Two novel homozygous mutations of CAPN1 in Chinese patients with hereditary spastic paraplegia and literatures review

Background Hereditary spastic paraplegias (HSP) are of great clinical and genetic heterogeneity. According to the clinical features, HSP can be divided into pure or complicated subtypes which combined with other neurological symptoms including cerebellar ataxia. Up to date, 78 loci or genes have been implicated in HSP. CAPN1 was a novel gene detected recently for spastic paraplegia 76 (SPG76). Methods Patients referred to our clinic with spastic or spastic-ataxic gait were collected. Genetic testing of the probands were performed by target sequencing of a panel containing over 4000 known virulence genes. And the candidate mutations were further confirmed by polymerase chain reaction (PCR) and Sanger sequencing. The clinical materials of these patients were demonstrated retrospectively. Results Two Chinese patients, both from consanguineous families, each carried a novel homozygous mutation of CAPN1, p.R48X and p.R339X. The male proband presented pure HSP subtype while the female proband presented complicated HSP symptoms with cerebellar ataxia. We then reviewed all the literatures of HSP patients carrying CAPN1 mutations and summarized the molecular spectrum and clinical characteristics of CAPN1-related SPG76. Conclusion These two SPG76 patients carrying CAPN1 mutations were the first reported in China. By reviewing the clinical manifestations of SPG76 patients, we validated the “spastic-ataxia” phenotype and emphasized the association between spasticity and ataxia, indicating the importance of CAPN1 screening in HSP patients.


Introduction
Hereditary spastic paraplegias (HSP) present great genetic and clinical heterogeneity, mainly manifesting as spasticity and weakness in the lower limbs [1]. On the basis of clinical features, HSP can be categorized into pure and complicated subtypes [2]. In addition to the dominant progressive spasticity and weakness, pure HSP can also present symptoms of hypertonic bladder and sensory disturbances. Complicated HSP is often accompanied by other neurological symptoms, including cerebellar ataxia, seizure, extrapyramidal signs, intellectual disability, peripheral neuropathy, amyotrophy, optic atrophy and others [3,4]. Among them, cerebellar ataxia occurs most frequently in complicated HSP, resulting in "spasticity-ataxia" phenotype [5]. The hereditary modes of HSP include autosomal-dominant (AD), autosomal-recessive (AR), X-linked and maternal trait of inheritance which due to mitochondrial impairment [4]. In all these hereditary modes, AR inheritance is the commonest one [6]. Up to date, a total of 78 loci have been implicated in HSP [5].
In this study, we reported two Chinese HSP probands, both from consanguineous family (Fig. 1), each carried a novel homozygous mutation of CAPN1. To our knowledge, they were the first SPG76 patients reported in China. Their clinical features and disease progressions were demonstrated retrospectively and would broaden the molecular and clinical spectrum of Chinese HSP patients.

Methods
The probands with walking problems such as spastic or spastic-ataxic gait were collected in the Neurogenetics clinic in Huashan Hospital (Shanghai, PRC). The clinical materials were investigated in both probands.
Genomic DNA was extracted from peripheral blood of both patients and their parents or siblings. Genetic testing of the probands were performed by target sequencing of a panel containing over 4000 known virulence genes. The sequencing was carried out by Illumina HiSeq X-ten platform. The variants screen protocol was as previously reported [18]. The candidate mutations were further confirmed by polymerase chain reaction (PCR) and Sanger sequencing. These mutations were also performed in the parents or siblings to confirm the family co-segregation.
Written informed consents were obtained from both patients and their relatives. This study was approved by the ethics committee of Huashan Hospital.

Results of genetic testing
In family A, the mean depth of target sequencing was 73.5X and the coverage was 100%. The percentage of the target region with mean depth > 20X was 97.0%. According to the screening criteria of low variants frequencies [< 1% in 1000Genome (http://www.1000genomes.org/ home), ExAC (http://exac.broadinstitute.org/)] and homozygous mapping, 38 variants were left. But after further screened by clinical manifestations, only one novel homozygous mutation of c.142C > T (p.R48X, NC_000011.10:g.64950314C > T) in CAPN1 (NM_ 001198868) was found with the depth of 84X.
In family B, the mean depth of target sequencing, the coverage and the percentage of the target region with mean depth > 20X was 107.8X, 99.3 and 97.4% respectively. After screened by the criteria mentioned above, one novel homozygous mutation of c.1015C > T (p.R339X, NC_000011.10:g.64956067 C > T) in CAPN1 was found with the depth of 70X.
Both mutations have been confirmed in the probands and their parents or siblings by Sanger sequencing. The unaffected parents and two unaffected elder sisters of the proband in Family A all carried c.142C > T in the heterozygous state. In family B, the mutation of c.1015C > T was found heterozygous in the unaffected parents of the proband.
Both variants were partly conserved across species (Fig. 2) and was predicted to be disease causing by mutationtaster (mutationtaster.org) since the truncated mutant took place in positions of R48 and R339 which might cause nonfunctional protein product or affect functional subdomains of the protein.

