Clinical characteristics
The proband (II:2) (Fig.1A) , a 63-years-old female, presented to the outpatient clinic in our hospital due to her progressive difficulty walking caused by moderate spasticity of the lower limbs for 24 years. She has felt unknown gait disorder as early as in 1992. For the last 11 years, this gait disorder has gotten worse, especially in cold weather. And she was confined to a wheelchair at the age of 52. In the last year, the proband suffered from the frequently urged to urinating and bowel functions. Physical examination showed that she had brisk deep tendon reflexes in all four limbs, simultaneously accompanied with obvious corticospinal tract signs (Babinski’s signs was positive), and decreased sense of pain, light touch and vibration in the lower limbs characterized with stocking pattern-distributed sensory loss. Muscle strength of the upper limbs was normal, while both the extensors and flexors in the lower limbs were 3/5. The results obtained from routine laboratory tests, electromyography, cranial and cervical MRIs did not reveal any obvious pathognomonic alteration. For her previous history, the proband had ever received some treatment on rheumatoid arthritis because of the pain in both hips and knees 26 years ago, but the uncomfortable symptom was not getting better. 9 years ago, a traumatic injury on her back further aggravated her discomfort though the cranial and cervical MRIs were both normal at that time.
With regard to the proband’s family history (Fig.1A), her parents were deceased, but her father had similar symptoms. In addition, she had three brothers and two sisters. One of her brothers who had similar symptoms has passed away and the rest were all asymptomatic. Details about her symptomatic father and brother are not clear. Physical examination gave the below results. Ⅲ:9 aged 32 years had an abnormal spine physiological curvature. The shoulders of Ⅳ:9 aged 11 years were not equal. The (Ⅲ:1) aged 46 years, Ⅲ:9 and Ⅳ:9 showed brisk deep tendon reflexes in all four limbs and positive Babinski signs. All the three symptomatic patients all could not run and squat since young age. Despite their motor symptoms, the proband’s nephew(Ⅲ:1) and the third daughter (Ⅲ:9) have frequent urge to urinate and to have bowel functions at the age of 40 years and 27 years.
The proband’s the third daughter (Ⅲ:9) and grandson(Ⅳ:9) suffer from other disease except HSP. The Ⅲ:9 patient who was diagnosed with pulmonary hypertension has been suffering from chest tightness and shortness of breath for three years, losing the ability to work. Similarly, the echocardiography of Ⅳ:9 aged eleven years showed mild reflux at mitral valve and tricuspid valve.
Clinical features of the four affected individuals in the family have been summarized in Table 1, and their clinical commonalities and personalities are exhibited respectively. The four symptomatic patients have the different degrees of disability. The disability score was evaluated according to a four-point scale (1: normal, 2: able to walk but not run, 3: need the help of a walking aid or support, 4: walk on wheelchair)[17]. In addition, the onset age ranges from 3 to 30 years old, although they have the same mutation in the exon 16.
Genetic findings and prediction results of protein structure and function by different methods
The Exome Sequencing analysis of the proband exhibited a novel disease-associated mutation in exon 16 of the already known disease-associated SPAST gene, and the in-frame deletion was identified in the three affected family members (Fig. 1B II:2, Ⅲ:1,Ⅲ:9,Ⅳ:9). It is an in-frame deletion mutation in the heterozygous state: the GAA nucleotides deletion at codon 1710-1712 position and the circled nucleotide represents the codon 1710 position, where the mutation starts. (Fig. 1B II:2, Ⅲ:1, Ⅲ:9,Ⅳ:9). According to Human Gene Mutation Database (HGMDpro), the pathogenic mutation site c.1710_1712delGAA has not been reported until now. Therefore, it is a novel mutation. And there are no mutations were identified when analysis of other genes associated with HSP was performed : PLP1, L1CAM, SPG11, SPG7, ATL1 and so on . While the rest asymptomatic family members had no mutations at this site (Fig.1B Ⅲ:5).
As highlighted in Figure1B, the results of RaptorX prediction showed that this new-found mutation we reported resulted in the synthesis of misfolded protein(Fig.2B) in comparison to native one (Fig.2A).We performed a protein sequence alignment across species showing the area of this in-frame amino acid deletion and the surrounding residues(https://www.uniprot.org/align/A20200502216DA2B77BFBD2E6699CA9B6D1C41EB2087CC0O). The result revealed that the spastin protein sequence across species is highly conserved at the position 570 of the protein(red frame) (Fig.2C). Therefore, the lysine deficiency at the position 570 of the protein has a significant impact on the function. Additionally, the result of PROVEAN demonstrated that the mutation site c.1710_1712delGAA has an functional impact on the SPAST protein sequence variations. Given a list of genomic coordinates and variants (2,32372308,AGAA,A), the amino acid change(p.K570del) can be quickly determined and PROVEAN score is computed to be -11.55, which is significantly lower than the score threshold (cutoff=-2.5). The deletion variant was predicted as deleterious (Table 2). According to American College of Medical Genetics and Genomics (ACMG) criteria[18], we score this variant as likely pathogenic PM1, PM2, PM4, PP3