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Comparative analysis and functional mapping of SACS mutations reveal novel insights into sacsin repeated architecture.
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PMID:  23280630     Owner:  NLM     Status:  MEDLINE    
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurological disease with mutations in SACS, encoding sacsin, a multidomain protein of 4,579 amino acids. The large size of SACS and its translated protein has hindered biochemical analysis of ARSACS, and how mutant sacsins lead to disease remains largely unknown. Three repeated sequences, called sacsin repeating region (SRR) supradomains, have been recognized, which contribute to sacsin chaperone-like activity. We found that the three SRRs are much larger (≥1,100 residues) than previously described, and organized in discrete subrepeats. We named the large repeated regions Sacsin Internal RePeaTs (SIRPT1, SIRPT2, and SIRPT3) and the subrepeats sr1, sr2, sr3, and srX. Comparative analysis of vertebrate sacsins in combination with fine positional mapping of a set of human mutations revealed that sr1, sr2, sr3, and srX are functional. Notably, the position of the pathogenic mutations in sr1, sr2, sr3, and srX appeared to be related to the severity of the clinical phenotype, as assessed by defining a severity scoring system. Our results suggest that the relative position of mutations in subrepeats will variably influence sacsin dysfunction. The characterization of the specific role of each repeated region will help in developing a comprehensive and integrated pathophysiological model of function for sacsin.
Alessandro Romano; Alessandra Tessa; Amilcare Barca; Fabiana Fattori; Maria Fulvia de Leva; Alessandra Terracciano; Carlo Storelli; Filippo Maria Santorelli; Tiziano Verri
Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Human mutation     Volume:  34     ISSN:  1098-1004     ISO Abbreviation:  Hum. Mutat.     Publication Date:  2013 Mar 
Date Detail:
Created Date:  2013-02-19     Completed Date:  2013-08-12     Revised Date:  2014-03-19    
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Nlm Unique ID:  9215429     Medline TA:  Hum Mutat     Country:  United States    
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Languages:  eng     Pagination:  525-37     Citation Subset:  IM    
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© 2012 Wiley Periodicals, Inc.
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MeSH Terms
Chromosome Mapping*
Computational Biology
Heat-Shock Proteins / genetics*,  metabolism
Muscle Spasticity / genetics*
Polymorphism, Single Nucleotide
RNA, Messenger / genetics,  metabolism
Sequence Alignment
Sequence Analysis, DNA
Spinocerebellar Ataxias / congenital*,  genetics
Grant Support
Reg. No./Substance:
0/Heat-Shock Proteins; 0/RNA, Messenger; 0/SACS protein, human

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Journal Information
Journal ID (nlm-ta): Hum Mutat
Journal ID (iso-abbrev): Hum. Mutat
Journal ID (publisher-id): humu
ISSN: 1059-7794
ISSN: 1098-1004
Publisher: Blackwell Publishing Ltd
Article Information
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Copyright © 2013 Wiley Periodicals, Inc., A Wiley Company
Received Day: 28 Month: 5 Year: 2012
Accepted Day: 06 Month: 12 Year: 2012
Print publication date: Month: 3 Year: 2013
Electronic publication date: Day: 24 Month: 12 Year: 2012
Volume: 34 Issue: 3
First Page: 525 Last Page: 537
PubMed Id: 23280630
ID: 3629688
DOI: 10.1002/humu.22269

Comparative Analysis and Functional Mapping of SACS Mutations Reveal Novel Insights into Sacsin Repeated Architecture
Alessandro Romano1
Alessandra Tessa2
Amilcare Barca1
Fabiana Fattori3
Maria Fulvia de Leva4
Alessandra Terracciano3
Carlo Storelli1
Filippo Maria Santorelli2*
Tiziano Verri1*
1Department of Biological and Environmental Sciences and Technologies, University of SalentoLecce, Italy
2Neurogenetics and Molecular Medicine, IRCCS Fondazione Stella MarisPisa, Italy
3Neuromuscular Unit, IRCCS Bambino Gesù HospitalRome, Italy
4Department of Neurology, Federico II UniversityNaples, Italy
Correspondence: *Correspondence to: Filippo M. Santorelli, Molecular Medicine and Neurogenetics, Children Neuropsychiatry Institute, University of Pisa, IRCCS Fondazione Stella Maris, Viale del Tirreno 331, I-56018 Pisa, Italy. E-mail: or Tiziano Verri, Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Via Provinciale Lecce-Monteroni, I-73100 Lecce, Italy. E-mail:
Communicated by David N. Cooper
Additional Supporting Information may be found in the online version of this article.
Contract grant sponsors: University of Salento (fondi ex-60% 2009–2012); Italian Ministry of Health; Telethon Italy (GGP10121A).


Autosomal recessive spastic ataxia of Charlevoix–Saguenay (ARSACS; MIM #270550) is an early-onset neurological disease presenting a founder effect in the Quebec regions of Charlevoix and Saguenay–Lac-St-Jean where the estimated carrier frequency is 1/22 [Bouchard et al., 1978; 1998]. The major clinical features of ARSACS include early-onset ataxia, later occurrence of spastic paraparesis, and brisk tendon reflexes, and an axonal sensory-motor peripheral neuropathy, with some instances of mental retardation or cognitive decline. Brain magnetic resonance imaging shows a distinct, tigroid appearance of the pons [Van Damme et al., 2009] and invariably an atrophied cerebellar vermis. Hypermyelination of the retinal nerve fibers [Bouchard et al., 1978, 1998] has long been considered a cardinal feature in Quebecois French–Canadian patients, and is not so obvious in cases from elsewhere [Criscuolo et al., 2004; Hara et al., 2005] or even absent. Several aspects including early appearance of abnormal pontocerebellar and retinal fibers seen at brain neuroimaging speak for a neurodevelopmental anomaly in ARSACS [Gazulla et al., 2012]. However, the progressive clinical course with involvement of the corticospinal tract and peripheral nerves in patients as well as studies in model mice questioned this hypothesis and suggested also the occurrence of a neurodegenerative process [Girard et al., 2012; Prodi et al., 2012].

The gene responsible for ARSACS (SACS) [Engert et al., 2000] is located on chromosome 13q12 and encodes sacsin, a protein whose canonic variant is described as a polypeptide of 4,579 amino acids (GenBank acc. no. NP_055178.3). The enormous size of the SACS gene and translated protein has considerably hindered biochemical studies to date, and currently much more is known about the genetics of ARSACS than about the function of sacsin in cells. Over the years, the number of ARSACS patients harboring mutations in the SACS gene has rapidly increased. They are distributed worldwide and are not limited to few ethnicities, and virtually any type of mutations has been discovered [Anheim et al., 2008].

