Branchio-Otic Syndrome Caused by a Genomic Rearrangement: Clinical Findings and Molecular Cytogenetic Studies in a Patient with a Pericentric Inversion of Chromosome 8

Branchio-oto-renal (BOR) syndrome is an autosomal dominantly inherited developmental disorder, which is characterized by anomalies of the ears, the branchial arches and the kidneys. It is caused by mutations in the genes EYA1,SIX1 and SIX5. Genomic rearrangements of chromosome 8 affecting the EYA1 gene have also been described. Owing to this fact, methods for the identification of abnormal copy numbers such as multiplex ligation-dependent probe amplification (MLPA) have been introduced as routine laboratory techniques for molecular diagnostics of BOR syndrome. The advantages of these techniques are clear compared to standard cytogenetic and array approaches as well as Southern blot. MLPA detects deletions or duplications of a part or the entire gene of interest, but not balanced structural aberrations such as inversions and translocations. Consequently, disruption of a gene by a genomic rearrangement may escape detection by a molecular genetic analysis, although this gene interruption results in haploinsufficiency and, therefore, causes the disease. In a patient with clinical features of BOR syndrome, such as hearing loss, preauricular fistulas and facial dysmorphisms, but no renal anomalies, neither sequencing of the 3 genes linked to BOR syndrome nor array comparative genomic hybridization and MLPA were able to uncover a causative mutation. By routine cytogenetic analysis, we finally identified a pericentric inversion of chromosome 8 in the affected female. High-resolution multicolor banding confirmed the chromosome 8 inversion and narrowed down the karyotype to 46,XX,inv(8)(p22q13). By applying fluorescence in situ hybridization, we narrowed down both breakpoints on chromosome 8 and found the EYA1 gene in q13.3 to be directly disrupted. We conclude that standard karyotyping should not be neglected in the genetic diagnostics of BOR syndrome or other Mendelian disorders, particularly when molecular testing failed to detect any causative alteration in patients with a convincing phenotype.

chial arch defects, preauricular pits/tags, and different renal disorders [Melnick et al., 1975;Fraser et al., 1978]. Affected individuals who lack renal dysplasia have been described as having branchio-otic (BO) syndrome (MIM 602588). The disease has a high penetrance; however, the clinical course of BOR/BO syndrome shows variable expressivity with a high inter-and intrafamilial variability [Ou et al., 2008]. Most of the described cases result from mutations in the EYA1 gene, which is located in 8q13.3 and encodes a member of the drosophila eyes absent (EYA) family of proteins. EYA1 was reported to act as a transcriptional coactivator and as a phosphatase [Vervoort et al., 2002;Krug et al., 2011]. Furthermore, EYA1 forms a complex with SIX proteins, members of the Dach family (dachshund) and presumably additional not yet known proteins [Ikeda et al., 2002;Li et al., 2003;Ahmed et al., 2012]. The formation of this complex leads to a coactivation of SIX transcription factors which are essential for normal development of several tissues and organs including the second branchial arch, ears, eyes, and kidneys [Ikeda et al., 2002;Li et al., 2003;Ahmed et al., 2012]. Up to date, more than 110 different mutations in the EYA1 gene (Molecular Otolaryngology Research Laboratory, Pendred/BOR homepage, http://www.healthcare.uiowa.edu/ labs/pendredandbor/) have been described to be associated with BOR/BO syndrome; they are randomly scattered over the entire gene . Few mutations have also been found in genes encoding the SIX1 and SIX5 proteins [Krug et al., 2011]. The mutation detection rate in patients with typical clinical features of BOR/BO syndrome has been reported with highest estimates of ∼ 70% [Krug et al., 2011]. This may be due to the genetic heterogeneity and clearly shows the difficulties in implementing a strategy for molecular testing in patients with BOR/BO syndrome. The spectrum of EYA1 mutations comprises missense, nonsense, frameshift, and splice-site mutations as well as small heterozygous deletions and complex genomic rearrangements. Therefore, the typical step-by-step diagnostic approach of BOR/BO syndrome includes sequencing of EYA1, SIX1 and SIX5 as well as multiplex ligation-dependent probe amplification (MLPA) analysis for the detection of possible deletions covering part or the entire EYA1 gene. Nevertheless, genomic rearrangements affecting EYA1 have been described to be a relatively frequent cause of BOR/BO syndrome and may be missed by standard diagnostic tools Smith et al., 1992;Vervoort et al., 2002]. Here, we report on a patient showing major features of BO syndrome but missing renal manifestations. Cytogenetic analysis revealed a pericentric inversion of chromosome 8. By fluorescence in situ hy-bridization (FISH), we demonstrate that one of the breakpoints directly disrupts the EYA1 gene and thus represents the genetic alteration causing BO syndrome in the affected female.

