J Nephrol DOI 10.1007/s40620-014-0112-x
ORIGINAL ARTICLE
SIX1 gene: absence of mutations in children with isolated congenital anomalies of kidney and urinary tract Susanna Negrisolo • Sonia Centi • Elisa Benetti Giulia Ghirardo • Manuela Della Vella • Luisa Murer • Lina Artifoni
•
Received: 8 January 2014 / Accepted: 13 May 2014 Ó Italian Society of Nephrology 2014
Abstract Background Mutations in human SIX1 gene cause branchiootorenal or branchiootic syndrome. Six1 deficient mice exhibit uni- or bilateral renal hypoplasia or kidney agenesis. Furthermore a lack of Six1 gene in the ureter leads to hydroureter and hydronephrosis. These murine malformations resemble human kidney and urinary tract congenital anomalies (CAKUT), a group of diseases with a diverse anatomical spectrum which includes duplex collecting system as much as urethra kidney and ureteropelvic anomalies. Our study focuses on whether mutations or deletion of this gene may be associated with nonsyndromic CAKUT. Methods Fifty unrelated patients (13–21 years) with nonsyndromic CAKUT were retrospectively recruited for SIX1 sequence variations analysis, and compared to three subjects without malformative nephrouropathies (controls). SIX1 coding sequence was screened by high resolution
melt analysis (HRMA) and by Sanger direct sequencing. A quantitative comparative real-time polymerase chain reaction (PCR) was later performed in order to detect the presence of SIX1 gene deletion. Results We did not find significant differences in the HRMA melting curves for each of the SIX1 coding exons between patients and controls, as also confirmed by Sanger direct sequencing. Moreover quantitative comparative realtime PCR for SIX1 and data normalization excluded total SIX1 gene deletion in our patients. Conclusions We did not find sequence variations in SIX1 coding regions or complete gene deletion in our CAKUT population. These results suggest that alterations in these sequences are unlikely to be a major cause of nonsyndromic CAKUT. Nevertheless, further studies are necessary to understand if altered SIX1 expression may play a role in human development of kidney and urinary tract congenital anomalies.
S. Negrisolo and S. Centi have contributed equally to this work.
Keywords CAKUT Children Kidney and urinary tract congenital anomalies Renal transplant recipients SIX1 gene
Electronic supplementary material The online version of this article (doi:10.1007/s40620-014-0112-x) contains supplementary material, which is available to authorized users. S. Negrisolo S. Centi M. Della Vella L. Artifoni (&) Laboratory of Immunopathology and Molecular Biology of the Kidney, Women’s and Children’s Health Department, University of Padua, Via Giustiniani 3, 35128 Padua, Italy e-mail: [email protected]
E. Benetti G. Ghirardo L. Murer Pediatric Nephrology, Dialysis and Transplant Unit, Women’s and Children’s Health Department, University of Padua, Via Giustiniani 3, 35128 Padua, Italy e-mail: [email protected]
S. Negrisolo e-mail: [email protected]
G. Ghirardo e-mail: [email protected]
S. Centi e-mail: [email protected]
L. Murer e-mail: [email protected]
M. Della Vella e-mail: [email protected]
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Introduction Human SIX1 gene, a member of the mammalian homeobox family (SIX1–SIX6), maps to chromosome 14q23.1 and consists of two exons, coding for a transcription factor of 284 amino acids that is widely expressed in many tissues in mammalian organogenesis (e.g. ear, branchial arch and kidney development) [1, 2]. SIX1 is a master regulatory protein which is required for the activation of multiple sets of genes during the earliest phase of kidney development. It is expressed in the metanephric mesenchyme differentiation (MM) before ureteric bud (UB) branching morphogenesis. Ureteric budding during MM differentiation represents a crucial step which is regulated by a complex network of genes that, if altered, may lead to impaired ureterorenal development and renal anomalies [3–5]. In Six1-/- mutant mice, UB grows out normally, elongates to differentiate into ureter, but it fails to form the collecting system [6–8]. Despite these observations, the mechanism by which Six1 induces epithelial branching to form the collecting duct system remains unclear. Kidney and urinary tract congenital anomalies (CAKUT) may vary phenotypically and may affect kidney(s) alone or the lower urinary tract. The spectrum of anomalies ranges from more common anomalies like vesicoureteral reflux (VUR) to more severe malformations such as uni- or bilateral renal hypo/dysplasia and renal agenesis. CAKUT are common in humans, occurring approximately in 1 out of 500 newborns [9]. Congenital renal anomalies can be considered a major cause of chronic renal failure in infants and young children, but little is known about their molecular pathogenesis. In humans, SIX1 mutations cause branchiootorenal (BOR) or branchiootic (BO) syndromes [10, 11]. BOR (MIM# 113650) is a clinically heterogeneous autosomal dominant form of syndromic hearing loss which is characterized by variable hearing impairment, pinnae malformations, branchial arch remnants and renal anomalies (e.g. collecting system duplication, renal hypoplasia, dysplasia and agenesis, hydroureter or megaureter, and hydronephrosis) [12]. BO (MIM# 608389) without renal anomalies is thought to be a variant of the same disorder. As Six1 deficient mice exhibit malformations that resemble human CAKUT (i.e. uni- or bilateral renal hypoplasia, kidney agenesis [13], hydroureter and hydronephrosis) [8], we evaluated whether SIX1 gene deletion or point mutations may be associated with nonsyndromic kidney and urinary tract congenital anomalies.