How mutant sacsin leads to neurodegeneration remains largely unknown. Earlier work had indicated that sacsin might be involved in chaperone-mediated protein-folding activity [Engert et al., 2000] and play a role in regulating the Hsp70 chaperone machinery [Parfitt et al., 2009]. Recent biological and comparative genomic evidence suggested that sacsin is organized in a repetitive supradomain structure of ∼360 amino acids, named sacsin repeating region (SRR) [Anderson et al., 2010], which in turn might drive its function. Biochemical characterization demonstrated that such repetitive supradomain possesses ATPase activity, which appears to be a requirement for sacsin function, as a disease causing mutation leads to an alternate conformation incapable of hydrolyzing ATP [Anderson et al., 2010]. As well, this structure has been shown to enhance the refolding efficiency of a client protein, maintain it in soluble folding-competent states, and cooperate with members of the Hsp70 chaperone family to increase the yield of correctly folded client [Anderson et al., 2011]. Even more recently, sacsin has been shown to operate as a dimer and bind GTP at its C-terminus [Kozlov et al., 2011], with mutations in this region also resulting in loss of function. In addition, sacsin has been indicated as a potential substrate of the ubiquitin ligase Ube3A protein, which is responsible for Angelman syndrome (MIM #105830), a neurodevelopmental disorder with a motor component that shares same clinical aspects with ARSACS [Greer et al., 2010]. Such observations onto the function(s) of sacsin mainly arise from preliminary analysis on single putative domains that have been recognized along the sacsin sequence and are presently considered hallmarks of its structure. Finally, the generation of a sacsin knockout mouse is opening intriguing perspectives in the exploration of the pathophysiological basis of ARSACS, having shown that sacsin localizes to mitochondria and participates in regulation of mitochondrial dynamics via its interaction with dynamin-related protein 1 [Girard et al., 2012].

In the present work, we aimed at expanding our knowledge on the structure of sacsin. Three very large (≥1,100 amino acids) repeated regions were detected along the sacsin amino-acid sequence, each characterized by the occurrence of at least three subrepeats. A fourth subrepeat occurred in the first and third repeated region only. Such organization in domains is common to sacsin in all vertebrates including mammals, birds, reptiles, and fish. The comparative analysis of vertebrate sacsins architecture in combination with the fine positional mapping of a large set of disease causing mutations in human SACS well supported the concept of the functional nature of these novel domains. Furthermore, the location of a small selection of genetic variants detected in ARSACS was put in relation with the phenotype adopting a Spastic Ataxia (SPAX) rating system of clinical severity. Scoring mutations suggested original structure–function paradigms for sacsin, with hints on the relative relevance of novel and known domains in the activity of the protein.

Materials and Methods
Human SACS Gene, mRNA, and Protein Sequences and SNPs

The reference sequences for human (Homo sapiens) SACS gene (GenBank acc. no. NC_000013.10), mRNA (GenBank acc. no. NM_014363.4), and protein (GenBank acc. no. NP_055178.3) were as reported in Entrez Gene at the National Center for Biotechnology Information (NCBI) ( The human SACS gene SNPs mapped in this study (missense and nonsense mutations only) were from dbSNP at NCBI ( and from literature [Engert et al., 2000; Guernsey et al., 2010; Vermeer et al., 2009]. Throughout the manuscript, we systematically used names for both DNA and protein variations whenever appropriate, and adopted a mutation numbering system based on cDNA sequence as suggested by the internationally agreed mutation nomenclature (

Pattern and Profile Searches

Putative domains were defined using the pattern and profile searches tools included in the ExPASy Proteomics Server (; in particular, the Simple Modular Architecture Research Tool 6 (SMART 6) ( [Letunic et al., 2009] and/or the ScanProsite tool ( [de Castro et al., 2006]. Internal repeats were detected by using the Prospero program, as included in SMART 6. Default parameters were always used for analyses and only domains above threshold were represented. SIM, an alignment tool for analysis of local similarity in nucleotide and amino-acid sequences ( [Huang and Miller, 1991], served to generate pairwise alignments of sacsin versus internal repeats using default parameters. The computed alignments were viewed using the graphical viewer program LALNVIEW ( [Duret et al., 1996]. Further details on the single computational tools and parameters used for analyses are reported in the legends to the figures as appropriate. Domains were drawn using the MyDomains image creator (

Protein Sequence Alignments and Phylogenesis

On the basis of the genomic analysis detailed in the Supporting Information, the deduced protein sequences of orangutan, dog, horse, mouse, rat, chicken, zebra finch, anole lizard, fugu, tetraodon, stickleback, medaka, and zebrafish were obtained and used for alignments. Pairwise alignments of human versus the other vertebrate sacsin proteins were obtained by using SIM, as detailed above. Multiple sequence alignment of vertebrate sacsin proteins was obtained by using ClustalW2 using default parameters ( [Larkin et al., 2007]. The phylogenetic reconstruction was generated by the neighbor-joining method [Saitou and Nei, 1987], as implemented in the Molecular Evolutionary Genetics Analysis 4 (MEGA4) software ( [Tamura et al., 2007].

Definition of a SPAX Scoring System (SPAX score) in ARSACS

Definition of a clinical score in ARSACS is lagging behind, although reliable and valid composite scores have been developed for the highly similar inherited ataxias [Trouillas et al., 1997] and the hereditary spastic paraplegias [Schüle et al., 2006]. To define a posteriori a measure of disease severity in ARSACS and to correlate scores with type and location of mutations in sacsin, we put together a measure of severity in SPAX score that takes into account the “core features” of ARSACS, including cerebellar ataxia, spastic paraplegia, and peripheral neuropathy. We are aware that SPAX scores are only an initial attempt to score disease severity, especially in the absence of functional tests, but the rating system has an intrinsic value in that it sums the gravity of the individual hallmarks of the disease through the use of validated scales. In particular, we used the parameters developed in the Scale for the Assessment and Rating of Ataxia [Schmitz-Hübsch et al., 2006] for cerebellar ataxia, the Spastic Paraplegia Rating Scale for motor symptoms and spasticity [Schüle et al., 2006], and the modified version of the Charcot–Marie–Tooth neuropathy score [Murphy et al., 2011] for peripheral neuropathy. In addition, cognitive impairment (0, absent to 3, if severe) and ocular findings (from 0, normal to 4, maximal abnormality) were assessed. When visual abnormalities were detected only at optical coherence tomography, a unit was subtracted from the subscore. The several items of the scales were reviewed by two independent investigators blind to the genotype, duplicated items removed, data on single items averaged, and then corrected for disease duration whenever possible (or for averaged disease duration in a family). A grade of functional severity in ARSACS varying from 0 to 2 (maximal severity) was then calculated.

