Identification of five novel BOR mutations in human EYA1 gene associated with branchio-oto-renal syndrome by a DHPLC-based assay.

To the Editor: Branchio-oto-renal (BOR) syndrome (OMIM 113650), an autosomal dominant disorder first described by Melnick et al. (1976), is defined by the presence of at least three of the four major features: hearing loss, branchial defects, ear pits and renal abnormalities, although other minor features can be present. There is considerable phenotypic variability between families (1, 2) and within family members (3–5). The prevalence is estimated at 1 : 40,000 in the general population and 2% among the profoundly deaf children (6). The BOR syndrome locus was mapped to chromosome 8q13.3 using linkage analysis (7, 8) and the EYA1 gene, yielding sequences homologous to the Drosophila ‘eyes absent’ (eya) gene, was shown to be the causative gene (9). The EYA1 gene is composed of 16 exons spanning 156 kb and codes for a 559 amino acid protein. Allelic mutations are responsible for branchiootic (BO) syndrome in some families (10, 11). This could indicate that BOR and BO syndromes are allelic disorders, although two BO families not linked to this gene support genetic heterogeneity (12, 13). A second locus was assigned to chromosome 1q31 (14) and a third locus to chromosome 14q21.3-q24.3 (15). In this study, mutation screening of the coding region of EYA1 in 21 patients and four relatives, belonging to nine unrelated families, and three sporadic cases was performed, using denaturing high performance liquid chromatography (DHPLC)-based protocol. Details of clinical features in the affected individuals are summarized in Table 1. The 16 exons of the human gene EYA1 were amplified using intronic primers flanking each exon, designed by Abdelhak et al. (9, 16). Sequencing was carried out for each abnormal elution profile, observed in heterozygosity in 12 patients (cases 2, 3, 6, 7, 10, 11, 12, 16, 17, 19, 20, 21), as shown in Fig. 1. Sequence variants were considered likely mutations when they caused a non-conservative amino acid change, were absent in 200 control chromosomes, and affected phylogenetically conserved residues. We have identified six different EYA1 mutations. All these mutations, except one, are novel and include deletions/insertions of a few nucleotides, exon skipping alterations and missense changes. The following four mutations affected the conserved exons 9–16, where most of the BO/BOR mutations have been found (http://www.medicine. uiowa.edu/pendredandbor/): c.868-1G>A, a point mutation in exon 9 splice acceptor site; c.10241027delTTTT, a deletion of four nucleotides in exon 10; c.1261G>C, a missense change in exon 12; and c.1456-1457insA, a single nucleotide insertion in exon 14. With the exception of the c.8681G>A mutation in exon 9, all of them were novel mutations. Two additional novel mutations (c.558delC in exon 7 and c.759delC in exon 8) were found in exons 7 and 8, where previously identified mutations had been located (17, 18). Our mutational screening was negative in three families and three sporadic cases, even after a complete cDNA sequence analysis of the six individuals. In these cases, the presence of the mutation and/or large rearrangements within intronic or regulatory sequences cannot be excluded. Alternatively, this result could indicate that other unidentified genes, such as modifier or unknown genes, could be linked to chromosomes 1q31 or 14q21.3-q24.3. The mutation detection rate of 50% in the EYA1 gene corroborates previous studies, confirming the genetic heterogeneity of BOR disease (9, 16, 19, 20). Compared to other conventional mutational screening strategies (Single Strand Conformational Clin Genet 2004: 66: 478–480 Copyright # Blackwell Munksgaard 2004 Printed in Denmark. All rights reserved CLINICAL GENETICS doi: 10.1111/j.1399-0004.2004. 00318.x