Alternative titles; symbols
HGNC Approved Gene Symbol: CSTB
SNOMEDCT: 230423006;
Cytogenetic location: 21q22.3 Genomic coordinates (GRCh38): 21:43,773,950-43,776,308 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 21q22.3 | Epilepsy, progressive myoclonic 1A (Unverricht and Lundborg) | 254800 | Autosomal recessive | 3 |
Stefin B (also called cystatin B) is a small protein that is a member of the superfamily of cysteine protease inhibitors (Jarvinen and Rinne, 1982; Turk and Bode, 1991). It has been isolated from human spleen and liver and its amino acid sequence has been fully determined. It is widely distributed and is localized mostly intracellularly, but has been found extracellularly. Its role is thought to be as a protector against the proteinases leaking from lysosomes.
In the course of positional cloning of the gene responsible for progressive myoclonus epilepsy (EPM1; 254800) that had been mapped to chromosome 21 in a segment of about 175 kb between D21S2040 and D21S1259, Pennacchio et al. (1996) found a cDNA that encoded cystatin B, which was previously known but had not been mapped to a specific chromosomal site. They confirmed previous reports that the gene encoding cystatin B is widely expressed by demonstrating that a probe made from the cDNA clone detected an mRNA approximately 0.8 kb in length in all tissues examined. On Northern blots, lymphoblastoid cells from affected individuals from 4 unrelated families showed reduced levels of cystatin B mRNA compared to those from unaffected, noncarrier individuals and the carrier parents of EPM1 patients.
Jerala et al. (1988) synthesized a gene coding for human stefin B by the solid-phase phosphite method and cloned it in the pUC8 cloning vector. Protein prepared in expression vectors was inhibitory to papain and reacted with antibodies against human stefin B. Pennacchio et al. (1996) stated that cystatin B is a tightly binding reversible inhibitor of cathepsins L (116880), H (116820), and B (116810).
Pennacchio et al. (1996) sequenced the STFB gene from affected and unaffected individuals whose families had EPM1. Sequencing revealed that the STFB gene is 2,500 bp in length and contains 3 small exons encoding the 98-amino acid protein, whose mature mRNA and amino acid sequence were previously known.
Pennacchio and Myers (1996) isolated and characterized the mouse homolog of cystatin B. The gene spans 3 kb of genomic DNA and contains 3 exons as in human and rat. The gene encodes a predicted 98-amino acid polypeptide identical in length to the rat, bovine, and human genes and bearing 86%, 71%, and 79% similarity, respectively, to the genes of the other species. By Northern analysis, they showed that mouse cystatin B is expressed in many tissues.
Using a yeast 2-hybrid system, Di Giaimo et al. (2002) identified 5 recombinant proteins interacting with cystatin B, none of which was a protease. Three of these proteins, RACK1 (176981), beta-spectrin (see 182790), and NFL (162280), coimmunoprecipitated with cystatin B in rat cerebellum. Confocal immunofluorescence analysis showed that the same proteins were present in the granule cells of developing cerebellum, as well as in Purkinje cells of adult rat cerebellum. The authors proposed that a cystatin B multiprotein complex might have a specific cerebellar function, and that the loss of this function might contribute to the etiopathogenesis of EPM1.
Using a monoclonal CSTB antibody and organelle-specific markers in human primary myoblasts, Alakurtti et al. (2005) showed that endogenous CSTB localizes not only to the nucleus and cytoplasm but also associates with lysosomes. Upon differentiation to myotubes, CSTB becomes excluded from the nucleus and lysosomes, suggesting that the subcellular distribution of CSTB is dependent on the differentiation status of the cell.
Pennacchio and Myers (1996) mapped the mouse cystatin B gene by interspecific backcross analysis to mouse chromosome 10, extending knowledge of the homology of synteny between this region of mouse chromosome 10 and human chromosome 21q22.3.