Identification of Novel Domains in Human SACS

Along with the original description of human SACS [Engert et al., 2000], it was suggested that repeating regions, two of which containing the putative ATP-binding domain of Hsp90, might have occurred in the sacsin protein. At that time, human SACS was considered to consist of a single gigantic exon spanning 12,794 bp [Engert et al., 2000]. With the identification of nine (one noncoding and eight coding) additional exons upstream of this gigantic exon, the presence of conserved amino-acid sequences occurring in triplicate along the encoded protein started to be foreseen, and very recently the formal description of the SRR supradomain has been proposed [Anderson et al., 2010]. In this study, a systematic analysis of domains along the human sacsin amino-acid sequence was performed. In particular, besides the well-known ubiquitin-like (ubiquitin; PFAM acc. no. PF00240), DnaJ (DnaJ molecular chaperone homology domain; SMART acc. no. SM00271), and HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain; SMART acc. no. SM00748) domains (see Fig. 1B), two large Prospero repeats, corresponding to the 61–1,371 and 2,473–3,893 protein fragments of the human sacsin, were detected along the polypeptide chain (Fig. 1A). Interestingly, both repeats shared similarity with a third homologous region in between them (along the 1,372–2,472 protein fragment), as detected by SIM analysis of sacsin protein versus each Prospero repeat (Fig. 1A). Similar results were also obtained by using the HHRepID program [Biegert and Söding, 2008] (data not shown). We named these three large homologous repeating regions Sacsin Internal RePeaTs (namely SIRPT1, SIRPT2, and SIRPT3; see Fig. 1). In spite of their low overall similarity (e.g., 16%–18% in human sacsin, with SIRPT1 vs. SIRPT2: 17%, SIRPT1 vs. SIRPT3: 16%, and SIRPT2 vs. SIRPT3: 18%), each SIRPT displayed, based on the degree of local similarity, at least three subrepeats (Fig. 1B) that were distanced by regions of extremely low similarity. We named these: subrepeat 1 (sr1), 2 (sr2), and 3 (sr3) (for position of the subrepeats along the protein, see Fig. 1B). Noteworthy, each sr1 contained a well-recognizable HATPase_c (Histidine kinase-like ATPases; SMART acc. no. SM00387) domain, which is adopted by the ATP-binding and catalytic domain of (among others) the members of the vast GHKL class of proteins (so-called after the founding members of the class: DNA Gyrase, Hsp90, bacterial histidine Kinases and MutL) [Dutta and Inouye, 2000] (Fig. 1B). Also, within the SIRPT architecture, sr1s and sr2s virtually corresponded to Anderson et al. SRR supradomain [Anderson et al., 2010] (Fig. 1B). On the other side, sr3 revealed no obvious relationships to any of the so far acknowledged domains included in databases. Besides the sr1, sr2, and sr3 domains described above, another repeated region could be identified in SIRPT1 and SIRPT3 in the area of very limited similarity. In fact, a long repeated region in SIRPT1 shared similarity with a homologous region in SIRPT3. We named this region srX (Fig. 1B). The srX domain had no obvious counterpart in the significantly shorter SIRPT2 (see Fig. 1A and B). Also, srX had no obvious similarity to any of the so far acknowledged domains in databases. Interestingly, in SIRPT3, the amino-acid sequence between srX and sr3 corresponded to a sacsin region previously reported to share limited homology with the Xeroderma Pigmentosum complementation group C binding (XPCB) domain of hHR23A [Kamionka and Feigon, 2004] and recently implicated in interactions with the ubiquitin ligase Ube3A [Greer et al., 2010] (Fig. 1B).

Conservation of Sacsin Structural Organization among Vertebrates

Comparative analysis of homologous proteins across phylogenetically distant species represents a powerful method for detecting conserved structural elements in proteins. Comparison of human sequences with sequences of other mammals, avians, reptiles, and teleost fish is valuable; in particular, teleosts offer maximal stringency for sequence comparisons among vertebrates. On this conceptual basis, we compared amino-acid sequence of sacsins from human with fish, having verified that: (1) genes encoding sacsin proteins are found in all vertebrate genomes sequenced so far, (2) sacsin proteins may have similar functional role(s) in all vertebrates, as supported by the evidence of similar expression patterns in mammals [Engert et al., 2000; Parfitt et al., 2009] and fish (such as zebrafish; see Supp. Fig. S1 and Supp. Table S1). In particular, as a result of a comprehensive gene analysis among vertebrates, sacsin proteins were deduced from human and other 13 vertebrate species, namely five mammals (orangutan, mouse, rat, horse, and dog), two birds (chicken and zebra finch), one reptile (anole lizard), and five fish (zebrafish, tetraodon, fugu, stickleback, and medaka). Then, the protein sequences were compared (for details, see Supp. Fig. S2), and the phylogenetic relationships among them are summarized in Figure 2A. With respect to the human protein, the other mammalian sacsins exhibited an overall degree of similarity (amino-acid identity) that varied from ∼99% to ∼93%, whereas the bird sacsins revealed an overall similarity of ∼84% and the reptilian sacsin of 83% (for details, see Supp. Table S2). Fish proteins shared an overall degree of similarity with human sacsin that varied from ∼70% to ∼68% (Supp. Table S2). As expected, degrees of similarity locally varied along the protein sequence. The local degree of similarity is depicted in Figure 2B. In spite of these differences, all vertebrate sacsins conserved the same structural architecture as the human sacsin (see Supp. Fig. S3).

Comparative Analysis of Vertebrate Protein Architecture and Positional Mapping of Human SACS Mutations Reveal the Functional Nature of the Sacsin Repeated Domains

On the basis of our SIRPT-centered protein architecture and sequence similarity data, the following intra/intersequence alignment strategy was played out to identify and typify unique conserved elements in the repeated domains of the vertebrate sacsin proteins. Namely, the amino-acid sequences corresponding to SIRPT1-sr1, SIRPT2-sr1, and SIRPT3-sr1 from the human and other vertebrate sacsins were aligned against each other (see Supp. Fig. S4). The same procedure was applied to the sequences corresponding to SIRPT1-sr2, SIRPT2-sr2, and SIRPT3-sr2 (see Supp. Fig. S5), to SIRPT1-sr3, SIRPT2-sr3, and SIRPT3-sr3 (see Supp. Fig. S6), and to SIRPT1-srX and SIRPT3-srX (see Supp. Fig. S7) of the human and other vertebrate sacsins. Notably, in spite of the highly selective alignment procedure, a number of amino-acid residues still kept appearing conserved in the same position of mate repeated domains.

If the sacsin repeated domains are functional, the amino acids that are found in these conserved positions should then be considered critical for sacsin function. Accordingly, in such repeated and conserved positions, one should expect to find more missense mutations associated with disease (missense pathogenic) than missense mutations not associated with disease (missense nonpathogenic) and/or nonsense (protein truncating) pathogenic mutations [Miller and Kumar, 2001; Miller et al., 2003]. To test this hypothesis, we collected missense (pathogenic and nonpathogenic) and nonsense mutations that have been reported to occur in human sacsin. In particular, Table 1 represents the recent update (January 2012) of all the acknowledged missense and nonsense mutations that are clearly pathogenetic in ARSACS patients of different geographic origins (Supp. Appendix I lists pathogenic missense and nonsense mutations identified later than January 2012 and frameshift mutations not used in this study). Furthermore, Supp. Table S3 represents the list of all the missense mutations that have been described as SNPs in humans up to January 2012; for the most part, these mutations were recognized as undoubtedly nonpathogenic and were used for analysis (for details, see legend to Supp. Table S3) (Supp. Appendix II and Supp. Appendix III report a recent update of SNPs from dbSNP and NHLBI Exome Sequencing Project, respectively). Detailed positional information and distribution of the mutations in the various domains along the human protein are summarized in Supp. Figure S2 and Supp. Table S4. All the mutations falling in positions within the sr1, sr2, sr3, and srX domains have been represented in Supp. Figures S4–S7. The relative amounts of missense pathogenic mutations, on one hand, and missense nonpathogenic mutations, on the other—expressed as percent of conserved vs. non-conserved mutations—are reported in Figure 3. As expected, with respect to the group of missense nonpathogenic mutations, missense pathogenic mutations were invariably over-represented in conserved positions in sr1, sr2, sr3, and srX (for details, see Supp. Table S5), thus suggesting that the four repeated domains of the SIRPT regions identified in this work (that include, with sr1 and sr2, and go beyond, with sr3 and srX, the SRR design; see Fig. 1B) [Anderson et al., 2010] do play a functional role in the sacsin protein.

