patient and his sister. 3.4.12. Family 12 The proband patient (Patient 13) was a 50-year-old man with a 18year-history of gait imbalance with pain and stiffness in the lower limbs. Past medical history recent behavioral changes with persecutory delusions. Family history (Fig. 3) showed a father and a paternal uncle with similar neurological pictures. Neurological examination showed moderate spastic paraparesis, dysphonia, mild bilateral ptosis, brisk tendon reflexes with bilateral extensor responses, pseudobulbar affect and cognitive impairment. Genetic studies disclosed a heterozygous missense mutation in the FIG4 gene (c.1207C N T [p.Gln403*]) in the proband patient and in his father.

Fig. 3. Pedigree charts from Patients 7 (A), 8 (B), 9 (C), 10 (D), 11 (E) and 12 (F).


4. Discussion Hereditary Spastic Paraplegia is a wide heterogeneous rare group of neurodegenerative disorders that was first described by Strümpell [7] and Lorrain [8] in the end of the nineteenth century, although the first classification was proposed in 1983 by Harding [9] and since then the number of phenotypes and genetic causes of HSP exponentially increased with some pitfalls and difficulties for the diagnosis and classification of this rare group of disease [10]. Currently, HSP is a syndromic designation for a clinically and genetically heterogeneous group of inherited neurodegenerative or neurodevelopmental disorders in which the main neurological symptoms and signs are weakness and spasticity in the lower limbs due to primary retrograde dysfunction of the long descending fibers of the corticospinal tract [1]. The pathophysiological mechanisms that lead to corticospinal tract dysfunction are multiple and heterogeneous with the most common molecular mechanisms described are membrane trafficking and organelle shaping, axonal transport, mitochondrial dysfunction, lipid metabolism disruption, ion channel dysfunction and myelination abnormalities with great overlapping with other neurodegenerative disorders such as ALS, SCA, CMT and NBIA [1,2,3,11]. The diagnosis of HSP is very extensive and in some contexts may represent a true odyssey with high financial costs and many years for the definitive genetic diagnosis. Generally, the diagnosis is based on the presence of a clinical picture of progressive spastic paraplegia in a pure or complex presentation, evidence of a genetic mutation in a locus related to a phenotype of HSP previously described in the literature, and exclusion of structural disorders or other genetic or acquired conditions that explain the clinical complaints [1]. Complex forms of HSP may be present with wide heterogeneous clinical and neuroimaging features with a growing number of mimicking differential diagnosis that is also impossible a definitive diagnosis without genetic tests [1,2]. SPG11 and SPG7 genes mutations are the most common complex forms of HSP worldwide and other forms can be extremely rare with no more than one family or few patients described. Thus, in a case of complex HSP with negative testing for SPG7 and SPG11 or with atypical phenotype for this conditions, new genetic techniques such as next-generation sequencing (mainly represented by whole-exome sequencing) are a notable tool for the definitive genetic diagnosis [1,2,12,13]. CHCHD10-related disorders is a broad, heterogeneous and complex spectrum of different neurological phenotypes such as mitochondrial myopathy, FTD, ALS, late-onset spinal motor neuropathy (SMAJ), cerebellar ataxia and axonal CMT that can occur alone or in any combination inherited in autosomal dominant trait or as sporadic condition [14–17]. Neuropathological studies with brain pathology have never been performed in patients with CHCHD10-related disorders and muscle pathology frequently exhibits typical features of mitochondrial myopathy including intracellular lipid accumulation with COX-negative and ragged-red fibers with or without multiple mitochondrial DNA deletions. Electron microscopy showed fragmentation of the mitochondrial network and a defect in cristae maintenance with paracristalline inclusions and energy production dysfunction that can affect many molecular mechanisms such as membrane vesicular