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Feb 4, 2010 - Branchio-oto-renal syndrome caused by partial EYA1 deletion due to LINE-1 insertion. Naoya Morisada & Nanna Dahl Rendtorff & Kandai ...

Pediatr Nephrol (2010) 25:1343–1348 DOI 10.1007/s00467-010-1445-x


Branchio-oto-renal syndrome caused by partial EYA1 deletion due to LINE-1 insertion Naoya Morisada & Nanna Dahl Rendtorff & Kandai Nozu & Takahiro Morishita & Takayuki Miyakawa & Tohru Matsumoto & Satoshi Hisano & Kazumoto Iijima & Lisbeth Tranebjærg & Akira Shirahata & Masafumi Matsuo & Koichi Kusuhara

Received: 11 April 2009 / Revised: 14 December 2009 / Accepted: 16 December 2009 / Published online: 4 February 2010 # IPNA 2010

Abstract A 7-year-old Japanese girl with conductive deafness and preauricular fistulae developed proteinuria. She had renal insufficiency, and ultrasound revealed bilateral small kidneys. These findings indicated that she had branchio-otorenal (BOR) syndrome. In the present patient, we identified, by using multiplex ligation-dependent probe amplification (MLPA) analysis, a heterozygous EYA1 gene deletion comprising at least exons 5 to 7. In her parents, we did not detect any deletion in EYA1 by MLPA, so the deletion was a de novo mutation. PCR analysis and sequencing of patient DNA revealed a heterozygous ∼17 kb EYA1 deletion starting from the eight last bases of exon 4 and proceeding to base 1,217 of intron 7. Furthermore, in place of this deleted region was inserted a 3756-bp-long interspersed nuclear elements-1 (LINE-1, L1). Accordingly, RT-PCR showed that exons 4–7 were not present in EYA1 mRNA expressed from the mutated allele. Although there are reports of L1 element insertion occurring in various human diseases, this is the first

report of a large EYA1 deletion in combination with L1 element insertion. Keywords BOR syndrome . EYA1 . Renal insufficiency . MLPA . LINE-1 (L1) . Retrotransposition . Insertion

Introduction Branchio-oto-renal (BOR) syndrome (OMIM 113650), which was first reported in 1975 by Melnick et al. [1], is characterized by branchiogenic malformation, deafness, and renal abnormalities. BOR syndrome is an autosomal dominant disorder and the prevalence is about 1 to 40,000 births, but sporadic cases are also seen [2]. The renal abnormalities reported in BOR patients include renal agenesis, hypoplasia, dysplasia, calyceal cyst or diverticulum, ureteral pelvic junction obstruction, pelviectasis, and

Naoya Morisada, Nanna Dahl Rendtorff and Kandai Nozu contributed equally to this work. N. Morisada (*) : T. Miyakawa Department of Pediatrics, Saiseikai Yahata General Hospital, 5-9-27, Haruno-machi, Yahatahigashi-ku, Kitakyushu, Fukuoka 805-0050, Japan e-mail: nmor[email protected]

K. Nozu : K. Iijima : M. Matsuo Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

N. Morisada : T. Morishita : T. Miyakawa : A. Shirahata : K. Kusuhara Department of Pediatrics, University of Occupational and Environmental Health, Kitakyushu, Japan

T. Matsumoto Matsumoto Clinic, Kitakyushu, Japan

N. D. Rendtorff : L. Tranebjærg Wilhelm Johannsen Centre for Functional Genome Research, Section of Genetics, Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, Copenhagen, Denmark

S. Hisano Department of Pathology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan L. Tranebjærg Department of Audiology, Bispebjerg Hospital, Copenhagen, Denmark