Subjects and methods Fifty unrelated patients (35 males and 15 females, aged 13–21 years) with nonsyndromic CAKUT (renal hypo/
123
dysplasia with or without VUR, oligomeganephronia, unior bilateral renal agenesis, ureteral anomalies with or without VUR, and hypoplasia of urethra) were retrospectively analyzed for SIX1 variations. All patients had been previously checked in terms of clinical signs and symptoms, as well as for family and personal medical history. They had also undergone diagnostic work-up for renal disorders through kidney and urinary tract ultrasound scan as well as contrast voiding cystourethrogram and diuretic renography. Renal dysplasia was defined as ‘poor cortical–medullary differentiation’ or ‘diffuse hyperechogenicity’ on sonography and renal hypoplasia was defined as ‘kidney length \ 2 standard deviations (SD) for age’ [14, 15]. Three subjects without malformative nephrouropathies (negative renal ultrasound nuclear scan and voiding cystourethrogram) were also analyzed for SIX1 encoding region by Sanger direct sequencing, and were employed as a control group. The study was approved by our Institutional Ethical Committee and informed consent for genetic screening was obtained from the patients themselves or their parents. Mutation and deletion screening Samples of frozen blood (200 ll) were used to isolate genomic DNA with a DNA extraction kit, according to manufacturer instructions. DNA concentration was determined by spectrophotometry (NanovueÒ). Exon 1 and the coding region of exon 2 (including exon–intron boundaries) of SIX1 gene were amplified using overlapping oligonucleotide primer pairs designed by the software Primer3 Input (http://frodo.wi.mit.edu/). The genomic reference sequences for SIX1 gene (GenBank: NC_000014.8 and cDNA GenBank: NM_005982.3) are available from The National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). SIX1 coding sequence was screened by high resolution melt analysis (HRMA) with RotorGeneTM 6000. Sanger direct sequencing of both strands was performed using a Big Dye Terminator v3.1 Cycle Sequencing Kit and analyzed on an ABI PRISMÒ 3100 Genetic Analyzer for all DNA samples. A quantitative comparative real-time polymerase chain reaction (PCR) using SIX1 coding exon 1 primers and MTR primers as reference gene [16] was later performed in order to detect the presence of SIX1 gene deletion. MTR codifies for methionine synthase, an enzyme that plays an important role in folate metabolism, since it catalyzes the reaction of homocysteine transmethylation to methionine. Deletion analysis was performed by RotorGeneTM 6000 using EvaGreenÒ DNA dye and experiments were done in triplicate for each data point.
J Nephrol
Results We retrospectively analyzed genomic DNA of 50 patients aged 13–21 years with CAKUT for SIX1 sequence variations. Mean age at examination was 14 years and males represented 70 % of the cohort. Clinical and instrumental analyses revealed the renal and urinary tract anomalies shown in Table 1. Twenty-four of the 50 patients had undergone renal transplantation for end-stage renal disease (ESRD) due to CAKUT at the Pediatric Nephrology, Dialysis and Transplantation Unit of the Women’s and Children’s Health Department of Padua (Italy): 6/50 Table 1 Congenital anomalies of kidney and urinary tract (CAKUT) observed in our study population CAKUT phenotype
Number patients/ total patients
Bilateral renal hypo/dysplasia
10/50
Bilateral renal hypo/dysplasia associated with primary vesicoureteral reflux
13/50
Unilateral renal agenesis and contralateral hypo/dysplasia
5/50
Bilateral renal agenesis
1/50
Bilateral oligomeganephronic renal hypoplasia Ureteropelvic junction obstruction
1/50 9/50
Megaureter
6/50
Ureteral duplicity
3/50
Hypoplasia of urethra
1/50
Hydronephrosis with bilateral IV grade vesicoureteral reflux
1/50
showed ESRD and 20/50 showed normal renal function. None had deafness, branchial anomalies or other extrarenal anomalies. No family history for renal anomalies or hearing loss was recorded. We did not find significant differences in the HRMA melting curves (Fig. 1) for each of the coding exons of SIX1 (including exon–intron boundaries) in our CAKUT cohort compared to controls. Sanger direct sequencing confirmed these results. None of the sequence variations reported in Ensembl Genome Browser (www.ensembl.org/) were present in our cohort, according to their very low frequencies. Quantitative comparative real-time PCR for SIX1 and relative data normalization to values obtained from our reference gene MTR also excluded total SIX1 gene deletion in our cohort.