trafficking, organelle morphogenesis and survival of motor neuron and other neuronal cells with corticospinal dysfunction [14,15]. Here, we reported a previously described heterogeneous missense mutation (p.Ser59Leu) in CHCHD10 gene in a Brazilian family with a novel and single neurological phenotype of complex HSP with deafness, polyneuropathy and chronic progressive external ophthalmoplegia. PLA2G6-associated neurodegeneration comprises a continuum of three major neurological phenotypes with large overlapping clinical and radiologic features that include: classic infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy and PLA2G6-related dystonia parkinsonism in which psychomotor regression, cerebellar ataxia,

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spastic tetraparesis, bulbar symptoms, seizures, hypotonia, neuropathy, parkinsonism, chorea, dystonia, nystagmus and optic atrophy with hypointense signal on T2-weighted brain MRI in the globus pallidus suggesting abnormal brain iron accumulation [18]. Cerebellar atrophy, white matter abnormalities and thin vertically oriented corpus callosum may occur in different combination with very early-onset (before age three years) as in classical form of infantile neuroaxonal dystrophy or in late childhood or adulthood as in PLA2G6-related dystonia-parkinsonism [18]. PLA2G6 gene encodes for a phospholipase A2 group IV that catalyze the release of free fatty acids from phospholipids with impaired activity of this enzyme leading to a phospholipid dyshomeostasis may alter the lipid composition of the plasma membrane, vesicles or endosomes with dysfunction of neuronal cells such as motor neurons in the corticospinal tracts and in glial cells [19,20]. Here, we present a case of two patients with early-onset spastic paraparesis, optic atrophy and cognitive impairment highly compatible with a complex HSP presentation, especially SPG7, SPG11 and SPG15 that are common causes of complex HSP with optic atrophy [1]. However, genetic testing showed mutations in PLA2G6 gene reinforcing the idea that this condition should always be remembered as a differential diagnosis for complex HSP with optic atrophy. Mutations in NARS2 gene that encodes the mitochondrial asparaginyl-tRNA synthetase cause Combined oxidative phosphorylation deficiency type 24 (OMIM #616239), an autosomal recessive mitochondrial disorder characterized by myopathy, epilepsy, intellectual disability, hypomyelination associated with systemic features such as hypochloremic metabolic alkalosis, kidney disease (tubulopathy and focal segmental glomerulosclerosis) and hepatopathy, resembling Alpers disease with just three patients reported in the literature [21, 22]. Here, we report a new patient with a complex phenotype of HSP with epilepsy, chronic kidney disease and intellectual disability with muscle biopsy showing ragged-red fibers with subsarcolemmal mitochondrial proliferation and type grouping formation in the absence of myopathic findings. SARS2 gene encodes the mitochondrial seryl-tRNA synthetase which provides serine aminoacylation to 2 mitochondrial tRNAs: tRNA-Ser (AGY) and tRNA-Ser (UCN). Mutations in this gene cause HUPRA syndrome, an acronym for Hyperuricemia, Pulmonary Hypertension, Renal Failure and Alkalosis Syndrome (OMIM #613845), an autosomal recessive multisystemic disorder described in four Pakistani patients characterized by progressive renal failure, metabolic alkalosis, pulmonary hypertension, global developmental delay, hematological disturbances (anemia, leukopenia, thrombocytopenia), diabetes mellitus with onset in early infancy and early death before the age of 2 years [23]. It was recently associated with a complex phenotype of HSP with intellectual disability [24]. Here, we report a new case of SARS2 mutations in a patient with spastic paraparesis with intellectual disability as already reported, but with some particular features such as diabetes mellitus and neuropathic features that have never been described. In clinical practice it may be difficult to decide whether the patient has an hereditary ataxia with spasticity (spastic-ataxia) or complex HSP with cerebellar features and additional clinical features, neuroimaging