vesico-ureteral reflux (VUR) [3]. BOR syndrome is associated with several genetic mutations in the eyes: absent homolog 1 gene (EYA1, 8q13.3) [4], the sinus oculis homeobox homolog 1 gene (SIX1, 14q23.1-q24.3) [5], the sal-like 1 gene (SALL1, 16q12.1) [6], the sinus oculisrelated homeobox homolog 5 gene (SIX5, 19q13.3) [7], and in an unidentified gene on 1q31 [8]. EYA1 mutations are considered to be a major cause of BOR syndrome in Japanese patients [2]. Multiplex ligation-dependent probe amplification (MLPA) has been developed as a novel method for the detection of deletions or duplication of one or more exons in various human disease genes [9], but there is only one report of EYA1 deletion detected by MLPA [10]. Recent whole genome studies revealed that mammalian genomes are littered with enormous numbers of transposable elements [11]. The long interspersed nuclear element-1 (LINE-1, L1) is the most abundant retrotransposon in the human genome, and its insertion into protein-coding sequences abolishes gene functions or creates novel gene function [12]. There have been some reports of the disorder by L1 element insertion, but few reports of L1 element insertion causing human disorder, especially hereditary kidney disease [13]. Here we report a case of BOR syndrome with a partial EYA1 deletion detected by MLPA analysis. Subsequent investigation showed that the mutation was caused by L1 element insertion into the EYA1 gene.

Fig. 1 Detection of deletion of EYA1 exons by multiplex ligationdependent probe amplification (MLPA) analysis. DNA from a normal individual (control) and the patient was subjected to EYA1 MLPA analysis, and the amounts of amplified target sequence were analyzed in an ABI Prism 3130XL genetic analyzer. The P153 probe mix for EYA1 includes probes for 14 EYA1 exons (exons numbers are

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Case report A girl was referred to our hospital for further examination of proteinuria at the age of 7, because proteinuria was detected in the annual urinary screening test for Japanese schoolchildren. Her growth, mental development and blood pressure (92/52 mmHg) were normal. She had had bilateral preauricular fistulae and cup-shaped pinnae since birth, but no other malformations were observed. Her hearing loss was confirmed as conductive by audiogram. Computerized tomography (CT) of the temporal bone showed bilateral developmental abnormalities of the mastoid and coalescence of the left auditory ossicles. She was one of a pair of dizygotic twins, but none of her relatives, including her cotwin, had renal disease, auditory disorder, or any other overt malformations. Ultrasound revealed bilateral small kidneys (left kidney: 63×32 mm, right kidney: 56×30 mm). Blood test showed elevated blood urea nitrogen (11.1 mmol/l, normal 2.9–7.9), serum creatinine (85.0 μmol/l, normal 35.4–70.8), and uric acid (512 μmol/l, normal 137–208) levels. Estimated creatinine clearance was calculated to be 55 ml/min/ 1.73 m2 (Schwartz formula) [14]. Hemoglobin (12.7 g/dl, normal 11.5–15.0), total cholesterol (4.09 mmol/l, normal 3.31–6.62), immunoglobulins (IgG 1,053 mg/dl, normal 870– 1,700, IgA 147 mg/dl, normal 110–410), and complement

indicated), and 14 control probes (indicated by asterisks). The patient is heterozygous for a deletion covering, at least, exons 5–7 in EYA1 (transcript variant NM_000503.4), as revealed by the lower peaks obtained from these three exons compared with peaks obtained with control individuals

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components (C3 9.9 g/l, normal 6.5–13.5, C4 2.2 g/l, normal 1.3–3.5) were normal. Total urinary protein obtained for 24 h was 0.23 g/day. Hematuria was not observed. Vesico-ureteral reflux was not observed in either side of the ureter by voiding cystourethrography (VCG). These findings were compatible with BOR syndrome. To confirm the diagnosis, we performed molecular genetic analysis after informed consent was obtained from the patient and her parents.

Materials and methods Genetic analysis by MLPA Genomic DNA was extracted from mononuclear cells from the patient’s peripheral blood with a Qiagen kit (Qiagen, Chatsworth, CA, USA), according to the manufacturer’s Fig. 2 Characterization of a ∼17-kb genomic deletion in combination with insertion of an L1 element into the EYA1 gene. a PCR amplifications of genomic DNA using primers annealing to introns 2 and 8 respectively. A band of 4,791 bp was seen in the patient DNA, but not in the control. The length from exon 3 to exon 8 was shortened by a genomic deletion and insertion of an L1 element in the patient. b Direct sequence revealed a deletion of the last eight bases of exon 4 and all sequences up to bp 1,218 of intron 7 and insertion of an L1 element of 3,756 bases. The L1 element does not have a poly (A) tail. c Schematic representation of the mutant allele compared with the normal allele