Discussion Studies on knockout mice and cell cultures have identified murine Six1 gene homolog of Drosophila sine oculis as an important factor in the early stages of renal development. This gene is expressed in the metanephric mesenchyme both before and after ureteric budding as well as during condensation. It is not expressed, however, in the Wolffian duct or in the ureteric bud epithelium. In mature kidney, Six1 expression is restricted to a subpopulation of collecting tubule epithelial cells [5, 17]. Mutations in human SIX1 gene are associated with BOR and BO syndromes [11, 18–20]. However, the frequency of SIX1 mutations is less than 4 % [18] and, in general, 40 %
Fig. 1 Example of the profiles obtained with HRMA: the melting curves of amplicons of 197 bp of exon 1 are shown. No differences were observed between 20 patients and 3 controls
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of patients with BOR/BO syndromes carry mutations in EYA1, the human homolog of the Drosophila eyes absent gene. Murine Eya1 gene and Six1 gene products are coexpressed during branchial arch system, otic, and kidney development. Six1 forms a complex with Eya1 and synergistically activates the target gene promoter through nuclear translocation of Eya1 by Six1 [21]. The experiments of Patrick et al. [22] demonstrated that the SIX1 BOR mutations contributed to the syndrome pathogenesis by abolishing the formation of the SIX1–EYA1 complex or by diminishing SIX1 ability to bind DNA. Moreover, Six1 is differentially expressed during ureter morphogenesis and is required for the maintenance and normal differentiation of smooth muscle progenitors. A lack of Six1 in the murine ureter leads to hydroureter and hydronephrosis. In the ureteral mesenchymal progenitors Six1 acts as a co-transcription factor for Tbx18 (an important T-box protein for smooth muscle cells differentiation) by forming a complex that regulates signaling pathways in order to control mesenchymal–epithelial interaction during ureter patterning [4]. This interaction was also confirmed in HEK293 cells in as much as the functional analysis of two missense mutations of SIX1 gene from BOR patients demonstrated a decrease or complete abolition of SIX1–TBX18 complex formation [4]. According to these findings, we included 19 patients with ureteral anomalies (i.e. hydroureter and hydronephrosis) in our study, but no variation of SIX1 sequence was found in these patients. More recently, however, high-throughput mutation analyses and next generation sequencing in nonsyndromic familial and isolated CAKUT [23–26] have identified new causative genes such as WNT4, FRAS1, FREM2, RET, BMP4, TRAP1 and DSTYK. These findings support a wide locus heterogeneity for CAKUT, but their frequency decreases from 10 % for FRAS1 mutations to 5 % for WNT4 mutations and even further down to 0.15 % for TRAP1 gene mutations. Only nine SIX1 causative mutations are reported in the Human Gene Mutation Database (HGMD) in patients with BOR/BO. Only one single patient showed both SIX1 missense substitution and PAX2 gene mutation. In the latter case, the authors suggested that SIX1 may act as a modifier gene in renal coloboma syndrome [27]. In our study no mutations and no gene deletion were identified in our patients with CAKUT, suggesting that alterations in the coding sequence of SIX1 gene is probably not a major cause of CAKUT. A lower frequency of mutations, however, cannot be excluded altogether. More recently, however, Hwang et al. [28] analyzed 17 dominant CAKUT-causing genes (BMP4, BMP7, CDC5L, CHD1L, EYA1, GATA3, HNF1B, PAX2, RET, ROBO2, SALL1, SIX1, SIX2, SIX4, SOX17, UMOD and UPK3A) by target exome sequencing in 749 individuals from 650
