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Fig. S1. Restriction fragment length polymorphisms to detect variants c.631T>C and c.1628A>T. Polymerase chain reaction (PCR) primers are depicted in bold and italic; recognition sites of the restriction enzymes applied in bold and underlined. (a) Upon restriction with PsiI a 321-bp PCR product from the normal allele yields two fragments of sizes 92 and 229 bp, respectively. Variant c.631T>C (nucleotide c.631 in red) abolishes the PsiI recognition sequence. PCR product from the altered allele, thus, remains undigested. (b) The forward primer introduces a C>T alteration at nucleotide c.1624 (in green) such as to create a second AgsI restriction site in the 395-bp product from the normal allele. Variant c.1628A>T (nucleotide c.1628 in red) abolishes this artificially created site. Restriction patterns for normal and altered allele, thus, differ (45 + 48 + 302 vs 93 + 302 bp, respectively).

Fig. S2. Genome-wide linkage analysis for the urofacial syndrome phenotype in the family presented. A total of three genomic regions (arrows) are homozygous in all patients but neither in their parents nor their healthy siblings. The corresponding LOD score of 2.78 is the maximum score expected considering the family structure (see Fig. 1b for pedigree).

Fig. S3. Haplotype analysis of the urofacial syndrome chromosome 10 critical region using microsatellites (see Table S2 for physical position of markers and of HPSE2 gene). (a) The disease haplotype (boxed) is present in heterozygosity in the parents. The affected children, but not unaffected siblings, are homozygous. (b) Exemplary raw data for the most terminal markers for all six children (order of individuals as in above pedigree).

Table S1. Localization of SNPs defining the chromosome 10 region linked to the phenotype, microsatellites applied to confirm this linkage and the HPSE2 gene

Table S2. Primers used to amplify and sequence the 12 exons of HPSE2

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