Case Report
The 43-year-old female patient presented with progressive sensorineural hearing loss. Since the age of 3 years, she has worn hearing devices. A computed tomography scan of the petrous bone showed inner ear malformations combined with deformities of the middle ear such as enlarged auditory tubes and absent mastoid cells. Bilateral preauricular fistulae were treated by surgery at the age of 21 years due to recurrent inflammation. She showed facial dysmorphism with arched and sparse eyebrows, periorbital oedema, sparse hair, a bulbous nose tip, a small upper lip, a receding chin, a high arched palate, and no ear lobes ( fig. 1 ). Renal ultrasound was normal. Her first pregnancy ended in a late-term abortion (34th week), and it became apparent that the male fetus had renal agenesis. Her 16-year-old daughter showed no clinical signs of BO or BOR syndrome; renal ultrasound showed no abnormalities, and preauricular fistulas were not found.

Laboratory Genetic Testing
After genetic counseling and collection of informed consents of the patient and her parents, we obtained blood (heparin and EDTA) samples of the trio. Genetic analyses were performed following the ethical guidelines of the institutes involved.

Molecular Genetic Testing
Genomic DNA was isolated from EDTA blood samples using standard methods. Mutation analysis of the 16 coding exons of EYA1 (GenBank accession number NM_172058), the 2 coding exons of SIX1 (GenBank accession number NM_005982), the 3 coding exons of SIX5 (GenBank accession number NM_175875), and their flanking intronic sequences was carried out by direct sequencing. Screening for deletions or duplications of the EYA1 gene was performed using the MLPA kit SALSA P153-A2 according to the manufacturer's instructions (MRC Holland, Amsterdam, The Netherlands).
Cytogenetic and Array Comparative Genomic Hybridization Analysis Routine cytogenetic investigations were performed on metaphase chromosomes derived from 72-h lymphocyte cultures according to standard protocols. A minimum of 15 metaphases were analyzed for each individual. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature [Shaffer et al., 2009]. Array comparative genomic hybridization (array CGH) was performed on genomic DNA of the patient using the Human Genome 180K CGH Microarray (Agilent Technologies, Waldbronn, Germany) according to the recommendations of the manufacturer. Interpretation was based on Human Genome Build 36 (NCBI36/hg18). 3 done as described before; method and MCB probe sets are specified in Weise et al. [2008]. Besides, a subcentromere-specific multicolor FISH probe set of chromosome 8 was used [Liehr et al., 2006]. Labeling and FISH were done according to Liehr et al. [2002].

High-Resolution Multicolor
Fluorescence in situ Hybridization FISH experiments for delineation of both breakpoints on chromosome 8 were performed with BAC and fosmid clones (online suppl. table 1; for all online supplementary material, see www.karger.com/doi/10.1159/000355436) as described previously [Najm et al., 2008]. BACs and fosmid clones were obtained from the BACPAC Resource Center, Children's Hospital (Oakland, Calif., USA).

Molecular Genetic Testing
Sequencing of the EYA1, SIX1 and SIX5 genes on DNA derived from the patient's white blood cells showed no mutation. MLPA of the EYA1 gene revealed no copy number change.

Cytogenetic and Array Comparative Genomic Hybridization Analysis
Cytogenetic analysis showed a pericentric inversion on one of the chromosomes 8 in the index patient ( fig. 2 ). The breakpoints were mapped close to 8pter and 8q13.

Mapping of the Breakpoints in 8p22 and 8q13.3 by FISH
By using various BAC and fosmid clones, we performed serial FISH analyses to delineate both breakpoints on chromosome 8. We first mapped the 8q13.3 breakpoint and identified the 2 BACs RP11-263H10 and RP11-242B23 and fosmid G248P8007F2, all covering part of EYA1 gene, as breakpoint-spanning clones: they gave a signal on wild type chromosome 8 as well as split signals on both arms of chromosome 8 with the pericentric inversion ( fig. 3 and data not shown). We narrowed the We also mapped the 8p22 breakpoint and identified BAC RP11-1069K22 and fosmid G248P87904B12 which overlapped the breakpoint (online suppl. fig. 1). The breakpoint is located within the PCM1 gene (online suppl. fig. 1).

