Leiden Muscular Dystrophy pages

Fukutin-related protein (FKRP)

( LGMD-2I / MDC1C )

(last modified February 27, 2005)


Contents



Summary


Brockington et al. (2001) isolated a gene which shows similarity to fukutin, the gene mutated in Fukuyama CMD (FCMD). The Fukutin-related protein gene (Gene Symbol FKRP) maps to human chromosome 19q13.3, between markers D19S219 and D19S606. The FKRP gene spans less than 12.5 kb and contains 4 exons; exons 1 to 3 contain 5' UTR sequences only, exon 4 covers the remaining part of the 5' UTR, the entire protein coding region and the full 3' UTR sequence. The encoded protein measures 495 amino acids and has a significant similarity to fukutin. FKRP and fukutin (FCMD) are type II membrane proteins (i.e. N-terminus outside, C-terminus inside) and putative glycosyltransferases that are probably localized to the Golgi apparatus.

Mutation analysis identified FKRP mutations first in MDC1C patients and later in LGMD-2I families. The range of phenotypic severity due to FKRP-mutations is large and has so far not been observed in other muscular dystrophies. LGMD-2I sometimes follows a Duchenne-like disease course, with a loss of ambulation in the early teens followed by the development of a cardiomyopathy (Brockington). Where muscle samples from LGMD-2I families were available, a secondary laminin-alpha2 deficiency and a variable reduction in alpha-dystroglycan expression were observed (Brockington).

FKRP and fukutin are required for the post-translational modification of dystroglycan and directly affect its processing. Wild-type FKRP and FKRP-Leu276Ile, the most common cause of the milder LGMD2I, are trafficked to the Golgi apparatus and are less susceptible to proteasomal degradation compared other mutant FKRP's. These latter proteins, incl. Ser221Arg, Pro448Leu and Ala455Asp are retained in the endoplasmatic reticulum. As a consequence of reduced FKRP function, the alpha-dystroglycan produced may have a reduced binding capacity to extracellular laminin and the extracellular matrix (ECM). This effectively weakens the bridge between the dystrophin-associated glyco-protein complex and the ECM and disrupts the basal lamina of the muscle.


The FKRP gene


Links to other databases:
Gene Symbol nomenclature   Entrez Gene   OMIM Gene Map

Brockington et al. (2001) isolated a gene which shows similarity to fukutin, the gene mutated in Fukuyama CMD (FCMD, OMIM:253800). The Fukutin-related protein gene (Gene Symbol FKRP) maps to human chromosome 19q13.3, between markers D19S219 and D19S606 (centromere - D19S219 - 1.2 Mb - D19S412 - 0.20 Mb - fkrp52 - 0.04 Mb - FKRP - 0.72 Mb - D19S606 - telomere, Louhichi [2004]), a 3cM region. The FKRP gene is transcribed from centromere to telomere. The gene is flanked on the centromeric side by the STRN4 gene (transcribed from telomere to centromere) and 15 kb downstream by the SLC1A5 gene (transcribed from telomere to centromere). Exon 1 of the STRN4 gene resides in intron 1 of the FKRP gene.

The FKRP gene contains 4 exons; exons 1 to 3 contain 5' UTR sequences only, exon 4 covers the remaining part of the 5' UTR, the entire protein coding region and the full 3' UTR sequence. Since the introns are rather small, the entire gene spans less than 12.5 kb.

Exon Exon size (bp) Intron size (bp) 5' cDNA position Splice after Remarks
1 33 1,936 (-285) - 5' UTR
2 62 426 -252 -
3 141 6,746 -190 -
4  3,071 - -39 - 39 bp 5' UTR, 1,488 bp full coding region and 1,540 3' UTR

Legend:
Exon: numbering of exons and intron/exon boundaries are as determined by Brockington, with the first base of the Met-codon counted as position 1 (see FKRP coding DNA Reference Sequence). Exon size: size of exon indicated in basepairs. Intron size: size of intron indicated in basepairs. 5' cDNA position: first base of the exon. Splice after: splicing occurs in between of two coding triplets (0), after the first (1) or the second (2) base of a triplet. Remarks: 5'UTR = 5' untranslated region, 3'UTR = 3' untranslated region.