Lalioti et al. (1997) identified 6 nucleotide changes in the CSTB gene in non-Finnish families with myoclonic epilepsy of Unverricht and Lundborg (EPM1; 254800) from northern Africa and Europe. One of these, a homozygous G-to-C transversion at nucleotide 426 in exon 1, resulted in a gly4-to-arg substitution (601145.0004) and was the first missense mutation described in association with EPM1. Molecular modeling predicted that this substitution would severely affect the contact of cystatin B with papain. The 6 mutations were found in 7 of the 29 unrelated EPM1 patients analyzed, in homozygosity in 1, and in heterozygosity in the others. They also found a tandem repeat of a dodecamer (CCCCGCCCCGCG) in the 5-prime untranslated region as a polymorphism (601145.0003). They identified 2 allelic variants with 2 or 3 tandem copies. The frequency of the 3-copy allele was 66% in the normal Caucasian population.
Lafreniere et al. (1997) presented haplotype and mutation analyses of 20 unrelated EPM1 patients and families from different ethnic groups. They identified 4 different mutations, the most common of which consisted of an unstable insertion of approximately 600 to 900 bp that was resistant to PCR amplification. This insertion mapped to a 12-bp polymorphic tandem repeat located in the 5-prime flanking region of the STFB gene in the region of the promoter (601145.0003). The size of the insertion varied between different EPM1 chromosomes sharing a common haplotype and a common origin, suggesting some level of meiotic instability over the course of many generations. Lafreniere et al. (1997) speculated that this dynamic mutation, which appeared distinct from conventional trinucleotide repeat expansions, may arise via a novel mechanism related to the instability of tandemly repeated sequences. Independently, Virtaneva et al. (1997) identified unstable minisatellite expansions in the promoter region of the cystatin B gene (symbolized CST6 by them). They stated that the mutation accounts for the majority of EPM1 patients worldwide. Haplotype data were compatible with a single ancestral founder mutation. The length of the repeat array differed between chromosomes and families, but changes in repeat numbers seemed to be comparatively rare events. Virtaneva et al. (1997) noted that unstable trinucleotide microsatellite repeat expansions are associated with at least 10 inherited neurologic disorders and are associated in all cases, except for Friedreich ataxia (229300), with strong anticipation. Unlike unstable trinucleotide repeats, which show a high degree of intergenerational instability, the EPM1-associated minisatellite mutation did not appear to be as unstable and anticipation has not been recognized in EPM1.
Antonarakis (1997) confirmed that the only EPM1-related point mutation in the cystatin B gene found in homozygous state was the gly-to-arg amino acid substitution (601145.0004). All other point mutations identified in EPM1 patients were found as compound heterozygotes with the 12-bp repeat expansion allele. The repeat expansion allele was also homozygous in some patients. Antonarakis (1997) found no patients with null point mutations (e.g., nonsense, frameshift, or splice site) in homozygous state; all EPM1 patients had residual gene activity. He proposed that homozygosity for null alleles was either nonviable or presented a different phenotype.
In an elaboration on their previous work, Lalioti et al. (1997) stated that the common mutation mechanism in EPM1 is the expansion of the dodecamer repeat (601145.0003), and considered this mutation to be the most likely source of the Finnish disorder described in 254800. An examination of 58 EPM1 alleles revealed that 50 of these contained the dodecamer repeat expansion. In addition to the expanded repeat mutation and the 2 or 3 repeats found in alleles considered to be normal, Lalioti et al. (1997) identified alleles with 12 to 17 repeats, which they termed 'premutational,' that were transmitted unstably to offspring. These 'premutational' alleles were not connected with a clinical phenotype of EPM1. Lalioti et al. (1997) stated that no correlation between number of repeat expansions and age of onset or severity had been found.
Most EPM1 alleles contain large expansions of the dodecamer repeat located upstream of the 5-prime transcription start site of the CSTB gene; normal alleles contain 2 or 3 copies of this repeat. All EPM1 alleles with an expansion were resistant to standard PCR amplification. To determine the size of the repeat in affected individuals, Lalioti et al. (1998) developed a detection protocol involving PCR amplification and subsequent hybridization with an oligonucleotide containing the repeat. In EPM1 patients, the largest detected expansion was approximately 75 copies; the smallest was approximately 30 copies. They identified affected sibs with repeat expansions of different sizes on the same haplotype, which confirmed the repeats' instability during transmissions. Expansions were observed directly; contractions were deduced by comparison of allele sizes within a family. In a sample of 28 patients, they found no correlation between age at onset of EPM1 and the size of the expanded dodecamer. This suggested that once the dodecamer repeat expands beyond a critical threshold, CSTB expression is reduced in certain cells, with pathologic consequences.
