Sequence of the human FKRP cDNA and analysis of the primary sequence. A, Partial nucleotide sequence and open reading frame are shown. The 4-kb cDNA encodes a protein of 495 amino acids, with a predicted molecular weight of 54.6 kDa. B,Multiple sequence alignment of fukutin homologues. Missense and nonsense mutations that were found to occur within the region aligned are indicated by red asterisks (*) and slashes (/), respectively. Amino acid residues are colored according to an 80% consensus: plus signs (+) indicate positively charged residues (H, K, and R; blue), minus signs (−) indicate negatively charged residues (D and E; red), black asterisks indicate serine or threonine residues (light blue on grey background), “a” indicates aromatic residues (F, H, W, and Y; blue on yellow background), “b” indicates big residues (E, F, H, I, K, L, M, Q, R, W, and Y; blue on yellow background), “c” indicates charged residues (DEHKR; pink), “h” indicates hydrophobic residues (A, C, F, G, H, I, L, M, T, V, W, and Y; black on yellow background), “l” indicates aliphatic residues (I, L, and V; grey on yellow), “p” indicates polar residues (C, D, E, H, K, N, Q, R, S, and T; blue), and s indicates small residues (A, C, D, G, N, P, S, T, and V; green). Residues that are conserved in >80% of sequences are shown as yellow on black. Residues excised from the alignment are indicated by numbers in parentheses. Predicted secondary structures are indicated below the alignment (e/E, extended or β-strand structure; h/H, helix). Residue numbers and GeneInfo identifiers (or else cosmid names) are shown following the alignment. Ca = Candida albicans; Ce = Caenorhabditis elegans; Dm = Drosophila melanogaster; HELP1 = haemolysin erythrocyte lysis protein 1; Hi = Haemophilus influenzae; Hs = Homo sapiens; Ll = Lactobacillus lactis lactis; Pi = Prevotella intermedia; Rp = Rickettsia prowazekii; Sc = Saccharomyces cerevisiae; Sp = Streptococcus pyogenes; Tb = Trypanosoma brucei. Fukutin homologues partially encoded by ESTs in T. brucei (EST AQ655742), T. cruzi(AI077222), and Leishmania major (AQ852136), as well as a Bacillus subtilis sequence similar to L. lactis TrpF C-terminal domain (GeneInfo number 1750106; bases 12108–12419), are not shown. Two C. elegans sequences, marked with a dagger (†), are alternative gene predictions that differ from published versions by the prediction of additional exons.

Schematic representation of the FKRP gene. The gene consists of 4 exons and spans no more than 12 kb. The entire coding sequence is located within exon 4. The 5′ UTR is blackened, the FKRP open reading frame is unblackened, and the 3′ UTR is hatched.

Northern blot analysis demonstrating the expression of FKRP mRNA. A 4.0-kb transcript is seen in all tissues studied. H = heart; B = brain; Pl = placenta; Lg = lung; Lv = liver; SM = skeletal muscle; K = kidney; Pc = pancreas. It is most highly expressed in skeletal muscle, placenta, and heart and is expressed relatively weakly in the remaining tissues. An additional transcript of ∼3.6 kb is visible in skeletal muscle, kidney, heart, and lung.

An example of mutation analysis in FKRP. a, The pedigree of family 1, showing the segregation of two heterozygous mutations, A926G and C1154A. Both affected children are compound heterozygotes, whereas the unaffected sib has inherited only one of the at-risk alleles. The father unexpectedly did not have the C1154A mutation in his lymphocyte DNA and most likely is a germline mosaic. Haplotype analysis at this and other loci confirmed paternity (data not shown). b, DNA sequence analysis of FKRP, identifying the mutations in family 1.

Muscle sections, from families 1 (a) and 2 (b), stained with haematoxylin and eosin. Immunolabeling for α- and β-dystroglycan is shown in c and e, repectively (family 1), and in d and f, respectively (family 2). α-dystroglycan was markedly reduced on most fibers in family 1, whereas >50% of fibers showed a reduction in family 2. β-dystroglycan was normally expressed in all fibers in both families. Immunolabeling for laminin α2 is shown in g (family 1) and h (family 2). A reduction in laminin α2 expression on the larger fibers was evident only in family 1. Bar = 100 μm.

a, Immunoblot analysis of muscle proteins extracted from families 1 and 2 (F1 and F2) showed a marked shift in the average molecular weight of α- but not β-dystroglycan. Control adult muscle (CTRL), together with muscle from patients with Duchenne muscular dystrophy (DMD), are included for comparison. The significant reduction in α-dystroglycan molecular weight was only observed in patients with MDC1C. b, The molecular weights of α- and γ-sarcoglycan are comparable with those of control and DMD muscle.