High frequency of SPG4 in Taiwanese families with autosomal dominant hereditary spastic paraplegia

This study included 20 unrelated AD-HSP kindreds (49 cases in total) of Han Taiwanese ethnicity collected from the Departments of Neurology, Rehabilitation and Pediatric, Chang Gung Memorial Hospitals, Taiwan (Additional file 1: Figure S1). Families 1 to 4 have been reported previously [12]. A diagnosis of AD-HSP was based on the following diagnostic criteria: (1) spastic paraplegia, or (2) spastic tetraparesis with earlier and more severe affliction of the lower limbs, or (3) spastic paraplegia as an early and prominent sign of a degenerative disease affecting the nervous system; (4) positive family history of spastic paraplegia with affected members in at least two generations; and (5) no other causes of the presenting symptoms [4]. Family history was recorded according to the patients' reports, and inheritance was ascertained in 14 families by examining affected family members. Clinical information was collected and neurological examinations were performed. Ambulatory disability was scored based on a five-point scale: 0 asymptomatic, 1 minimally impaired (able to run), 2 mildly impaired (able to walk independently but not run), 3 moderately impaired (walk with an aid), and 4 severely impaired (wheelchair bound).

Genomic DNA was extracted from peripheral blood leukocytes for genetic analysis, and mutation screenings were conducted in the following order. First, nucleotide substitutions and small deletions or insertions in all exons and their adjacent splicing sites of the SPG4 gene (SPAST) were analyzed by polymerase chain reactions and direct sequencing. If a mutation was not detected, multiplex ligation-dependent probe amplification (MLPA) was performed to detect exonic deletions in SPAST. Finally, for the cases with no detected mutations in SPAST, sequence analysis and MLPA of the SPG3A gene (ATL1) and sequence analysis of the SPG31 gene (REEP1) were performed. Detected mutations were checked in 100 control DNA samples collected from unrelated ethnic Han Taiwanese.

MLPA was performed using SALSA MLPA kits (P165-B1 HSP probemix; MRC-Holland, Amsterdam, Netherlands) according to the manufacturer's instructions. For each sample, a normalized value ratio for a relative peak area between 0.8 and 1.2 was considered normal. A heterozygous deletion was expected with a ratio between 0.3 and 0.7, and a heterozygous duplication between 1.3 and 1.7. Total mRNA from the blood leukocytes was extracted using Trizol procedure (Invitrogen, Carlsbad, CA, United States). mRNA analysis was performed as described previously [12] to assess alternations in gene transcription due to SPAST mutations.

Novelty of a detected mutation was checked with the Human Gene Mutation Database [13]. Amino acid conservation in orthologues was assessed using the HomoloGene database [14]. Prediction of pathogenicity of a missense mutation was analysed using the PolyPhen-2 program [15].

Data were presented as mean ± SD unless otherwise specified. Disease severity was defined as “mild” with an ambulation score of 1 or 2, or “severe” with a score of 3 or 4. The progression rate of the disease was assessed with a disease-progression score (DPS) calculated as the current disability score divided by disease duration. Mutations were classified according to the consequence, namely, whether they caused a complete loss of the AAA cassette (i.e. nonsense and frameshift mutations proximal to the encoding sequence of the AAA cassette, whole gene deletion, and mRNA decay) or not. Age at onset (AAO) with respect to mutation consequence was tested with t-tests (asymptomatic cases were excluded from the analysis). The relationships between disease severity and current age, gender, disease duration and mutation consequence were tested using the chi-square test or t-test. The relationships between DPS and AAO, gender and mutation consequence were tested using the Pearson test or t-test. Independence of the association between disease severity (or DPS) and clinical and genetic factors was examined using logistic (or linear) regression analysis. A p value <0.05 was considered to be statistically significant.

