In our current endeavor, we attempted to expand the mutational spectrum of SPAST
. To this end, we screened for mutations in the SPAST
gene in 200 HSP patients and identified 47 different mutations, out of which 29 were novel mutations. The overall frequency of SPAST
mutations in our cohort was 28.5% (57/200). The mutation rate did not change significantly when we only considered pure HSP, which was 29.4% (32/109). Interestingly, in case of complex HSP a high mutational rate of 21.7% (5/23) was detected in our HSP cohort, which highlights the need to screen for the SPAST
mutation in complex HSP cases. However, owing to lack of family history, we were unable to show segregation of additional symptom(s) with paraplegia, therefore, it is also possible that the complex phenotype could also be because of an independent locus other than the SPAST
. The mutation detection rate in our cohort is consistent with the range of 15–44%, which was observed previously in other populations.8, 9, 10, 11, 13
The frequency of mutations increased if we only considered the autosomal dominant HSP cases (36.4%) suggesting that prevalence of SPAST
mutation is higher in the familial cases. Among the sporadic cases of HSP, the frequency of mutations was 6.5% (3/46), which was lower than the previously reported rate of 12–18%.13, 14
This discrepancy could be because of the different population type and size. Nevertheless, from a diagnostic point of view, our and other reports emphasize the need to screen for SPAST
mutations in the sporadic HSP cases.
In our HSP cohort, we identified 51 mutations in the SPAST
gene by direct sequencing of all the 200 HSP patients, which left 149 HSP cases in which no mutations could be detected by conventional sequencing. To determine gross deletion/insertion in the SPAST
gene, we performed MLPA analysis in these 149 HSP cases and detected six additional mutations, which accounts for 4% (6/149) of remaining HSP cases. Previously, two independent studies used same SPAST
-specific MLPA assay and reported a much higher proportion (18–20%) of deletion in HSP patients.19, 20
The observed disparity in the proportion of large deletions between our HSP cohort and others could be because of the divergence and ethnic variability in these cohorts. Nevertheless, our report of much lower proportion of gross deletions in the SPAST
gene in the HSP patients stresses the need to perform MLPA in various HSP cohorts to determine the incidence rate of gross deletions in worldwide HSP populations.
It is remarkable that 22 (85%) out of the 26 novel mutations (excluding the gross deletions) were located in the AAA domain of spastin. Previously, our group reported clustering of mutations in the AAA domain of spastin in a German HSP cohort12
and this clustering in AAA domain was also observed in several other HSP cohorts.6, 7, 8, 9, 10
Moreover, the distribution of mutations reported in the database over the structural domains of spastin outside the AAA domain were also not uniform; rather they were concentrated in certain regions of the protein, which constituted various functional domains, such as MIT and MTBD. In prior studies, exon 1, exon 5 and exon 8 of SPAST
was recognized as hot spot regions;32, 33, 34
however, no correlation to functional domain of spastin was implicated. Overall, it appears that different functional domains of spastin are target regions for mutations, which underlines their functional significance. Identification of these cluster regions highlights the need to set these regions as priority in the molecular diagnostic screens.
Beside a few exceptions, almost all the missense mutations in spastin are located in the AAA domain and recent studies suggest that these missense mutations might exert a dominant-negative effect on the molecular function of spastin.35, 36
Utilization of a recently modeled structure of the AAA domain of spastin,22
as a framework, enabled us to classify the identified missense mutations from our cohort into different functional groups such as active site, protomer–protomer interaction, pore loop and unknown structural group of mutations. The functional categorization of the novel missense mutations, based upon the structural model of spastin will enable us in future to predict any identified sequence variant in a HSP-SPAST
patient as disease-causing mutation with greater level of certainty. These structural predictions of various functional classes of missense mutations need to be validated by biochemical/cellular studies and data from the structural model should be interpreted with cautiousness. However, in a recent study, we could validate at the cellular level the functional effect of two sequence variants (E442Q and R499C) of spastin, which were predicted as active site mutations from the structural model of spastin.22
The rare S44L polymorphism is considered to act as a modifier of the HSP phenotype.10, 31, 37
S44L is not considered as a susceptibility factor for HSP because its frequency rate is similar in HSP patients and controls.14
In our study, we could not ascertain the role of S44L (heterozygous state alone) on manifestation of HSP. It is possible that the patients heterozygous for S44L might have another mutation in spastin, which could not be identified by our screen or might have a mutation in a different HSP gene.
No apparent genotype–phenotype correlation is evident among missense mutations and other SPAST
mutations.8, 14, 38
Although several studies indicated that missense mutation might act in a dominant-negative fashion in contrast to other mutations, which lead to a loss of function. To determine, whether missense mutation leads to early onset of HSP, we assorted our HSP cohort into two different groups based upon AAO (≤35 and >35 years). The rational behind sorting our HSP cohort into these two age groups was derived from Harding's classification of HSP patients into two distinct groups, early age onset (≤35 years) and late age onset (>35 years).39
This AAO (≤35 and >35 years) classification was also used by Fonknechten and coworkers for determination of genotype–phenotype correlation.8
We observed an obvious difference in the proportion of mutations between the missense group as compared with the other types of mutations in age group of >35 years. However, the observed difference was not statistically significant (P
>0.05) because of a very small sample size. Remarkably, we could reject a null hypothesis that there is no difference in the proportion of subjects for missense mutations between two age groups, ≤35 and >35 years, which was statistically significant (P
<0.0124). Our data show a tentative genotype–phenotype correlation and suggest that in case of missense mutations the onset of phenotype is earlier. Owing to a small sample size, this correlation between AAO and missense mutation should be interpreted with discretion. Previously, early AAO in patients with missense mutation was also reported; however, this study only accounted for two missense mutations out of a total five mutations.40
Moreover, in a meta-analysis38
no significant correlation between AAO and mutational class was evident, but one limitation of this study was the sample size. Nevertheless, these different pathomechanism modes, such as loss of function and dominant-negative function for different classes/types of spastin mutations need to be carefully resolved by experimental means; otherwise there will be repercussions on the likely success of any therapeutical approach devised for spastin-associated HSP.