signs and molecular mechanisms helping in the diagnostic workup [25]. If the patient presents with more proeminent or isolated pyramidal signs (clinically or determined by appropriated scales) with a genetic evaluation revealing a cause of hereditary ataxia or HSP, the phenotype may be better classified as a complex form of HSP for workup and management [1,2,25]. VAMP1 gene codes for a vesicle-associated membrane protein-1, also recognized as synaptobrevin-1, which is an integral membrane protein involved in the synaptic vesicle cycle (particularly exocytosis) at the presynaptic nerve terminal. Mutations in VAMP1 are involved with Autosomal Dominant Spastic Ataxia 1 SPAX1 (OMIM #108600) which presents with a picture of progressive spastic-ataxia before age 20 years [25–27]. MTPAP gene encodes for nuclear-encoded mitochondrial poly(A) RNA polymerase responsible for polyadenylation of mRNA in human mitochondria which is essential

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for the maintenance of mitochondrial gene expression and its mutations cause Autosomal Recessive Spastic Ataxia 4 – SPAX4 (OMIM #613672) [28]. Here, we presented two families with a complex HSP with minor or absent cerebellar dysfunction features and prominent extrapyramidal manifestations due to VAMP1 and MTPAP mutations, reinforcing the idea that genes involved with hereditary spastic-ataxia should always be remembered as a differential diagnosis for complex HSP with or without cerebellar features, such as KIF1C mutations that cause Autosomal Recessive Spastic Ataxia 2 – SPAX2 (OMIM #611302) [29] or SPG58 [5]. Giant Axonal Neuropathy type 1 (OMIM #256850) caused by homozygous or compound heterozygous mutations in the GAN gene is an infancy-onset neurodegenerative disorder characterized by peripheral (sensory-motor axonal neuropathy) and central nervous system (cerebellar features and pyramidal signs) involvement and abnormalities of the hair (curly and kinky hair). The presence of giant axonal swellings filled with neurofilaments on nerve biopsy with axonal loss is the main pathological hallmark of the disease [30]. Although, the phenotype of spastic paraplegia has also been described due to GAN mutations [31], this condition is rarely thought as a differential diagnosis for complex HSP with prominent neuropathic findings and here we described a case that disclosed this situation and proved the idea that genes responsible for inherited neuropathies with central nervous system involvement may also be a cause of HSP. VCP gene codes the valosin-containing protein which is a member of the AAA-ATPase superfamily involved with multiple cellular functions such as substrate extraction of ubiquitin-proteosome systems, Golgi complex biogenesis, in chlatrin-mediated membrane trafficking, protein degradation at the outer mitochondrial membrane, peroxisomal assembly, autophagosome maturation, regulation of cell cycle and also plays a role in motor neuron function and survival [32]. VCP mutations can cause a wide spectrum of neurological and systemic phenotypes that include [33]: hereditary Inclusion Body Myopathy, familial or sporadic ALS, FTD, ALS-FTD, CMT type 2Y, Inclusion Body Myopathy with early-onset Paget disease and Frontotemporal Dementia type 1, lateonset Alzheimer's Disease, Paget Disease of the Bone, Parkinson's Disease, Limb-Girdle Muscular Dystrophy, Distal Myopathy and HSP [32, 33]. Here, we describe a new case of complex HSP due to a novel mutation in VCP gene, establishing in association with two previous reports [34,35] that mutations in the VCP are a causative gene for HSP. Polyglucosan is an amylopectin-like polysaccharide associated with defective glycogen metabolism present in some glycogen storage disorders (GSD), such as GSD type 0B (muscle GSD type 0), GSD type IV (Andersen disease), GSD type VII (Tarui disease) and GSD type XV (glycogenin deficiency) due to mutations, respectively, in GYS1, GBE1, PFKM and GYG1 genes or in other diseases such as Polyglucosan body myopathy 1 with or without immunodeficiency, Progressive Myoclonic Epilepsy (Lafora' disease) type 2A or 2B, Progressive Myoclonic Epilepsy type 10 and PRKAG2-related disorders (gamma-2 subunit