The functional nature of sr1, sr2, sr3, and srX is also sustained by the observation that in human sacsin pathogenic missense mutations were found to be over-represented in these domains with respect to the regions between domains (interdomains), as qualitatively assessed by calculating the likelihood of occurrence of missense pathogenic mutations, that is, the ratio of the percentage mutations on a given region and the percentage of amino acids of the protein on the same region (for details, see Table 2). In particular, the calculated likelihood was 1.97, 1.42, 0.79, and 0.51 for sr1, sr2, srX, and sr3 domains, respectively, with respect to 0.35 for the interdomains.

Functional Relevance of sr1, sr2, srX, and sr3 in Sacsin Protein Based on Composite SPAX Scores Analysis

To investigate on the putative functional relevance of the various repeated domains that result from the proposed new sacsin architecture, we analyzed the clinical phenotype in patients selected for having, in a given domain (i.e., sr1, sr2, srX, and sr3), a missense pathogenic mutation (1) in homozygosis or (2) in heterozygosis with a frameshift mutation, a stop mutation, or a macrodeletion (see Table 3). It is reasonable to think that in an autosomal recessive disorder, such as ARSACS, frameshift mutations, stop mutations, and macrodeletions can abolish sacsin function, although other mechanisms, such as dominant-negative effects, cannot totally be excluded until functional tests are performed. Under these conditions, we expect that differences in the clinical phenotypes observed in patients (1) are due to the effect(s) of the missense mutation on protein function and (2) provide (at least in part) information on the functional relevance of the protein domain where the missense mutation acts. In fact, although the nature of the substituted amino acid may contribute per se to the severity of the phenotype, it cannot be ignored that the effect of an amino-acid substitution depends on the protein domain where the substitution occurs. As a means to evaluate the pleomorphic clinical phenotype of ARSACS, we defined a composite SPAX score, which takes into account the major core features (cognitive, cerebellar, spasticity, peripheral nerve, and retinopathy) that are part of the disease. This scoring system is largely based on validated rating scales for spasticity, peripheral neuropathy, and cerebellar function, corrected for disease duration and used to evaluate the severity of the clinical phenotype (see Table 3).

As a way to define the maximal severity of the disease, in our analysis, we initially calculated SPAX scores from patients in which both alleles were predicted to generate truncated sacsins (due to presence on both alleles of either a frameshift mutation or a stop mutation or a macrodeletion; for a description of various combinations of alleles, please see Supp. Table S6). As it results from the analysis of Figure 4, these patients formed a homogeneous group that ranked at the highest SPAX scores among those calculated in this study (for comparison, see also Table 3), with values varying from 1.48 to 1.84. Conversely, when SPAX scores were calculated from patients carrying a pathogenic missense mutation in sr1, sr2, srX, or sr3 (in homozygosis or heterozygosis with a frameshift mutation, a stop mutation or a macrodeletion, as described above), it was evident that the severity of the clinical phenotype largely varied (see Fig. 4 and Table 3) from values similar to those observed in patients carrying (a) truncated protein(s), for example, 1.69 for the c.1420C>T (p.R474C)/c.5719C>T (p.R1907X), which suggests nearly complete abolition of protein function, to significantly lower values, for example, 0.69 for c.3932T>A (p.M1311K)/c.3932T>A (M1311K), which suggests subsistence of partial or residual protein activity. Overall, the presence of the missense pathogenic mutations in sr1, sr2, srX, or sr3 established on average a set of phenotypes (i.e., SPAX scores) significantly less severe (i.e., lower) than those observed for mutations that generated truncated proteins (ANOVA; P < 0.0001). We assumed that such a behavior correlated to the relevance that the domain in which the mutation falls had for sacsin activity. In particular, a trend to lower SPAX scores passing from sr1 and sr2 to srX and sr3 could be observed, suggesting (1) that alterations in srX and sr3 do cause less harmful, although measurable, effects on the function of the protein with respect to sr1 and sr2, and thus (2) that srX and sr3 play a “minority” role in the operational mechanism of the protein with respect to sr1 and sr2.


In this study, a systematic inspection of vertebrate sacsins has been carried out to identify repeated domains along the protein. By using a combination of standard databank consulting tools and bioinformatics methods, three large (≥1,100 amino acids) repeated regions have been identified. Such internal repeats, named SIRPT1, SIRPT2, and SIRPT3, cover ∼84% of the protein sequence, and each contains three subrepeats, named sr1, sr2, and sr3, with sr1 and sr2 falling into Anderson et al. SRR supradomain [Anderson et al., 2010]. In addition, a fourth subrepeat, named srX, occurs in the first and the third internal repeat only, in a region between sr2 and sr3. Our SIRPT-based architectural structure is invariably conserved in all vertebrate sacsins. This is not unexpected, as vertebrate sacsins share a high degree of similarity at both global and local level (this study), and most probably exert similar functional roles, a notion also supported by the observation that similar expression patterns can be found in both mammals [Engert et al., 2000; Parfitt et al., 2009] and fish (this study).

All the different subrepeats identified within the SIRPT architecture most likely represent regions involved in sacsin function. To answer this question, we have developed a strategy that combines very stringent alignments of the vertebrate sacsin domains with positional mapping of the human SACS mutations (for details, see Results). As a matter of fact, at least two pieces of evidence come out from our analyses indicating that the different subrepeats identified do represent functional regions. First, in sr1, sr2, srX, and sr3, missense pathogenic mutations are invariably over-represented in conserved positions with respect to missense nonpathogenic mutations. Second, missense pathogenic mutations are over-represented in sr1, sr2, sr3, and srX with respect to the regions between domains [Miller et al., 2003], this scheme being fully applicable also to the well-known DnaJ and HEPN domains. All together, these findings indicate that there is a strong tendency in the sacsin protein to gather the missense mutations associated with disease within the newly identified or the already known domains.

Sacsin is considered to operate in a chaperone-like manner, but very limited information is available on its activity, mainly due to the technical difficulties of managing with such an unusually long protein by means of standard biochemical, cellular, or molecular biology assays [Anderson et al., 2010; Kozlov et al., 2011; Parfitt et al., 2009). Under such circumstances, achieving information on the functional role(s) of our sr1, sr2, srX, and sr3 domains represents a difficult task. In the effort to obtain new hints on the impact of the newly identified domains in the activity of the protein, we have developed a procedure that allows evaluation of the functional relevance of the domains by measuring the severity of the clinical phenotype, quantified in terms of SPAX score, in patients selected for carrying missense pathogenic mutations in sr1, sr2, srX, and sr3 in homozygosity or heterozygosity with a null allele (for details, see Results). In spite of the limits of this experimental approach, essentially because of the so far limited number of patients composing each group, from our analysis it is evident that: (1) patients carrying missense pathogenic mutations in homozygosity or heterozygosity with a null allele exhibit significantly milder phenotypes, that is, lower SPAX scores, than patients carrying a null mutation on each allele (a condition that is predicted to fully abolish protein function; for details, see Results); (2) mean SPAX scores decrease passing from sr1 to sr3, with sr1 (1.06) = sr2 (1.10) > srX (0.83) > sr3 (0.71), which suggests that alterations in srX and sr3 are less damaging in patients than those in sr1 and sr2, and thus that srX and sr3 play a less determinant role in the operational mechanism of the protein with respect to sr1 and sr2. Nonetheless, we recognize that our data should be weighted cautiously and that additional determinants of severity might come out from future functional tests. In this context, it has to be underlined that our simplified approach cannot take into account in a simple way the yet possible contribution of the nature of the amino-acid substitution on the severity of the phenotype. Thus, we considered that the effect of an amino-acid substitution depends on the protein domain where the substitution falls and comes to operate rather independently of the nature of the mutation. That this may hold true comes from the observation that the same type of amino-acid change (see, e.g., R-to-C, that occurs thrice in sr1 and once in sr2) may result in either high (in sr2) or medium-to-low (in sr1) SPAX scores (for details, see Table 3).