Haplotype analysis in a previous study suggested that 3 of 4 independent EPM1 mutations were present in 4 families. By sequence comparison, Pennacchio et al. (1996) identified 2 different mutations in the cystatin B gene. One was a G-to-C transversion at the last nucleotide of intron 1 (601145.0001), altering the sequence of the 3-prime splice site AG dinucleotide that is in this position in almost all introns. The second mutation, which was found in alleles of the cystatin B gene from 2 of the 4 families, changed CGA to TGA, generating a translation stop codon at amino acid position 68 (601145.0002). Despite identifying these 2 mutations in affected chromosomes from 3 of the 4 families, Pennacchio et al. (1996) were unable to detect any sequence difference in the gene encoding cystatin B from the remaining 1 or 2 alleles they had available for study. Specifically, the Finnish ancestral mutation was not identified. However, studies indicated that the expression of the Finnish gene, as well as that of all the other mutant alleles, was defective. Pennacchio et al. (1996) stated that, despite ubiquitous expression of this protein, it is not understood why mutation of the gene encoding cystatin B causes the symptoms of EPM1, an apparent tissue-specific phenotype.
Alakurtti et al. (2005) transiently expressed 4 mutations altering the CSTB polypeptide in BHK-21 cells. The 2400_2402delTC (601145.0005)-truncated mutant protein showed diffuse cytoplasmic and nuclear distribution, whereas R68X (601145.0002) was rapidly degraded. Two missense mutations, G4R (601145.0004), affecting the highly conserved glycine that is critical for cathepsin binding, and Q71P (601145.0006), failed to associate with lysosomes. Alakurtti et al. (2005) concluded that CSTB has an important lysosome-associated physiologic function and suggested that loss of this association contributes to the molecular pathogenesis of EPM1.
Pennacchio et al. (1998) found that mice lacking cystatin B as a result of targeted disruption of the gene develop myoclonic seizures and ataxia similar to the symptoms shown in EPM1. The principal cytopathology appeared to be loss of cerebellar granule cells, which frequently display condensed nuclei, fragmented DNA, and other cellular changes characteristic of apoptosis. This mouse model of EPM1 was thought to provide evidence that cystatin B, as a noncaspase cysteine protease inhibitor, has a role in preventing cerebellar apoptosis.
Lieuallen et al. (2001) identified 7 genes with consistently increased transcript levels in neurologic tissues from Cstb-deficient knockout mice: cathepsin S (116845), C1q B-chain of complement (120570), beta-2-microglobulin (109700), glial fibrillary acidic protein (137780), apolipoprotein D (107740), fibronectin-1 (135600), and metallothionein II (156360). These proteins are expected to be involved in increased proteolysis, apoptosis, and glial activation. The molecular changes in Cstb-deficient mice were consistent with the pathology found in the mouse model.
By complete sequencing of the cystatin B gene in affected members of 4 families with myoclonic epilepsy of Unverricht and Lundborg (EPM1A; 254800), Pennacchio et al. (1996) identified 2 different mutations in the cystatin B gene. One of these found in an American family was a G-to-C transversion at the last nucleotide of intron 1, altering the 3-prime splice site AG to AC.
In 2 of 4 families studied with progressive myoclonic epilepsy of Unverricht and Lundborg (EPM1A; 254800), Pennacchio et al. (1996) found that affected individuals carried a CGA- (arg) to-TGA (stop) mutation in the cystatin B gene.
Alakurtti et al. (2005) transiently expressed the R68X mutation in BHK-21 cells. The altered CSTB polypeptide was rapidly degraded.