instructions. In order to investigate whether an EYA1 deletion or duplication was causing the BOR syndrome symptoms in the patient, MLPA was performed. MLPA detects larger deletions or duplications of one or more exons, but not inversions. The MLPA-EYA1 kit (P153) has recently become available and was purchased from MRC-Holland (Amsterdam, The Netherlands). The EYA1 probe mix contains 17 probes for 14 of the 18 EYA1 exons (for three exons two different probes are present) and 14 control probes. In the current version of the kit, no probes are included for exon 8, 13, and 16 (transcript variant NM_000503.4). However, these exons are in close proximity to neighboring exons, with introns 8, 13, and 16 spanning 404, 112, and 105 bp respectively. MLPA experiments were performed essentially as described by MRC-Holland (http://www.mrc-holland. com/pages/indexpag.html). Amplification products were identified and quantified by capillary electrophoresis on an ABI 3130XL genetic analyzer (Applied Biosystems, Foster


City, CA, USA). Peak heights were normalized and deletions were suspected when the peak height was lower than 62% of the controls. In the present case, the MLPA analysis identified a heterozygous EYA1 deletion confined to, at least, exons 5–7 compared with transcript variant NM_000503.4 (Fig. 1). Among the neighboring exons, exons 4 and 9 were amplified normally. However, since the EYA1 MLPA kit contains no probe for exon 8, we could not exclude the possibility that exon 8 is deleted, although exon 8 is close to exon 9 at a distance of 404 bp. In the parents of the patient we did not detect any mutation in EYA1 by MLPA, so the deletion was found to be a de novo mutation. Long-range PCR and sequencing of patient DNA Based on the MLPA result, we conducted further analysis of the patient DNA by polymerase chain reaction (PCR) in order to obtain a fragment spanning the aberrant chromosomal junctions. Specific exons of EYA1 were amplified by PCR using the primers 5′-AGGCAGATGTGCTGAAG TTGT-3′ and 5′-ACACAGCCCTTCCTTGTGTA-3′, in intron 3 and intron 7 respectively. PCR products from the patient DNA showed a band of 4,791 bp (Fig. 2a). The PCR-amplified products were purified and sequenced by primer walking using the Dye Terminator Cycle Sequencing kit (Amersham Bioscience, Piscataway, NJ, USA) and Fig. 3 Transcript abnormality in the EYA1 gene. a RT-PCR using primers in exons 2 and 9 respectively. The sample from the patient (right) shows two bands of 268 bp and 700 bp respectively, while the control sample (left) clearly shows a single band of 700 bp. b Direct sequence of the lower RT-PCR band revealed that exon 3 is immediately followed by exon 8 in the present case, which means that exons 4 to 7 have been skipped

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an ABI Prism 310 DNA sequencer (Applied Biosystems). Sequencing revealed a 17,770-bp EYA1 deletion starting from the last eight bases of exon 4 and proceeding to bp 1,217 of intron 7. Furthermore, in place of this deleted region was inserted an L1 element of 3,756 bases (Fig. 2b). These results confirmed the deletion of exons 5–7 detected by MLPA and additionally identified the exact deletion break point junctions. Since the deletion starts from the eight last bases of exon 4, the deletion thereby includes the splice donor site for this exon. The MLPA did not detect the eight bases deleted in the 3′ end of exon 4, as the MLPA probe for this exon is located in the 5′ end of exon 4. The inserted L1 element contains no poly (A) tail and is integrated in antisense orientation with respect to EYA1. Interestingly, the break point in intron 7 was located within a 316-bp repeat element (L1ME4a) belonging to the L1 element class of repeats. RT-PCR analysis Finally, we performed mRNA expression analysis using peripheral blood mononuclear cells from the patient. Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed onto cDNA by using random hexamers and the Superscript III kit (Invitrogen). After 40 cycles of PCR amplification using the primers 5′TCAAGCCAGTTCAGATGTTGC-3′, and 5′-ATGTGCTG