Our results extend and refine the current knowledge on the organization of some sacsin domains. In particular, the sr1 and sr2 domains identified in this work substantially form the SRR supradomain recently defined by others [Anderson et al., 2010]. This supradomain is composed of an N-terminal portion (∼160 residues), which is homologous to the HATPase_c domain of Hsp90, and a C-terminal portion (∼200 residues), which consists of a novel sequence invariably connected to the HATPase_c domain [Anderson et al., 2010]. Our bioinformatics approach divides this SRR supradomain in two well-defined repeated domains, that is, sr1 and sr2, which are separated by an evident nonrepeated linker segment. This organization is coherent with a system that works as an Hsp90-like protein. In fact, in Hsp90-type chaperones, the ATP binding domain is connected to the middle domain via a divergent linker region. In particular, in our sacsin organization, sr1 represents the ATP binding domain and sr2 the middle domain. Notably, in Hsp90 the middle domain invariably contains an arginine residue accepting phosphate after ATP hydrolysis [Pearl and Prodromou, 2006]. This phosphoacceptor arginine, already observed by Anderson et al. (2010) as invariably conserved in each C-terminal region of their SRR supradomains, does occur in each sr2 domain. Interestingly, our study clearly demonstrates the crucial role of this arginine in the operational mechanism of sacsin. In fact, a mutation occurring on one of such conserved arginines, namely c.1420C>T (p.R474C) in SIRPT1-sr2, associates to one of the highest SPAX scores (1.69) found in this survey.

In this article, we report for the first time the occurrence of two novel repeated domains, namely srX and sr3, downstream the Hsp90-type regions discussed above. Such domains share no similarity to any domains reported so far in databanks, and no obvious role can be assigned to them. However, in the context of an Hsp90-like scheme of function, srX and/or sr3, located near the sr1/sr2 “biochemical clamp” that allows ATP binding and hydrolysis, may participate (via dimerization, client binding, cochaperone interaction, regulation, etc.) to sacsin chaperone activity. In this respect, there has been recent demonstration that a large sacsin region (RegA), virtually corresponding to our SIRPT1, do exhibit a chaperone-like activity that can be detected in vitro by standard biochemical approaches [Anderson et al., 2011]. Such protein module is composed of the Hsp90-like region and of a large undefined downstream region. However, our study identifies srX and sr3 as functional elements in that large undefined region and likely involved in the chaperone activity of the whole module.

In conclusion, we used a functional comparative genomics approach that combines bioinformatics sequence examination tools to mapping and phenotypical analysis of human mutations, to provide novel information on the organization in repeated domains of sacsin. In particular, our results establish that large portions of the protein can be arranged in a few and well-defined repeated domains. The demonstration of the functional nature of sr1, sr2, srX, and sr3 suggests that these regions contribute to the activity of the protein. Further studies are needed to define the specific role(s) of such domains, in the perspective of developing a comprehensive and integrated model of function for sacsin in the context of cell pathophysiology. In a larger perspective, our approach that combines comparative analysis of vertebrate protein sequences/architecture, positional mapping of human mutations, and severity of clinical phenotype can be tentatively applied in the biomedical field to shed light on the functional nature of other proteins associated to disease but of yet unknown function.

Disclosure statement: The authors declare no conflict of interest.

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Supplementary material

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

Click here for additional data file (humu0034-0525-SD2.txt)


[Figure ID: fig01]
Figure 1 

Identification of domains in human sacsin. A: (upper panel) Internal repeats above threshold were detected by Prospero. (Lower panel) Pairwise sequence alignments of human sacsin versus the first and the second Prospero repeat (corresponding to amino acids 61–1,371 and 2,473–3,893, respectively) were generated by SIM. The computed alignments were visualized by LALNVIEW. Percent identity is reported in the figure. Different colors indicate different degrees of similarity (amino-acid identity) along the aligned sequences (black: 100%; white: nothing detected). B:Sacsin Internal RePeaTs (SIRPTs) and relevant subrepeats 1 (sr1), 2 (sr2), 3 (sr3), and X (srX) within SIRPTs are indicated, spanning along the protein sequence as follows (amino-acid range in parentheses): SIRPT1 (amino acids 84–1,374), SIRPT2 (1,444–2,443), SIRPT3 (2,512–3,896), SIRPT1–sr1 (84–339), SIRPT1–sr2 (400–557), SIRPT1–sr3 (1,212–1,374), SIRPT1–srX (644–1,162), SIRPT2–sr1 (1,444–1,747), SIRPT2–sr2 (1,826–1,968), SIRPT2–sr3 (2,287–2,443), SIRPT3-sr1 (2,512–2,768), SIRPT3–sr2 (2,826–2,960), SIRPT3–sr3 (3,736–3,896), SIRPT3–srX (3,081–3,659). The sacsin repeating region (SRR) supradomains defined by Anderson et al. (2010, 2011) are indicated as SRR1 (amino acids 107–505), SRR2 (1,471–1,921) and SRR3 (2,539–2,922), with each supradomain composed of an sr1, an sr2, and an sr1sr2 connecting (linker) region. Please note that sr1 starts 23–27 amino acids upstream the C-terminus of the SRR domain (with SRR virtually starting with the HATPase_c domain) and sr2 ends 38–52 amino acids downstream the N-terminus of the SRR domain. Putative domains above threshold as detected by using SMART 6 and/or ScanProsite are also indicated: ubiquitin-like (ubiquitin; PFAM acc. no. PF00240), HATPase_c (histidine kinase-like ATPases; SMART acc. no. SM00387), DnaJ (DnaJ molecular chaperone homology domain; SMART acc. no. SM00271), HEPN (higher eukaryotes and prokaryotes nucleotide-binding domain; SMART acc. no. SM00748). For sake of clarity, the putative sacsin XPCB domain is also shown. Domains were drawn using the MyDomains image creator.

[Figure ID: fig02]
Figure 2 

Comparative analysis of vertebrate sacsin proteins. A: Unrooted phylogenetic tree depicting the evolutionary relationship of mammalian (orangutan, dog, horse, mouse, and rat), bird (chicken and zebra finch), reptilian (anole lizard), and fish (zebrafish, stickleback, medaka, fugu, and tetraodon) sacsin proteins. The unrooted tree was constructed using the neighbor-joining (NJ) method based on the alignment of the amino-acid sequences of the vertebrate sacsins. Bootstrap values (1,000 replicates) indicating the occurrence of nodes are reported above each branch in the figure. B: Schematic alignment of human versus the above listed vertebrate sacsin proteins. Pairwise sequence alignments and scores were generated using SIM. The computed alignments were visualized by LALNVIEW. Species are aligned according to their overall similarity (amino-acid identity) with respect to the human protein (from the highest to the lowest degree of overall similarity). Percent identity is reported in the figure. Different colors along the sequences are indicative of different degrees of similarity along the aligned sequences (black: 100%; white: nothing detected).

