Figure 1: Identification of GUCA1A mutations in two Spanish pedigrees. (a), (d) Pedigrees of two unrelated families affected by adRD. Individuals are identified by pedigree number. Squares indicate males, circles indicate females, slashed symbols indicate deceased, solid symbols indicate affected individuals, open symbols indicate unaffected individuals, and arrow indicates the proband. In (a), pedigree of the Family 141 is shown with haplotypes of STR markers spanning the linked interval on chromosome 6. Markers and their physical positions (Mb) are indicated at the left of each row. Ten members indicated with asterisks were genotyped by SNP markers. C/C indicates two copies of wild-type GUCA1A, and C/T indicates one copy of wild-type and one copy of mutant GUCA1A. In (d), T/C indicates one copy of wild-type and one copy of mutant GUCA1A found in the proband III:2 in Family 387. (b), (e) Sequencing chromatograms showing the comparison of DNA sequences of normal control (top) to the heterozygous C-to-T transition in exon 4 of GUCA1A (bottom) resulting in a leucine-to-phenylalanine change (p.L84F; GenBank accession number JQ924784) at position 250 (b) and to the heterozygous T-to-C transition in exon 4 of GUCA1A (bottom) resulting in a isoleucine-to-threonine change (p.I107T; GenBank accession number JQ924785) at position 320 (e). These mutations segregated with the disease phenotype and were not found in 200 normal controls. (c) Restriction fragment length analysis confirmed the c.250C>T mutation showing that the transition from C to T results in the loss of restriction site of SmlI. Wild-type samples produced two fragments of 276 bp and 241 bp, while the restriction target site (5′-CTCAAG-3′) in exon 4 of GUCA1A was destroyed by the mutation. Analyzed individuals are identified by pedigree number, bp: base pair, and M: 100 bp DNA ladder. (f) Multiple amino acid alignment of known vertebrate showing the evolutionary conservation of guanylate cyclase-activating proteins (only the region containing EF2 and EF3 are shown). Amino acid residues are colored according to the similarity of their physicochemical properties. The conserved 12-amino-acids Ca2+-binding loop of EF3 is highlighted in grey. The two mutations occurring in high conserved amino acid region across the species are localized next to EF2 (p.L84F) and within EF3 (p.I107T). (g) The region comprising the exonic splicing enhancer (ESE) and silencer (ESS) motifs in the wild type (WT) exon 4 is compared to the corresponding region in the mutant (250T) exon 4 of GUCA1A. The SF2/ASF binding ESE as well as two ESS recognition sites for hnRNP I (ESS1) and SRp20 (ESS2) are depicted. In the context of GUCA1A exon 4, we have hypothesized that a binding ESE is disrupted in the case of 250T allele and thus the antagonizing function of SF2/ASF is abolished. Instead, cryptic ESS sites for binding of hnRNP I and SRp20 exon inclusion suppressors are activated which results in exon 4 skipping. The strengthened motif TCTT binding hnRNP I is indicated in dark red. c.C250T substitution is depicted by underlying of the nucleotide. (h) Cartoon representation of chicken Gcap1 (PDB ID: 2R2I) with the Ca2+ binding EFhands rendered by VMD software. Residues affected by mutation in the human orthologue are colored in red for Leu84 (corresponding to Leu83 in chicken Gcap1) and green for Ile107 (corresponding to Ile106 in chicken Gcap1).