A total of 14 different SPAST mutations were identified in 18 families (Table 1). In this cohort, SPG4 accounted for 90% of the AD-HSP families. Mutation types were quite heterogeneous, including nonsense (2), missense (2), insertion (2), deletion (2), insertion-deletion (1), splicing site (2), and large exonic deletion (3) (Figure 1). Four mutations (c.1361_1363 ins GGG, c.1005-1 G > C, c.1004 + 1 G > T, and c.1738_1740 del ATT/ins GA) were novel mutations. These mutations were neither included in the data derived from the 1000 Genome Project (http://​www.​1000genomes.​org/​) [16] nor detected in the 100 control DNA samples. The residue leucine affected by the missense mutation p.L461P and the residue aspartate affected by D555G were highly evolutionarily conserved according to the public genome database (Additional file 2: Figure S2). The substitutions of amino acids caused by the two missense mutations were predicted to be damaging (a score of 1.000 and 0.988 respectively) by the PolyPhen-2 program. In general, nonsense and missense mutations were located in the 3′ half of SPAST exons, while frameshift (mini-insertions and mini-deletions) and splicing site mutations were more evenly distributed in the gene. Co-segregation of mutations with the disease was confirmed in 14 families, in which at least two affected members underwent genetic testing. Mutations in SPAST, ATL1 and REEP1 were not detected in two families (Families 14 and 19).

For the in-frame insertion c.1361_1363 ins GGG detected in Family 6, only the wild type of mRNA was detected in mRNA analysis and the decay of mutant mRNA is suspected (Additional file 3: Figure S3). For the other insertions and mutations of missense, nonsense, and deletion types, mRNA analysis showed nucleotide changes identical to those in genomic DNA. The two splicing site mutations in intron 6 led to skipping of transcription of the first 8 bases of exon 7 (Family 9) and the whole of exon 6 (Family 10), respectively. For the exonic deletion revealed by MLPA in Family 2, cDNA analysis showed a deletion of 1090 bases at the 5′-end of exon 17, as reported previously [12]. In Family 4, the effects of complete loss of exon 17 on translation could not be determined due to failure to amplify the mutant transcript.

In this AD-HSP cohort, AAO in the probands ranged from 1 to 48 years, and six (30%) families had at least one case whose AAO was less than 10 years. The clinical characteristics of the SPG4 cases are summarized in Table 2. The onset of disease ranged from 1 to 69 years of age. Among the 47 cases carrying SPAST mutations, 44 (94%) showed spastic paraparesis and 3 (6%) were asymptomatic. All of the symptomatic SPG4 patients manifested a pure phenotype, except for a case in Family 7 who presented with distal amyotrophy in the upper limbs. The most common clinical presentation was gait disorder due to spasticity or increased tendon reflexes in the lower limbs. Neurological examinations of the cranial nerves and upper limbs were normal except for an accentuated jaw-jerk and tendon reflex in some patients. Considerable variations in AAO, rate of progression or severity of disease were noted, even in mutation carriers from the same family (Table 1). There was no clear phenotype correlation with respect to the different types of mutations. For the two families without detected mutations, one had a pure phenotype while the other presented with deafness in the affected members.

For the SPG4 cases, mutations causing complete loss of the AAA cassette were associated with an earlier onset of disease (20 ± 18 years, n =16) compared with those with preservation of partial or total AAA cassette (32 ± 19 years, n = 28; p = 0.041). In univariate analysis, disease severity was related with the patients’ current age (37 ± 19 years for mild disease vs. 55 ± 12 years for severe disease, p <0.001), and DPS was correlated with AAO (r = 0.564, p <0.001) (Additional file 4: Table S1 and Additional file 5: Table S2). After adjusting for gender, disease duration and consequence of mutation, the association of age (odds ratio 1.10, 95% confidence interval 1.04-1.16 for every increase of 1 year, p = 0.002) with disease severity, and that of AAO (β = 0.038, standard error 0.010, p <0.001) with DPS remained significant. Consequence of SPAST mutation was not related with either disease severity or DPS.