(A) Pedigree of the family in which specific members suffer from IRH. Filled symbols represent affected individuals, half-filled symbols are heterozygous for the mutation C3209T, and open symbols represent clinically unaffected family members not screened for the mutation. Slash marks indicate deceased individuals, and double lines show consanguinity. (B) Schematic representation of the critical interval between polymorphic markers D4S2623 and D4S1575 on chromosome 4q and the intron-exon structure of the EGF gene, depicting the identified mutation. The position of the polymorphic markers is indicated by vertical bars. The horizontal arrow below the schematical boxed representation of EGF depicted between the polymorphic markers indicates the localization and orientation of the EGF gene. Cen, centromeric; tel, telomeric. (C) Genomic mutation sequence analysis of EGF in wild-type, heterozygous, and affected individuals. The mutated nucleotide and resulting amino-acid change is shown under the affected individual’s sequence. The black bar under that sequence indicates the mutated codon. The black bars under the sequences of the WT and heterozygous specify codon 1070 of EGF. The affected individuals both have a homozygous mutation C3209T in exon 22, resulting in the amino acid substitution P1070L. (D) Schematic model of pro-EGF, which resides as a type I membrane protein at the plasma membrane. The position of the P1070L mutation is depicted. (E) Sequence homology analysis of juxtamembrane domain and flanking residues. P1070 is strongly conserved among different species and forms the second proline of a basolateral PXXP sorting motif. The indicated colors represent functional conservation of the amino acids: red, small plus hydrophobic; blue, acidic; magenta, basic; green, hydroxyl plus amine plus basic; gray, others. TM, transmembrane domain.