primers for DNA-amplification


The FKRP mRNA


Links to other databases:     RefSeq: NM_024301     UniGene: Hs.193261

Northern blot analysis revealed a transcript of 2.8 kb (Esapa 2002, 4.0-kb acc. to Brockington 2001) in all tissues studied. The FKRP transcript is expressed highest in skeletal muscle, placenta, kidney, liver, brain, lung and heart and relatively weakly in the other tissues (brain, lung, liver, kidney and pancreas). Brockington (2001) described an additional 3.6 kb transcript in skeletal muscle, kidney, and lung. FKRP mRNA expression is similar in distribution and level to that of fukutin (FCMD). 

primers for RNA-amplification


The FKRP protein


Links to other databases:    RefSeq: NP_077277

Fukutin-related protein (FKRP) is a 495 amino acid type II membrane protein (i.e. N-terminus outside, C-terminus inside). At its N-terminus FKRP contains a hydrophobic Golgi signal anchor sequence (amino acids Met1-Asn33, Esapa et al. 2002) coinciding with the transmembrane region. This region includes an RxxR-motif (aa Arg2 to Arg5) conserved in several Golgi-resident proteins. The glycosyltransferase DxD-motif is present from aa 362-364. FKRP has two potential sites for N-linked glycosylation, Asn172 and Asn209. 

FKRP (as wel as fukutin) is thought to be a phospho-ligand tansferase and possibly a glycosyl transferase, a hypothesis supported by the finding that MDC1C and LGMD2I are associated with a secondary deficiency in the glycosylation of alpha-dystroglycan (DAG1). Esapa et al. (2005) do not agree with Matsumoto et al. (2004) who suggest that FKRP is localized in the rough ER where they believe it might be involved in the initial step of O-mannosylation on a-dystroglycan.

Murine and humane FKRP shows 94% sequence identity. FKRP shows significant similarity to fukutin and a family of protiens involved in modifying cell surface molecules such as glycoproteins and glycolipids (Brockington et al. 2001). All members of this protein family contain a conserved "DxD"-motif (Breton & Imberty). 

FKRP antibodies

A polyclonal rabbit antibody was described by Esapa et al. (2002). Antibodies against FKRP do not detect endogenous protein in murine C2C12 cells (Esapa et al. 2005). FKRP is targeted to the Golgi apparatus through an N-terminal signal anchor (Esapa et al. 2002, Esapa et al. 2005). FKRP expressed in COS-7 cells was shown to have a half-life of >24h. This half-life is decreased significantly by some disease-related mutations (Esapa et al. 2005). Immunoprecipitation of wild-type and mutant FKRP from transfected COS-7 cells identified calnexin (CANX) as bound to FKRP in a glycan-independent manner and especially in ER-retained mutant FKRP.

FKRP and fukutin are required for the post-translational modification of dystroglycan. Over-expression of FKRP gives a reduction of the 160 kDa alpha-dystroglycan isoform and a concomittant increase in the 90 and 60 kDa alpha-dystroglycan immuno-reactive proteins. FKRP mutants in the RxxR and DxD-motifs are not able to affect dystroglycan processing (Esapa et al. 2002). Based on its similarity to yeast Mnn4p, FKRP may be a non-catalytic regulator of mannosylation.


FKRP and disease: LGMD-2I / MDC1C


After excluding the loci for Limb-Girdle muscular dystrophy  types 2A to 2H, Driss et al. mapped a new LGMD locus in a large consanguineous Tunesian family with 13 patients to human chromosome region 19q13.3. The locus was designated LGMD type 2I. A maximum LOD-score of 4.36 at recombination fraction 0.00 was obtained with marker D19S606. A stretch of homozygosity was detected around this marker. Recombination events between siblings place the LGMD-2I locus in a 9 cM interval flanked by D19S412 and D19S879.

The FKRP gene mapped to a region on chromosome 19q13.3 where also the gene for LGMD-2I had been mapped, between markers D19S219 and D19S606, suggesting that MDC1C and LGMD-2I are allelic disorders. Brockington et al (2001) collected 25 LGMD families, including 14 consistent with linkage to the LGMD-2I locus, and tested the FKRP gene for mutations. 


Disease-causing FKRP mutations


Links to other databases:   OMIM: 607155 (LGMD-2I)  /  OMIM:606612 (MDC1C)  /  OMIM:253280 (MEB)  /  OMIM:236670 (WWS)

MDC1C (OMIM) is a rare recessive autosomal disorder characterized by severe muscular dystrophy presenting at birth or in the first few weeks of life (Topaloglu et al. 2003). LGMD2I (OMIM) is a more common disease, generally mild with a variable age of onset ranging from childhood to adulthood (Brockington et al. 2001, Poppe et al 2003). Recently, FKRP mutations have been found in patients with CMD, mental retardation and brain abnormalities (Topaloglu et al. 2003, Louhichi et al. 2004). Recently, two patients diagnosed with Muscle-Eye-Brain disease (MEB) and Walker-Warburg syndrome (WWS) have been found to carry homozygous FKRP mutations; c.919T>A (p. Tyr307Asn) and c.953G>A (p.Cys318Tyr) resp. (Beltran-Valero de Bernabe et al. 2004).