Clinical characteristics
The proband from family A gradually developed walking difficulties and stiffness in the lower limbs at age 18. The symptoms deteriorated slowly. He came to our clinic at age 38 and shown a typical spastic gait.
Neurologic examination revealed that the muscle tone in both lower limbs was extremely high. Knee and ankle hyperreflexia were also found in both lower limbs. Bilateral Hoffmann sign and Babinski sign were positive. He could still walk along a straight line. Finger-to-nose test and diadochokinesia were performed well. The examination of ocular movement was fine. No distal sensory impairment, cognitive deterioration, bladder dysfunction, or dysarthria was complained. Nerve conduction study and electromyography did not reveal any neurogenic and myogenic damages. The results of head and spinal cord magnetic resonance image (MRI) were also negative.
The proband from family B referred to our clinic for progressive walking difficulties at age 41. Five years ago, she reported weakness in the lower limbs and there was a slight tiptoe when she was walking. She felt slight imbalance and could not walk along a straight line well. The neurological examination revealed hyperreflexia in four limbs and positive bilateral Hoffmann sign. She presented with a moderately spastic-ataxic gait. She had slight bilateral dysmetria when performed finger-to-finger test and mild dysdiadochokinesia. The heel knee test was fine. The ocular pursuit and saccades were normal. She scored 6/40 on the Scale for the Assessment and Rating of Ataxia (SARA). The mini-mental state examination score was 29/30 (education year of 14) suggesting the normal cognitive function. No dysarthria, distal sensory impairment or bladder dysfunction was reported. Neither cerebellum nor spinal cord showed significant atrophy on MRI.

Literatures review
A total of nineteen pedigrees including 35 patients (24 Female,11 Male patients) with CAPN1 mutations reported till recently were reviewed (Table 1). All the patients showed a pattern of AR inheritance and 85.7% pedigrees were consanguineous. Thirty patients carried homozygous mutations and five patients carried compound heterozygous mutations. The onset age ranged from five to 39 years old. With all the available clinical materials, lower limbs spasticity, presenting with stiffness, hyperreflexia and pathological signs, developed in around 80% patients, followed by cerebellar ataxia developing in 62.9% of the cases, dysarthria in 51.4%, skeletal or tendon deformity in 31.4%. Weakness in lower limbs and ocular movement disorder could also be seen. Some patients developed abnormal bladder function, dysphagia, peripheral neuropathy, intention tremor and even other uncommon symptoms.

Discussion
With the wildly application of next generation sequencing, more and more classical "HSP genes" causing cerebellar ataxia were found and vice versa. So, these genes could be categorized as "spasticity-ataxia" spectrum. According to a review in 2017, genes related to "spasticity-ataxia" spectrum was expanded to 69 members [5]. CAPN1 was one of them, manifesting as pure HSP or complicated HSP. The mutations in CAPN1 causing autosomal recessive HSP have been found since 2016 by whole exome sequencing in three pedigrees. Among these patients carrying CAPN1 mutations, lower limbs spasticity was the predominant symptom combined with cerebellar ataxia or not. Therefore, "spasticity-ataxia" phenotype might conduce to the diagnosis of SPG76.
The protein product of CAPN1, calpain-1, also known as μ-calpain, contains four domains: the N-terminal anchor helix region, the CysPc protease domain (including two protease core domains of PC1 and PC2), the C2 domain-like domain and the penta-EF-hand domain (PEF). As a calcium-activated cysteine protease, calpain-1 binds to calcium through PEF domain [19]. It has been proved that the activation of calpain-1 is required for its neuro-protective role in CNS [20]. Several mechanisms for the protective role were suggested by interacting with CDK5 and NR2B to control NMDA-receptor degradation [21] or affecting AMPA receptors through degradation of its substrate, glutamate receptor-interacting protein [22]. In calpain-1 deficient mice, dysfunction of calpain-1 reduced dendritic branching complexity and led to spine density deficits [23]. In zebrafish embryos, knockdown of calpain-1 induced disruption of microtubule network in brain and spinal cord [24], which indicated that dysfunction of calpain-1 could result in neurodegeneration or disorganization of neurons [25]. Immunohistochemistry study revealed that calpain-1 was the major calpain isoform in cerebellar neurons [26], and the activity of it in cerebellum was higher than that in cortex or hippocampus [27], suggesting that calpain-1 played a key role in maintaining the normal cerebellar function.
All the reported mutations scattered in exons 2-6, 8-10, 13, 14 and 21of CAPN1, potentially damaged the normal structure of calpain-1 or led to early termination of protein coding, causing the dysfunction of calpain-1. In this current study, the probands were in accordance with two HSP subtypes: the male patient presented with pure HSP subtype with normal cerebellar function, while the female patient manifested as classical complicated HSP subtype showing symptoms of both HSP and cerebellar ataxia. Two novel homozygous mutations c.1176G > A and c.675C > A of CAPN1 were detected respectively. These two mutations were situated in exon 2 and exon 10 and brought on a premature stop codon at the positions of R48 and R339, causing the destruction of calpain-1 normal structure. The structural incompleteness of calpain-1 would interfere with its neuro-protective role in CNS and induce neurodegeneration or disorganization of neurons, which might lead to SPG76.

Conclusion
Together with previously reported cases, our study broadened the clinical and molecular spectrum of CAPN1-related SPG76 and exemplified the concept of "spasticity-ataxia" phenotype, further increasing our understanding of complicated HSP form and its connection with cerebellar ataxia. All these observations indicated that CAPN1 screening is necessary in HSP patients, especially when patients suffer from spasticity-ataxia phenotype.