Discussion
In this study, we present clinical and genetic data of a female patient presenting with signs of BOR syndrome. According to improved clinical diagnostic criteria which have been introduced by Chang et al. [2004], there are 4 major criteria, i.e. branchial anomalies, deafness/hearing loss, preauricular pits, and renal anomalies, as well as several minor criteria, such as anomalies of the external, middle or inner ear, preauricular tags, facial asymmetry, and palate abnormalities [Chang et al., 2004]. Our patient displayed 2 major clinical features, namely sensorineural hearing loss and preauricular tags, and several minor features, e.g. anomalies of the outer, the middle and the inner ear as well as a long and narrow face. Altogether, she meets the diagnostic criteria of BO syndrome due to the lack of renal abnormalities. Additionally, our patient reported a stillborn child displaying renal agenesis in her first pregnancy which is in line with the observation that an extensive clinical variability exists, even between family members harboring the same mutation [Chang et al., 2004]. Unfortunately, we neither have detailed information on additional malformations nor the chromosomal status of the fetus. Thus, although we can only speculate on the genetic cause of the late-term miscarriage, we assume the presence of the same chromosome-8 inversion as seen in the mother.
Molecular genetic testing failed to detect a mutation in the genes EYA1 (8q13.3), SIX1 and SIX5 . Using highest clinical diagnostic standards, the detection rate of mutations in EYA1 was almost 70% in patients with a clear-cut phenotype [Chang et al., 2004;Krug et al., 2011]. Mutations in SIX5 and SIX1 are causative in 5% of the cases [Sanchez-Valle et al., 2010]. Several studies have demonstrated that in ∼ 80% of mutation-positive cases, mutations could be detected by direct sequencing, whereas in ∼ 20% of affected individuals, complex genomic rearrangements of chromosome 8, which likely affect expression of EYA1 on one allele, caused the phenotype [Chang et al., 2004;Sanchez-Valle et al., 2010]. The latter applies to the female patient reported here, as we identified a balanced pericentric chromosome-8 inversion with direct interruption of the EYA1 gene by 1 of the 2 breakpoints.
Various chromosomal rearrangements in patients with BOR syndrome have been reported so far, ranging from inversions via small insertions to large deletions. However, most of the reported rearrangements are unbalanced, and in some cases, other genes in addition to EYA1 were deleted leading to a contiguous gene syndrome in patients with BOR syndrome and additional clinical features [Chang et al., 2004]. Therefore, most of these rearrangements cannot be compared with our case. Vervoort et al. [2002] characterized and summarized genomic rearrangements detected by the means of sequencing, single-strand conformation polymorphism, Southern blot, and transcript analysis. They reported a single inversion in chromosome 8, which was associated with BOR syndrome [Vervoort et al., 2002]. This paracentric inversion was found in several members of a family with clinical signs of BOR syndrome. Breakpoint analysis was performed by Southern blot analysis and revealed one of the breakpoints in intron 1 and the second breakpoint outside the EYA1 gene. The study was published in 2002. Meanwhile, standard diagnostic methods for the detection of large genomic rearrangements have been improved by introducing tools to identify abnormal copy numbers, such as MLPA. However, MLPA does not allow a detection of balanced translocations and inversions, similar to array CGH. In our case, we narrowed down the inversion breakpoints on the p and q arm of chromosome 8 and found that the one in 8q13.3 disrupts the EYA1 gene and thus caused classical BO(R) syndrome in the patient. The second breakpoint in 8p22 is located within the PCM1 gene (online suppl. fig. 1). PCM1 encodes the pericentriolar material 1 protein, which is a component of centriolar satellites [Kubo et al., 1999]. PCM1-containing centriolar satellites move along microtubules, i.e. toward centrosomes, and are involved in the microtubule-and dynactin-dependent recruitment of proteins to the centrosome [Kubo et al., 1999;Dammermann and Merdes, 2002]. Mutations in PCM1 have not been associated with any Mendelian disorder so far. However, loss of both the PCM1 gene and the encoded protein has been observed in breast and ovarian carcinomas [Armes et al., 2004;Pils et al., 2005;Venter et al., 2005] and papillary thyroid carcinoma [Corvi et al., 2000]. Thus, according to the current data, it seems unlikely that the phenotype of the patient with BO syndrome is caused or modified by direct interruption of PCM1 . Considering these results, we conclude that conventional cytogenetic approaches should be taken into account in the genetic diagnostics of patients showing clinical signs of BOR syndrome as well as other autosomal dominantly inherited conditions when molecular genetic analysis was negative.