of AMP-activated protein kinase), respectively, due to mutations in RBCK1, EPM2A, NHLRC1, PRDM8 and PRKAG2 genes [36,37]. Adult Polyglucosan Body Neuropathy (APBN) is an autosomal recesssive disorder caused by mutations in GBE1 gene characterized by a slowly progressive disease with peripheral and central nervous system involvement usually starting after age 40 years with neurogenic bladder, spastic paraplegia with vibration loss, axonal neuropathy and cognitive decline with white matter abnormalities on brain MRI [38]. Although spastic paraplegia represents a major hallmark of APBN, this disorder is rarely highlighted as a differential diagnosis of HSP and here we provide a case description that proves that APBN can present as complex HSP phenotype and that new inherited metabolic dysfunctions such as glycogen metabolism defects can also be involved in the pathophysiology of HSP. DNMT1 gene encodes for a DNA (cytosine-5) methyltransferase responsible for the methylation of cytosine residues in human genome and this pattern of methylation is responsible for the repression of parasitic sequence elements in the genome and expression status of genes

subject to genomic imprinting and X inactivation [39]. Mutations in the DNMT1 gene in an autosomal dominant pattern are linked to two different conditions: Autosomal Dominant Cerebellar Ataxia, Deafness and Narcolepsy (or ADCADN; OMIM #604121) [40] and Hereditary Sensory Neuropathy type IE (OMIM #614116) [41]. Although spasticity represents a neurological feature described in patients with DNMT1 mutations, a phenotype of complex HSP was described only in one patient in a recent study of a large cohort with 97 patients with complex HSP [13] and here we described a new case with the same genetic etiology corroborating the idea that DNMT1 mutations can present phenotypically as HSP. CAV1 gene encodes for the caveolin-1, an integral membrane protein in whom mutations can cause three different disorders: Congenital Generalized Lipodystrophy type 3 (OMIM #601047) [42]; Partial lipodystrophy, congenital cataracts and neurodegeneration syndrome (OMIM #606721) [43], and Primary Pulmonary Hypertension type 3 (OMIM #615343) [44]. Only one family has been reported with Partial lipodystrophy, congenital cataracts and neurodegeneration presenting with adolescence-onset spasticity and ataxia [43]. Herein, we described a family with two brothers with a picture of HSP complicated by multiple endocrinopathies and ophthalmological findings, suggesting that CAV1 mutations may be a rare cause of HSP. FIG4 gene codes for Fig4 protein, a Sac domain-containing inositol phosphatase 3, also called phosphatidylinositol 3,5 bisphosphate 5phosphatase which is a lipid phosphatase located in the cytoplasmic membrane of vacuoles and endosomal membranes involved with the cycling process of intracellular organelles, vesicular trafficking and the dynamic changes related to fission and fusion of intracellular transport vesicles that plays a crucial role in the pathophysiology of HSP [1,45]. The clinical spectrum of FIG4-related disorders comprise neurological and systemic features that can present with four different phenotypes: CMT type 4 J (OMIM #611228), familial ALS type 11 (OMIM #612577), Yunis-Varon syndrome (OMIM #216340) and Bilateral Temporooccipital Polymicrogyria (OMIM #612691) [45]. In the first description of FIG4 mutations associated with ALS, some patients do not present with definite ALS according to El Escorial World Federation of Neurology criteria but with the phenotype of Primary Lateral Sclerosis (PLS) [46] which is a major differential diagnosis for HSP and sometimes almost impossible to differentiate both conditions. In our family description the presence of symmetrical progressive spastic paraparesis of the lower limbs, absence of muscle weakness, slow progression of the disease, and the presence of peripheral neuropathy and cognitive impairment, which are inconsistent findings in patients with PLS, supports the idea that this family has a complex phenotype of HSP. 5. Conclusions Our manuscript evinces that new genetic techniques such as wholeexome sequencing are revolutionary and useful tools for the accurate diagnosis