[Figure ID: fig03]
Figure 3 

Relative amount of conserved versus nonconserved missense mutations in SIRPT sr1, sr2, sr3, and srX domains. When mapped on our multiple alignments (see Supp. Figs. S4–S7), in each domain “conserved” (i.e., identical, conserved and semi-conserved, as assessed by ClustalW) pathogenic missense mutations were invariably over-represented with respect to missense nonpathogenic mutations (for details, see Supp. Table S5). Unclear mutations (i.e., variants not yet clearly associated with disease; for details, see Supp. Tables S3–S5) were omitted from the analysis.

[Figure ID: fig04]
Figure 4 

Composite SPAX (Spastic Ataxia) scores versus sacsin repeated domains. This scatter dot plot shows the assortment of SPAX scores from patients carrying a pathogenic missense mutation in sr1, sr2, srX, or sr3 in homozygosis or heterozygosis with a frameshift mutation, a stop mutation or a macrodeletion (for details, see Table 3). SPAX scores from patients in which both alleles were predicted to generate truncated proteins (for the presence on both alleles of either a frameshift mutation or a stop mutation or a macrodeletion) were also represented (for details, see Supp. Table S6). For comparison, SPAX scores from patients carrying a pathogenic missense mutation in DnaJ or HEPN in homozygosis or heterozygosis with a frameshift mutation, a stop mutation or a macrodeletion were also drawn (for details, see Table 3). Within each category, the horizontal line indicates the calculated mean value.

[TableWrap ID: tbl1] Table 1 

Human SACS Gene (Missense, Nonsense, and Frameshift Leading to Immediate Premature Protein Truncation only) Pathogenic Mutations Mapped in this Study

Mutation Amino-acid change

Origin Exon Original nucleotide position reporteda (see Reference, last column) Newly assigned nucleotide position in CDSb Original amino-acid position reportedc (see Reference, last column) Newly assigned amino-acid position in proteind Reference
Synthetic construct 3 sdm sdm R50A p.R50A Parfitt et al., 2009
Synthetic construct 3 sdm sdm R51A p.R51A Parfitt et al., 2009
Synthetic construct 4 sdm sdm L58A p.L58A Parfitt et al., 2009
Synthetic construct 4 sdm sdm D60A p.D60A Parfitt et al., 2009
The Netherlands 7 c.502G>T c.502G>T p.D168Y p.D168Y Vermeer et al. (2008, 2009)
Synthetic construct sdm sdm D168Y p.D168Y Anderson et al. (2010)
Belgium 7 c.602C>A c.602C>A p.T201K p.T201K Baets et al. (2010)
Italy 8 c.815G>A c.815G>A p.R272H p.R272H In house database
Maritime Canada (Acadian descent) 8 c.814C>T c.814C>T p.R272C p.R272C Guernsey et al. (2010)
Italy 8 c.826C>T c.826C>T p.R276C p.R276C Prodi et al. (2012)
Aragona-Spain/Croatia 8 c.832C>T c.832C>T p.Q278X p.Q278X Gazulla et al. (2012)
Japan 8 922C>T c.922C>T L308F p.L308F Takado et al. (2007)
The Netherlands 8 c.961C>T c.961C>T p.R321X p.R321X Vermeer et al. (2008, 2009)
Italy 8 c.1373C>T c.1373C>T p.T458I p.T458I In house database
Italy 8 c.1420C>T c.1420C>T p.R474C p.R474C In house database
The Netherlands 8 c.1475G>A c.1475G>A p.W492X p.W492X Vermeer et al. (2008, 2009)
France 8 c.1607C>T c.1607C>T p.P536L p.P536L Anheim et al. (2008)
Morocco 8 c.1667T>C c.1667T>C p.L556P p.L556Pe Baets et al. (2010)
Aragona-Spain 8 c.1894C>T c.1894C>T p.R632W p.R632W Gazulla et al. (2012)
The Netherlands 9 c.2182C>T c.2182C>T p.R728X p.R728X Vermeer et al. (2008, 2009)
UK 10 c.2224C>T c.2224C>T p.R742X p.R742X Terracciano et al. (2010)
Japan 10 g.2405T>C c.2405T>C L802P p.L802P Kamada et al. (2008)
Belgium 10 c.2971T>C c.2971T>C p.C991R p.C991R Baets et al. (2010)
Japan 10 987T>C c.3161T>C F304S p.F1054S Shimazaki et al. (2005)
Aragona-Spain 10 c.3198T>A c.3198T>A p.C1066X p.C1066X Gazulla et al. (2012)
Belgium 10 c.3491T>A c.3932T>A p.M1164K p.M1311K Ouyang et al. (2008)
Japan 10 3774C>T c.4033C>T Q1198X p.Q1345X Okawa et al. (2006)
Italy 10 1858C>T c.4108C>T Q620X p.Q1370X Grieco et al. (2004)
Turkey 10 g.2018T>C c.4182T>C C648R p.C1398R Richter et al. (2004)
Italy 10 c.4198T>A c.4198T>A p.Y1400N p.Y1400N In house database
Italy 10 c.4567T>C c.4567T>C p.W1523R p.W1523R In house database
Serbia 10 c.4724G>C c.4724G>C p.R1575P p.R1575P Baets et al. (2010)
Spain 10 c.4748C>G c.4748C>G p.P1583R p.P1583R In house database
Belgium 10 c.4760A>G c.4760A>G p.H1587R p.H1587R Baets et al. (2010)
Algeria 10 c.4934G>A c.4934G>A p.R1645Q p.R1645Q In house database
The Netherlands (Turkish descent) 10 c.4957G>T c.4957G>T p.E1653X p.E1653X Vermeer et al. (2008, 2009)
The Netherlands 10 c.5125C>T c.5125C>T p.Q1709X p.Q1709X Vermeer et al. (2008, 2009)
The Netherlands 10 c.5143A>T c.5143A>T p.K1715X p.K1715X Vermeer et al. (2008, 2009)
Italy 10 c.5629C>T c.5629C>T p.R1877X p.R1877X Anesi et al. (2011)
Italy 10 c.5639C>T c.5639C>T p.T1880I p.T1880I In house database
Algeria 10 c.5719C>T c.5719C>T p.R1907X p.R1907X Prodi et al. (2012)
Tunisia 10 3662T>C c.5836T>C W1196R p.W1946R El Euch-Fayache et al. (2003)
Japan 10 6355C>T c.6355C>T R2119X p.R2119X Hara et al. (2007)
Algeria 10 c.6409C>T c.6409C>T p.Q2137X p.Q2137X H'mida'Ben Brahim et al. (2011)
Italy 10 c.6680T>C c.7121T>C p.L2374S p.L2374S Terracciano et al. (2009)
Belgium 10 c.7276C>T c.7276C>T p.R2426X p.R2426X Baets et al. (2010)
Quebec 10 g.5254C>T c.7504C>T R1752X p.R2502X Engert et al. (2000)
France 10 c.7673C>T c.7673C>T p.A2558V p.A2558V Anheim et al. (2008)
Spain 10 7848C>T c.8107C>T R2556C p.R2703C Criscuolo et al. (2005)
France 10 c.8289_8291delTTC c.8289_8291delTTC p.Y2763X p.Y2763X In house database
Morocco 10 c.8393C>A c.8393C>A p.P2798Q p.P2798Qe Baets et al. (2010)
Aragona-Spain/Italy 10 c.8677A>T c.8677A>T p.R2893X p.R2893X In house database
Aragona-Spain 10 c.9670C>T c.9670C>T p.R3224X p.R3224X Gazulla et al. (2012)
Japan 10 7492T>C c.9742T>C W2498R p.W3248R Ogawa et al. (2004)
Tunisia 10 c.10290C>G c.10290C>G p.Y3430X p.Y3430X H'mida'Ben Brahim et al. (2011)
The Netherlands (English descent) 10 c.10442T>C c.10442T>C p.L3481P p.L3481P Vermeer et al. (2008, 2009)
The Netherlands 10 c.10906C>T c.10906C>T p.R3636X p.R3636X Vermeer et al. (2008, 2009)
Belgium 10 c.10907G>A c.10907G>A p.R3636Q p.R3636Qf Baets et al. (2010)
Belgium 10 c.10934T>C c.10934T>C p.L3645P p.L3645P Baets et al. (2010)
Belgium 10 c.10954C>A c.10954C>A p.P3652T p.P3652Tf Baets et al. (2010)
Belgium 10 c.10517T>C c.10958T>C p.F3506S p.F3653S Breckpot et al. (2008)
Italy 10 c.10743C>T c.11185C>T p.Q3582X p.Q3729X Kamionka and Feigon (2004), Masciullo et al. (2008)
Tunisia 10 c.11374C>T c.11374C>T p.R3792X p.R3792X Bouhlal et al. (2008)
Maritime Canada (Acadian descent) 10 c.11707C>T c.11707C>T p.R3903X p.R3903X Guernsey et al. (2010)
The Netherlands 10 c.12160C>T c.12160C>T p.Q4054X p.Q4054X Vermeer et al. (2008, 2009)
Tunisia 10 c.10046G>C c.12220G>C p.A3324P p.A4074P El Euch-Fayache et al. (2003), H'mida-Ben Brahim et al. (2011)
Italy 10 c.12428_12429insA c.12428_12429insA p.Y4143X p.Y4143X Prodi et al. (2012)
Japan 10 10723C>T c.12973C>T R3575X p.R4325X Takiyama (2006), Yamamoto et al. (2006)
The Netherlands 10 c.12992G>A c.12992G>A p.R4331Q p.R4331Q Vermeer et al. (2008, 2009)
Synthetic construct sdm sdm R4331Q p.R4331Q Parfitt et al., 2009
Italy c.12991C>T c.12991C>T p.R4331W p.R4331W Prodi et al. (2012)
Synthetic construct 10 sdm sdm H4337Q p.H4337Q Parfitt et al., 2009
Belgium 10 c.13027G>A c.13027G>A p.E4343K p.E4343K Baets et al. (2010)
Italy 10 c.13132C>T c.13132C>T p.R4378X p.R4378X Anesi et al. (2011)
UK 10 c.13237C>T c.13237C>T p.Q4413X p.Q4413X Terracciano et al. (2010)
Aragona-Spain 10 c.13405G>C c.13405G>C p.A4469P p.A4469P Gazulla et al. (2012)
Belgium 10 c.13523A>C c.13523A>C p.K4508T p.K4508T Baets et al. (2010)
Turkey 10 g.11471A>G c.13645A>G N3799D p.N4549D Richter et al. (2004)
Synthetic construct sdm sdm N4549D p.N4549D Kozlov et al. (2011)

