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Received date: 16/06/2024 Published date: 05/07/2024 Case Report

A novel mutation of CAPN1 in patients with complicated hereditary spastic paraplegia

Lilu Kuang^1,†, Liang Deng^1,†, Jianhuang Chen^1,†, Runcheng He²*

¹Department of Neurology, Jili Hospital, Changsha, Hunan 410399, China.
²Department of Neurology, the Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China.
†These authors have contributed equally to this work and are co-first authors.

*Corresponding author

*Runcheng He. Department of Neurology, the Second Xiangya Hospital, Central South University, Changsha, Hunan, P.R. China, 410011. E-mail: heruncheng_xy@163.com

DOI: 10.55920/JCRMHS.2024.07.001305

Abstract

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous disease. Based on the phenotype, HSP is distinguished as pure or complicated forms. To date, more than 80 loci or genes have been reported to cause HSP. CAPN1variants are responsible for a complex inherited form of spastic paraplegia 76 (SPG76) with a combination of corticomotor tract disorder and cerebellar ataxia. We here report clinical and molecular findings of a Chinese patient with a novel homozygote c.1439T>C variant (p.Leu480Pro) in the CAPN1 gene. Causative mutation was identified by target sequencing of a custom panel containing over 730 known virulence genes and confirmed by Sanger sequencing. Our report emphasizes the importance of the “spasticity-ataxia” phenotype in the diagnosis of SPG76 and broadenes the clinical and molecular spectrum of “CAPN1-associated neurodegeneration”.

Introduction

Hereditary spastic paraplegia (HSP) is an umbrella group of clinically and genetically heterogeneous disorders characterized by progressive weakness and spasticity of the lower extremities as a result of corticospinal dysfunction(Fink et al. 2013). Based on the phenotype, HSP is distinguished as pure or complicated forms. Pure form HSP is characterized by signs of involvement of the pyramidal tract, such as spasticity and weakness restricted to the lower limbs. Hypertonic bladder and sensory disturbance are also frequently reported. Complicated form HSP often accompanied by other neurological symptoms, including cerebellar ataxia, seizure, cognitive impairment, peripheral neuropathy, amyotrophy, dysarthria and others(Shribman et al. 2019). The genetic classification for HSP is based on sequential numbering of chromosomal loci or specific genes. There are more than 80 genetic types of HSP. Autosomal dominant (AD), autosomal recessive (AR), or X-linked modes of inheritance are reported, and AR inheritance is the commonest one(Shribman et al. 2019).

Recently, loss‐of‐function mutations in the CAPN1, the gene encoding calpain-1, have been identified as causative for spastic paraplegia 76 (SPG76)(Gan-Or et al. 2016). Calpain-1 is a calciumactivated intracellular proteinase involved in processes of synaptic plasticity, synaptic restructuring, axon migration, axon maintenance and necrosis, among others(Wang et al. 2016). Loss of calpain-1 function will lead to neuronal and axonal dysfunction and degeneration. CAPN1 variants were initially identified in AR inherited complex HSP pedigrees in 2016, and they had drawn increased attention within the last 5 years.

In this study, we reported the clinical and genetic characteristics of a Chinese HSP proband carrying a novel homozygous mutation of CAPN1.

Methods

The proband, a 28-year-old male, was admitted to Department of Neurology, Jili Hospital due to gait ataxia and weakness of legs for 8 years. He presented with truncal and gait ataxia at age 20. The symptoms deteriorated slowly. At age 23, he noticed a stretched feeling of the left lower limb. He needed a cane for walking at age 26. In neurological examination, he exhibited slight spasticity and extensor plantar responses in the lower limbs, with a moderate vibration sense decrease in the lower limbs. He had moderate bilateral dysmetria when performed finger-to-finger test and moderate dysdiadochokinesia. He had wide-base gait, positive Romberg’s test, and impossible tandem. The ocular pursuit and saccades were normal. The ophthalmologic examination revealed a decrease in visual acuity. He presented pes cavus. No dysarthria, or bladder dysfunction was complained. He scored 19/32 on the Scale for the Assessment and Rating of Ataxia (SARA). Routine laboratory tests were in the normal range. The ophthalmologic examination did not find macular damage or optic atrophy. Motor evoked potentials (MEP) were markedly slowed while sensory evoked potentials showed a mild increase of central conduction time from lower limbs. Cerebellar and thoracic cord showed atrophy on magnetic resonance imaging (MRI) (Figure 1).

Figure 1: Brain Magnetic resonance imaging (MRI) showed the atrophy of the cerebellum, and spinal cord MRI showed thoracic cord atrophy

Figure 2: Pedigree of the family. Patient I-1 is the proband.