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GATACGGTGAGC-3′, nested PCR was performed using the primers 5′-CAGATGTTGCTGTTTCCTCAA-3′ and 5′TGGCCAAAACTGGGATAAGAC-3′, annealing to exon 2 and 9 respectively. PCR products were separated on 2% agarose gel and also sequenced with a DNA sequencer. We used the Human Kidney cDNA library (Invitrogen) to obtain normal control kidney cDNA. PCR products from the patient’s sample showed two bands, one having the same size as the control and the other one being shorter (Fig. 3a). Sequencing of the shorter PCR product revealed the complete absence of exons 4 to 7, and consequently, exon 3 was directly joined to exon 8 (Fig. 3b). These results demonstrated that the allele with the EYA1 deletion in combination with L1 element insertion on the mRNA level caused skipping of EYA1 exons 4 to 7 and that this mutation created no novel splice donor site. On the mRNA level deletion of exons 4–7 is predicted to lead to deletion of 432 bases; c.124_432del309 (according to transcript variant NM_000503.4). On the protein level this is predicted to lead to an in-frame deletion of 144 amino acids, p.Val42_Gln185del in EYA1.

Subsequent to MLPA analysis we analyzed genomic DNA by PCR in order to precisely determine the break points of this large deletion. Interestingly, we detected, in combination with the deletion, the presence of an L1 element inserted between the final 9 bp of exon 4 and bp 1,218 of intron 7 in the EYA1 gene. L1 belongs to the retrotransposons that are mobile elements, and the most abundant and the only active autonomous non-long terminal repeat (non-LTR) retrotransposons in the human genome [14]. There are some reports of L1 element insertion causing various human diseases, but L1 retrotransposon-associated large genomic deletions have rarely been reported. EYA1 gene mutation by short interspersed nuclear elements (SINE) retrotransposon was reported by Abdelhak et al. [20]. They reported that the Alu sequence was inserted into the exon 10 of the EYA1 gene. Therefore, to the best of our knowledge, our patient is the first case of EYA1 mutation by L1 element insertion. In conclusion, we report the first case of BOR syndrome caused by a large EYA1 gene deletion associated with L1 insertion. We believe that this report provides useful insight into the association of L1 transposition and human disease.


Acknowledgements The authors thank Dr. N. Fujimoto and Prof. T. Matsumoto, Department of Urology, Dr. T. Miyamoto and Dr. M. Tamura, Kidney Center, University of Occupational and Environmental Health, Japan, and laboratory technician Marianne Lodahl, Wilhelm Johannsen Centre for Functional Genome Research, The Panum Institute, University of Copenhagen, Denmark. We would like to acknowledge financial support of these studies from the Lundbeck Foundation and Widex AS.

Our patient with conductive hearing loss, preauricular fistulae, and renal insufficiency was diagnosed as having BOR syndrome caused by EYA1 mutations due to L1 retrotransposon insertion by genetic analysis. EYA1 encodes a protein tyrosine phosphatase that plays a critical role in signal transduction during the process of human ear and kidney formation [15]. Although recent studies indicate a lower frequency of EYA1 gene mutation [16], deletions and duplications in EYA1 have been identified in up to 20% of BOR patients [17]. Therefore, prior to sequencing of EYA1 (17 PCRs per patient), we performed EYA1 MLPA analysis (one ligation and one PCR per patient). When applied in this order, the two methods provide the most time- and cost-efficient strategy for molecular diagnosis of EYA1 [16] compared with previous methods used for identifying EYA1 rearrangements including Southern blotting [4, 18] and semiquantitative PCR [16]. The advantage of MLPA analysis compared with other methods such as semiquantitative and long-range PCR is that all exons can be analyzed at the same time in a single reaction tube. One previous study identified deletion of exon 9 in a familial case and deletion of exons 9–10 in a sporadic case respectively by applying MLPA analysis of EYA1 [11]. The present study is the second report identifying partial EYA1 deletion by MLPA analysis. As MLPA is more rapid and sensitive compared with previous technologies, we believe that it is the most appropriate method for the first step of mutational screening of the EYA1 gene [19].

Disclosure A Health Labour Sciences Research Grant for the Research on Measures for Intractable Diseases (H21-nanchi-ippan103 to K.I. and K.N.).

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