In non-Finnish families from northern Africa and Europe with progressive myoclonic epilepsy of Unverricht and Lundborg (EPM1A; 254800), Lalioti et al. (1997) identified a tandem repeat of a dodecamer (CCCCGCCCCGCG) in the 5-prime untranslated region of the cystatin B gene. They identified 2 allelic variants with 2 or 3 tandem copies. The frequency of the 3-copy allele was 66% in the normal Caucasian population.
In cases of EPM1A, Lafreniere et al. (1997) identified homozygosity for an unstable insertion of approximately 600 to 900 bp that was resistant to PCR amplification. This insertion mapped to a 12-bp polymorphic tandem repeat located in the 5-prime flanking region of the STFB gene. The insertion mutation was identified in 38 unrelated EPM1 chromosomes of various ethnic origins. The size of the insertion varied between different EPM1 chromosomes sharing a common haplotype and a common origin, suggesting some level of meiotic instability. The most striking attributes of the mutation in the 5-prime flanking region were its unstable nature (size variation) and its resistance to PCR amplification. Both of these attributes have been documented in fragile X syndrome (300624), which represents an expansion of up to 4 kb of a polymorphic CGG trinucleotide repeat in the 5-prime untranslated region of the FMR1 gene (309550).
Independently, Virtaneva et al. (1997) reported unstable 15- to 18-mer minisatellite repeat expansions within the cystatin B promoter region in EPM1 patients. The repeat units were flanked by the 12-mer tandem repeat and were similar to the 12-mer in sequence. Haplotype data were compatible with a single ancestral founder mutation. The length of the repeat array differed between chromosomes and families, but changes in repeat numbers seemed to be comparatively rare events. They stated that the mutation accounts for a majority of EPM1 cases worldwide. Lalioti et al. (1997) presented further evidence that the common mutation mechanism in EPM1 is expansion of the dodecamer repeat, not the expansion of de novo 15- or 18-mer minisatellites, as had been suggested by Virtaneva et al. (1997).
Mazarib et al. (2001) studied a 5-generation Arab EPM1 family lacking photosensitivity, i.e., myoclonic jerks were not precipitated by photic stimulation. Three living affected individuals were homozygous for repeat expansions and 11 of the 16 unaffected family members were heterozygous. Instability was demonstrated by the presence of expansions of different sizes occurring on the same haplotype background in this inbred family. The lack of photosensitivity in this family was unexplained.
Lalioti et al. (1997) identified a homozygous G-to-C transversion at nucleotide 426 in exon 1 of the cystatin B gene in non-Finnish families from northern Africa and Europe with progressive myoclonic epilepsy of Unverricht and Lundborg (EPM1A; 254800). The mutation resulted in a gly4-to-arg substitution and was the first missense mutation described in association with EPM1. Molecular modeling predicted that this substitution severely affected the contact of cystatin B with papain.
Alakurtti et al. (2005) transiently expressed the G4R mutation in BHK-21 cells. The mutant protein failed to associate with lysosomes.
Bespalova et al. (1997) described the complete sequence of the CSTB coding region and splice junctions of a patient with progressive myoclonus epilepsy (EPM1A; 254800) who had decreased cystatin B mRNA levels but lacked previously characterized mutations. The patient was found to be heterozygous for a 2-bp deletion (2404delTC) in the third exon of the CSTB gene. The mutation caused a translational frameshift and protein truncation after 74 amino acids. The patient (EP6) had been described in detail by Pranzatelli et al. (1995). This mutation was also found by Lalioti et al. (1997) and Lafreniere et al. (1997). Alakurtti et al. (2005) transiently expressed the 2400delTC mutation in BHK-21 cells. The truncated mutant protein showed diffuse cytoplasmic and nuclear distribution.
In a Dutch patient with progressive myoclonic epilepsy of Unverricht and Lundborg (EPM1A; 254800), de Haan et al. (2004) identified a 2398A-C transversion in the CSTB gene, resulting in a gln71-to-pro (Q71P) substitution.
Alakurtti et al. (2005) transiently expressed the Q71P mutation in BHK-21 cells. The mutant protein failed to associate with lysosomes.
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