His sister and parents were asymptomatic (Figure 2). Genomic DNA was extracted from peripheral blood of both patients and his parents or sister through a standard phenol-chloroform method. All family members signed a written informed consent. Multiple molecular genetic tests of the proband were used, including long-range polymerase chain reaction (PCR), target sequencing, and Sanger sequencing. A total of 10 fragments including CAG/CTG expansion sequences were amplified by PCR in different reactions using an ABI Prism Thermal cycler. At the same time, the proband was performed by target sequencing of a custom panel containing over 730 virulence genes known to cause HSP, SCA and other movement disorders. The sequencing was carried out by Illumina HiSeq X-10 platform. The mean depth of the targeted regions was 161.43X and the coverage was 99.88%. Subsequently, we performed Sanger sequencing to confirm the candidate variants detected through targeted sequencing analysis. These variants were also performed in his parents and his sister to confirm the family co-segregation.

Results

The mutation screening results of Spinocerebellar ataxia 1 (SCA1), SCA2, SCA3, SCA6, SCA7, SCA8, SCA12, SCA17, SCA36 and DRPLA were negative. Target sequencing showed the presence of the novel homozygous mutation of c.1439T>C (p.Leu480Pro) in the CAPN1 gene, and sanger sequencing confirmed this mutation. We confirmed cosegregation of the mutation with the disease in the pedigree. The unaffected parents carried c.1439T>C in the heterozygous state (Figure 3).

Figure 3: DNA sequence at codon 480 of CAPN1 gene from the proband (A), I-1(B), I-2(C),and II-2 (D). The red arrow indicates the c.1439T>C mutation causing a substitution of Leucine by Proline at codon 480 of the protein.

The mutation is predicted to be “deleterious” by sorts intolerant from tolerant (SIFT), and “probably damaging” by Polyphen-2. The variant meets the criteria of PS2, PM2, and PP3 of the American College of Medical Genetics (ACMG) guideline, and its pathogenicity is further supported.

Therefore, we conclude that this missense variant is a “likely pathogenic” variant for SPG76.

Discussion

HSP is a clinically and genetically heterogeneous disease. The clinical features of the proband with both spasticity and cerebellar ataxia in this report is similar to those reported in previous studies(Lai et al. 2020, Méreaux et al. 2021, Peng et al. 2019). “Spasticity-ataxia” phenotype would be conducted to the diagnosis of SPG76. The overlapping of spastic paraplegia and ataxia might be caused by shared vulnerability of corticospinal tracts and cerebellar circuits toward disturbances of the same molecular pathways(Synofzik et al.2017). These overlapping phenotypes present extremely challenging obstacles for the correct clinical diagnosis of HSP.

For a long time, HSPs and ataxia have been considered clinically disparate syndromes. However, with the wildly application of next generation sequencing (NGS), we have increasingly realized that they largely share the same genes. For example, several HSP‐causative genes, including CYP7B1 (SPG5), SPG7 (SPG7), SPG11 (SPG11), ZFYVE26 (SPG15), PNPLA6 (SPG39), GBA2 (SPG46), KIF1C (SPG58), CAPN1 (SPG76), ATP13A2 (SPG78), and UCHL1 (SPG79), have been associated with cerebellar ataxia. Likewise, “traditional” ataxia genes were often recognized to result in HSP phenotypes(Khateeb et al. 2006, Rahimi et al. 2020, Synofzik et al. 2017).

CAPN1, located in chromosomal region 11q13, encodes calpain 1, a calcium-activated cysteine protease that is important for several biological roles in corticospinal and cerebellar tracts. In 2013, it was reported for the first time that loss-of-function mutations in CAPN1 are associated with spinocerebellar ataxia in dogs(Forman et al. 2013). Homozygous or compound-heterozygous mutations in CAPN1 have been found to be responsible for complicated HSP with cerebellar ataxia since 2016(Lai et al. 2020).

The mutation of c.1439T>C (p.Leu480Pro) in the CAPN1 gene was defined as novel, likelypathogenic missense mutation. It is one of the so called “HSP–ataxiaspectrum disease genes. On the other hand, mutations of CAPN1 can lead to pure HSP without cerebellar ataxia as well. Therefore, a term ‘CAPN1-associated neurodegeneration’ has been proposed to describe the HSP and other related neurodegenerative diseases caused by the CAPN1 variants (Méreaux et al. 2021, Rahimi et al.2020). It shows that spastic paraplegia and ataxia share overlapping phenotypes and pathogenic genes.

Statement: The subjects gave written informed consent in accordance with the Declaration of Helsinki. And the they gave written informed consent for publication of this report as well.

Disclosure: The authors report no interest relevant to the manuscript.

Authors contribution: LL, LD : drafting the manuscript. LL, LD and YX: analysis and interpretation of data. RH, YX: revising the manuscript. RH: study concept and design.

Funding: No

Conclusions

The results of this nationwide, representative health researchin schoolchildren showed that the prevalence ofhealthy habits decreased from childhood to adolescence. Also, healthy habits were associated with decreased odds of obesity and low PF measurements. This knowledge could be helpful in recommendations to make certain healthy habits in children and adolescents.

Acknowledgments: All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by KDT, DBP,DV, GA,and LSS. The first draft of the manuscript was written by KDT, DV, and GA, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

All authors agree with the manuscript and declare that the content has not been published elsewhere.

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.

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