CDS, coding sequence; sdm, site-directed mutagenesis.

This table represents the recent update (January 2012) of the acknowledged (41 missense, 28 nonsense, and two frameshift leading to premature protein truncation) mutations that associate to pathogenicity in ARSACS patients of different geographic origins. A total of 10 variants (eight missense, one nonsense, and one frameshift leading to premature protein truncation) are new and as yet unpublished, and were identified upon a large collaborative clinical-genetic work performed in the laboratory of one of us (Filippo M. Santorelli, in house database) on behalf of SPATAX, the Euro-Mediterranean clinical network on inherited ataxias and spastic paraplegias. Detailed information on the associated clinical and paraclinical features in patients as well as on mutation analyses will be presented elsewhere. This table also lists eight missense mutations artificially generated by molecular biology approaches in the ubiquitin-like domain (four variants) [Parfitt et al., 2009], HATPase_c domain (one variant) [Anderson et al., 2010], DnaJ domain (two variants) [Parfitt et al., 2009], and HEPN domain (one variant) [Kozlov et al., 2011] that helped defining the functional nature of such domains.

aNucleotide positions and changes are indicated as reported in the original article and refer to different NCBI Reference Sequences (see Reference, last column). DNA mutation numbering system in use, based on cDNA sequence (with a “c.” symbol before the number) (, can be found for more recent mutations only. For elder mutations, numbering system based on genomic sequences can be found (with a “g.” symbol before the number).

bOn the basis of the following NCBI Reference Sequence: GenBank acc. no. NM_014363.4. DNA mutation numbering system in use is based on cDNA sequence (with a “c.” symbol before the number). Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, with the initiation codon being codon 1 (

cAmino-acid positions and changes are indicated as reported in the original article and refer to different NCBI Reference Sequences (see Reference, last column). Amino-acid change numbering system in use, based on protein sequence (with a “p.” symbol before the letter) (, can be found for more recent variants only.

dOn the basis of the following NCBI Reference Sequence: GenBank acc. n. NP_055178.3. Amino-acid change numbering system in use is based on protein sequence (with a “p.” symbol before the letter) (

eDouble mutant alleles.

fDouble mutant alleles.

gNew variants not mapped in this study are reported in Supp. Appendix I.

[TableWrap ID: tbl2] Table 2 

Percentage of the Amino Acids in a Given Region (% protein), Percentage of the Mutations in the Same Region (% mutation) and Likelihood of a Mutation Occurring in the Region (% Mutation/% Protein), Calculated as the Ratio of the Percentage Mutations on a Given Region and the Percentage of Amino Acids of the Protein on the Region

Region Whole sacsin Ubiquitin-like sr1 sr2 sr3 srX XPCB DnaJ HEPN Interdomains
Protein (fragment) length (aa) 4,579 72 817 436 481 1098 76 60 117 1,422
% Protein 100 1.57 17.84 9.52 10.51 23.98 1.66 1.31 2.56 31.05
Missense mutations 37 0 13 5 2 7 0 3 3 4
% Mutation 100 0 35.13 13.51 5.41 18.92 0 8.11 8.11 10.81
Likelihood (% mutation/% protein) 1.00 0.00 1.97 1.42 0.51 0.79 0.00 6.19 3.17 0.35

aa, amino acids.