Mutation analysis (Brockington) identified 7 different mutations in 17/25 LGMD-families. Fifteen families carried an identical 826C>A (Leu276Ile) mutation; five were homozygous (not from consanguineous marriages), the other 10 compound heterozygotes. In 8 families, no mutations were found; four cases were compatible with linkage to 19q13.3, the other four represented isolated cases. The frequent 826C>A mutation was not found on a conserved haplotype. Brockington et al. note that FKRP-mutations account for a significant proportion of patients with LGMD.

The range of phenotypic severity due to FKRP-mutations is large and has so far not been observed in other muscular dystrophies. LGMD-2I sometimes follows a Duchenne-like disease course, with a loss of ambulation in the early teens followed by the development of a cardiomyopathy (Brockington). Where muscle samples were available, a secondary laminin-alpha2 deficiency and a variable reduction in alpha-dystroglycan expression were observed (Brockington). Immunohistochemical staining and Western blot analysis for laminin-alpha2 (using an antibody directed against the 80kDa fragment) may give conflicting results with near normal staining on tissue sections but virtual absence of staining on Western blot. This indicated that FKRP-mutations result in altered processing, epitope masking or abnormal folding of the laminin-alpha2 protein chain. Staining for dystrophin, sarcoglycans (alpha, beta and gamma), caveolin, dysferlin, emerin, lamin A/C, calpain-3 and beta-dystroglcan were normal. 

After identification of the FKRP gene as altered in LGMD-2I (Brockington, 2001), Driss et al. (2003) studied the FKRP-gene in a family and revealed a homozygous c.1486T>A [p.X496Argext21] change (always in combination with a c.341C>G change). Immunohistochemical analysis of muscle fibers showed a normal expression of dystrophin, sarcoglycan subunits, sarcospan, utrophin, dysferlin, caveolin-3, dystrobrevin, laminin-alpha-2 and collagen IV and VI. alpha-Dystroglycan, beta-dystroglycan was preserved. Compared to normal muscle, Western blot analysis showed a narrower alpha-dystroglycan band with a marked lower molecular mass (alpha-dystroglycan normally 150 to 200-kDa); beta-dystroglycan staining was normal. Staining of the 300-kDa LAMA2 fragment was reduced, while the 80-kDa fragment was near absent.

No patients have been found with two FKRP null alleles suggesting that the complete lack of FKRP might result in embryonic lethality. There are no obvious phenotypic correlations between the nature of the missense mutation and the severity of the disease. In addition to patients homozygous for the 826C>A (L276I) mutation, several compound heterozygotes have been identified that have the 826C>A (L276I) change combined with a different change, sometimes even a potential null allele, on the other chromosome. Since all these patients have LGMD-2I, the 826C>A ( Leu276Ile) seems to protect the individual from a severe form of the disease. Please note that these patient have often almost normal alpha-dystroglycan immunoreactivity (Brockington 2001).

FKRP sequence variations
(mutations and polymorphisms)

Esapa et al. (2005) show that upon expression in COS-7 and C2C12 cells, the protein variants Ser221Arg, Ala455Asp and Pro448Leu localize to the ER. Protein variant Leu276Ile, like normal FKRP, predominantly localizes to the Golgi, with some protein detectable in the ER. Consequently, FKRP protein mutants associated with the severe forms of the disease (MDC1C) do not seem to reach the Golgi. Expressed in COS-7 cells normal FKRP has a half-life of >24h while the Pro448Leu variant has a half-life of only ~14h. Esapa et al. (2005) suggest that difference in disease severity can be explained by differential FKRP-trafficking of mutants such that some mutants, although retaining (some of their) function, are unable to act on their substrates because they cannot reach the Golgi apparatus. As a consequence of reduced FKRP function, the alpha-dystroglycan produced may have a reduced binding capacity to extracellular laminin and the extracellular matrix (ECM). This effectively weakens the bridge between the dystrophin-associated glyco-protein complex and the ECM and disrupts the basal lamina of the muscle.


Miscellaneous



FKRP sequences


Similar sequences

For a multiple sequence alignment see Brockington (2001) 



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