of heterogeneous and complex inherited neurological disorders such as HSP and provide new genetic association and molecular mechanisms for motor neuron degeneration in the endless universe of HSP. New genetically-proven mechanisms linked to HSP have been provided and thus expanded new ideas regarding pathophysiological pathways linked to previously unidentified causes of HSP. Authors' contributions PVSS participated in conception and design of study, acquisition and analysis of data, and participated in the writing of the first draft. TB participated in conception and design of study and acquisition of data. RBD participated in conception of study and acquisition and analysis of data. MAT participated in organization of study and acquisition and analysis of data. SB participated in execution of study and acquisition and analysis of data. FGMN participated in conception and organization of study and review and critique of the manuscript. WBVRP participated in


conception and design of study, acquisition and analysis of data, and writing of the first draft. ASBO participated in review and critique of the manuscript. 1- Conception and design of study: A. Conception, B. Organization, C. Execution; 2- Acquisition and analysis of data: A. Acquisition; B. Analysis of data. 3- Manuscript: A. Writing of the first draft; B. Review and Critique. Souza PVS: 1A, 1B, 1C, 2A, 2B, 3A (Nothing to disclose). Bortholin T: 1A, 1B, 1C, 2A (Nothing to disclose). Dias RB: 1A, 2A, 2B (Nothing to disclose). Chieia MAT: 1B, 2A, 2B (Nothing to disclose). Burlin S: 1C, 2A, 2B (Nothing to disclose). Naylor FGM: 1A, 1B, 3B (Nothing to disclose). Pinto WBVR: 1A, 1B, 2A, 2B, 3A (Nothing to disclose). Oliveira ASB: 3B (Nothing to disclose). Disclosures Dr. Pinto ([email protected]) reports no disclosures. Dr. Souza ([email protected]) reports no disclosures. Dr. Oliveira (acary. [email protected]) reports no disclosures. Dr. Bortholin (thiago.bort@ yahoo.com.br) reports no disclosures. Dr. Chieia ([email protected]) reports no disclosures. Dr. Dias ([email protected]) reports no disclosures. Dr. Burlin ([email protected]) reports no disclosures. Conflict of interests The authors declare that they have no conflict of interest. Financial disclosure and funding support The authors have nothing to disclose regarding supports for the work related to drugs, grants or equipments. Ethical statement Full consent was obtained from the patients for the case report. This study was approved by our Ethics Institution. Acknowledgements The authors have no funding sources or other co-authors to describe. References [1] P.V.S. Souza, W.B.V.R. Pinto, G.N. de Rezende Batistella, T. Bortholin, A.S. Oliveira, Hereditary spastic paraplegia: clinical and genetic hallmarks, Cerebellum (2016) http://dx.doi.org/10.1007/s12311-016-0803-z. [2] S. Klebe, G. Stevanin, C. Depienne, Clinical and genetic heterogeneity in hereditary spastic paraplegias: from SPG1 to SPG72 and still counting, Rev. Neurol. (Paris) 171 (2015) 505–530. [3] C. Blackstone, C.J. O'Kane, E. Reid, Hereditary spastic paraplegias: membrane traffic and the motor pathway, Nat. Rev. Neurosci. 12 (2010) 31–42. [4] T. Lo Giudice, F. Lombardi, F.M. Santorelli, T. Kawarai, A. Orlacchio, Hereditary spastic paraplegia: clinical-genetic characteristics and evolving molecular mechanisms, Exp. Neurol. 261 (2014) 518–539. [5] G. Novarino, A.G. Fenstermaker, M.S. Zaki, et al., Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders, Science 343 (2014) 506–511. [6] J. Finsterer, W. Löscher, S. Quasthoff, J. Wanschitz, M. Auer-Grumbach, G. Stevanin, Hereditary spastic paraplegias with autosomal dominat, recessive, X-linked, or maternal trait of inheritance, J. Neurol. Sci. 318 (2012) 1–18. [7] A. Strümpell, Beiträge zur pathologie des rückenmarks I spastische spinalparalysen, Arch. Psychiatr. Nervenkr. 10 (1880) 676–717. [8] M. Lorrain, Contribution à l'étude de la paraplégie spasmodique familiale: travail de la clinique des maladies du système nerveux à la Salpêtrière, G. Steinheil, Paris, 1898. [9] A.E. Harding, Classification of the hereditary ataxias and paraplegias, Lancet 1 (1983) 1151–1155.

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