[TableWrap ID: tbl3] Table 3 

Composite SPAX Score Assigned to Selected ARSACS Patients

SACS mutation Severity of clinical phenotype

Allele 1b Allele 2b Referencea Sacsin internal repeat (SIRPT) subregion (sr) or interdomain where the mutation is located Duration (yrs) Onset Cognitive Cerebellar Spasticity Peripheral neuropathy Retinal Composite SPAX score = total score corrected for years disease duration/100
c.4182T>C (p.C1398R) c.4182T>C (p.C1398R) Richter et al. (2004) Interdomain between SIRPT1–sr3 and SIRP2–sr1 16 3 1 1 1 1 1 0.88
c.4198T>A (p.Y1400N) c.5719C>T (p.R1907X) In house database Interdomain between SIRPT1–sr3 and SIRP2–sr1 11 2 0 1 2 2 1 0.55
c.4567T>C (p.W1523R) c.11303insG (p.T3768fsX1) In house database SIRPT1–sr1 18 3 1 2 2 2 0 0.93
c.502G>T (p.D168Y) c.502G>T (p.D168Y) Vermeer et al. (2008) SIRPT1–sr1 33 3 0 2 2 0 0 1.16
c.602C>A (p.T201K) c.7276C>T (p.R2426X) Baets et al. (2010) SIRPT1–sr1 26 3 0 2 2 2 0 0.77
c.4748C>G (p.P1583R) c.8677A>T (p.R2893X) In house database SIRPT1–sr1 15 3 1 3 2 1 1 1.24
c.4760A>G (p.H1587R) c.3421_3422insAC (p.L1141fsX9) Baets et al. (2010) SIRPT1–sr1 15 1 1 3 1 3 3 1.34
c.815G>A (p.R272H) c.815G>A (p.R272H) In house database SIRPT1–sr1 34 3 2 2 3 3 0 1.30
c.814C>T (p.R272C) c.814C>T (p.R272C) Guernsey et al. (2010) SIRPT1–sr1 24 3 1 2 2 3 0 1.10
c.4934G>A (p.R1645Q) c.2224C>T (p.R472X) In house database SIRPT1–sr1 21 3 2 3 3 2 2 1.20
c.826C>T (p.R276C) c.826C>T (p.R276C) Prodi et al. (2012) SIRPT1–sr1 28 2 2 3 3 3 0 0.78
c.8107C>T (p.R2703C) c.8107C>T (p.R2703C) Criscuolo et al. (2005) SIRPT2–sr1 29 1 0 3 3 3 0 0.80
c.922C>T (p.L308F) c.922C>T (p.L308F) Takado et al. (2007) SIRPT1–sr1 32 2 0 2 3 3 0 1.07
c.1373C>T (p.T458I) Δ (1.5 Mb macrodeletion) In house database SIRPT1–sr2 24 2 0 3 1 1 3 0.70
c.5639C>T (p.T1880I) c.8289_8291delTTC (p.Y2763X) In house database SIRPT2–sr2 24 2 0 3 2 1 2 0.70
c.1420C>T (p.R474C) c.5719C>T (p.R1907X) In house database SIRPT1–sr2 13 3 1 3 3 2 1 1.69
c.5836T>C (p.W1946R) c.5836T>C (p.W1946R) El Euch-Fayache et al. (2003) SIRPT2–sr2 34 2 0 3 3 1 1 1.30
c.1894C>T (p.R632W) c.12973C>T (p.R4325X) Gazulla et al. (2012) Interdomain between SIRPT1–sr2 and SIRPT1–srX 17 2 1 1 2 1 0 0.67
c.9742T>C (p.W3248R) c.9742T>C (p.W3248R) Ogawa et al. (2004) SIRPT3–srX 22 2 2 1 2 1 3 0.84
c.2405T>C (p.L802P) c.482delA (p.N161fsX14) Kamada et al. (2008) SIRPT1–srX 17 1 2 3 1 2 0 0.88
c.10442T>C (p.L3481P) c.9910insT (p.L3304fsX14) Vermeer et al. (2008) SIRPT3–srX 40 2 0 1 2 1 0 0.76
c.3161T>C (p.F1054S) c.3161T>C (p.F1054S) Shimazaki et al. (2005) SIRPT1–srX 36 3 0 3 0 2 0 0.81
c.10934T>C (p.L3645P) c.7374delT (p.L2458fsX16) Baets et al. (2010) SIRPT1–srX 26 1 0 3 3 3 0 0.83
c.10958T>C (p.F3653S) Δ (1.5 Mb macrodeletion) Breckpot et al. (2008) SIRPT3–srX 15 2 1 2 2 1 0 0.87
c.7121T>C (p.L2374S) Δ (1.5 Mb macrodeletion) Terracciano et al. (2009) SIRPT2–sr3 26 1 1 2 2 1 0 0.73
c.3932T>A (p.M1311K) c.3932T>A (p.M1311K) Ouyang et al. (2008) SIRPT2–sr3 17 2 1 1 2 1 0 0.69
c.12220G>C (p.A4074P) c.12220G>C (p.A4074) El Euch-Fayache et al. (2003), H'mida-Ben Brahim et al. (2011) Interdomain between SIRPT3–sr3 and DnaJ 39 3 0 3 2 3 0 0.46
c.12992G>A (p.R4331Q) c.5143A>T (p.K1715X) Vermeer et al. (2008) DnaJ 37 2 0 3 2 1 0 1.36
c.12991C>T (p.R4331W) c.5719C>T (p.R1907X) Prodi et al. (2012) DnaJ 13 3 2 2 3 3 1 1.72
c.13405G>C (p.A4469P) Δ (1.5 Mb macrodeletion) Gazulla et al. (2012) HEPN 18 2 1 3 3 1 0 0.94
c.13645A>G (p.N4549D) c.13645A>G (p.N4549D) Richter et al. (2004) HEPN 11 3 0 3 3 2 1 1.77

del, base deletion (microdeletion); Δ, deletion (macrodeletion); fs, frameshift; ins, insertion; yrs, years.

aSee also Table 1.

bNumbering based on the following NCBI Reference Sequences: GenBank acc. no. NM_014363.4, for nucleotide, and GenBank acc. n. NP_055178.3, for protein. DNA mutation numbering system in use is based on cDNA sequence (with a “c.” symbol before the number). Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, with the initiation codon being codon 1. Amino-acid change numbering system in use is based on protein sequence (with a “p.” symbol before the letter) ( Original nucleotide and amino-acid positions and changes can be found in the original articles (see Reference).(Continued)

[TableWrap ID: tbl4]

Table 3. Continued Individual Items to Score Disease Severity

Score Onset Cognitive Cerebellara Spasticityb Peripheral neuropathyc Retinal
0 Adult Absent Absent Absent Absent Absent
1 Juvenile Mild decline Mild Mild Mild No functional impairment but aware of worsened acuities
2 Teen IQ lower than peers Moderate Moderate Moderate Reduced night vision
3 Early-onset Marked mental retardation Severe Severe Severe Abnormal fundoscopy or ERG

IQ, intelligence quotient; ERG, electroretinogram.

Note: Total scoring is corrected for time of disease (yrs) under the assumption that disease severity worsen with disease duration, and it is expressed as percent.

aOn the basis of SARA: Scale for the Assessment and Rating of Ataxia and IACRS (Inherited Ataxia Clinical Rating Scale).

bOn the basis of SPRS: Spastic Paraplegia Rating Scale.

cOn the basis of CMT (Charcot–Marie–Tooth) neuropathy score (second version).

Article Categories:
  • Research Articles

Keywords: autosomal recessive spastic ataxia of Charlevoix–Saguenay (ARSACS), comparative protein analysis, functional mapping of human mutations, neurodegeneration, protein domain architecture, repeated domains, SACS, sacsin.

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