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families with CAKUT. The authors detected 37 heterozygous mutations in 12/17 genes but, in line with our findings, no causative mutations were identified in coding regions of SIX1. Recent animal studies in vivo and in vitro [29] identified porcine Six1 gene core promoter region and demonstrated that DNA methylation level of the region is critical for promoter activity. No studies, however, have to date been carried out to investigate transcriptional regulatory mechanisms of human SIX1. The gene promoter is a critical cis-regulatory element for transcription, and sequence variations in this region can be a putative cause of disturbed transcriptional regulation that leads to the disease. Depending on the genetic defect location and nature, a mutation in the promoter region of a gene may disrupt the normal process of gene activation and may decrease or increase the level of mRNA and thus of its protein [30]. Taken together, these data suggest that SIX1 coding sequence mutations are not responsible for the CAKUT phenotype in our cohort. Therefore, the frequency of SIX1 mutations in nonsyndromic CAKUT may be assumed to be very low and alterations of transcriptional regulatory mechanisms of human SIX1 gene may be decisive for the CAKUT phenotype. Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
References 1. Ohto H, Takizawa T, Saito T, Kobayashi M, Ikeda K, Kawakami K (1998) Tissue and developmental distribution of six family gene products. Int J Dev Biol 42(2):141–148 2. Noguchi Y, Ito T, Nishio A, Honda K, Kitamura K (2011) Audiovestibular findings in a branchio-oto syndrome patient with a SIX1 mutation. Acta Otolaryngol 131(4):413–418 3. Nie X, Xu J, El-Hashash A, Xu PX (2011) Six1 regulates Grem1 expression in the metanephric mesenchyme to initiate branching morphogenesis. Dev Biol 352(1):141–151 4. Nie X, Sun J, Gordon RE, Cai CL, Xu PX (2010) SIX1 acts synergistically with TBX18 in mediating ureteral smooth muscle formation. Development 137(5):755–765 5. Xu PX, Zheng W, Huang L, Maire P, Laclef C, Silvius D (2003) Six1 is required for the early organogenesis of mammalian kidney. Development 130(14):3085–3094 6. Dressler GR (2006) The cellular basis of kidney development. Annu Rev Cell Dev Biol 22:509–529 (Review) 7. Costantini F (2006) Renal branching morphogenesis: concepts, questions, and recent advances. Differentiation 74(7):402–421 Review 8. Shah MM, Sampogna RV, Sakurai H, Bush KT, Nigam SK (2004) Branching morphogenesis and kidney disease. Development 131(7):1449–1462 (Review) 9. Nakanishi K, Yoshikawa N (2003) Genetic disorders of human congenital anomalies of the kidney and urinary tract (CAKUT). Pediatr Int 45(5):610–616
J Nephrol 10. Ruf RG, Xu PX, Silvius D et al (2004) SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1–SIX1–DNA complexes. Proc Natl Acad Sci USA 101(21):8090–8095 11. Krug P, Morinie`re V, Marlin S et al (2011) Mutation screening of the EYA1, SIX1, and SIX5 genes in a large cohort of patients harboring branchio-oto-renal syndrome calls into question the pathogenic role of SIX5 mutations. Hum Mutat 32(2):183–190 12. Izzedine H, Tankere F, Launay-Vacher V, Deray G (2004) Ear and kidney syndromes: molecular versus clinical approach. Kidney Int 65(2):369–385 (Review) 13. Kobayashi H, Kawakami K, Asashima M, Nishinakamura R (2007) Six1 and Six4 are essential for Gdnf expression in the metanephric mesenchyme and ureteric bud formation, while Six1 deficiency alone causes mesonephric-tubule defects. Mech Dev 124(4):290–303 14. Dinkel E, Ertel M, Dittrich M, Peters H, Berres M, SchulteWissermann H (1985) Kidney size in childhood. Sonographical growth charts for kidney length and volume. Pediatr Radiol 15(1):38–43 15. Han BK, Babcock DS (1985) Sonographic measurements and appearance of normal kidneys in children. Am J Roentgenol 145(3):611–616 16. Harmon DL, Shields DC, Woodside JV et al (1999) Methionine synthase D919G polymorphism is a significant but modest determinant of circulating homocysteine concentrations. Genet Epidemiol 17(4):298–309 17. Yu J, McMahon AP, Valerius MT (2004) Recent genetic studies of mouse kidney development. Curr Opin Genet Dev 14(5): 550–557 18. Kochhar A, Orten DJ, Sorensen JL et al (2008) SIX1 mutation screening in 247 branchio-oto-renal syndrome families: a recurrent missense mutation associated with BOR. Hum Mutat 29(4):565–577 19. Ruf RG, Berkman J, Wolf MT et al (2003) A gene locus for branchio-otic syndrome maps to chromosome 14q21.3-q24.3. J Med Genet 40(7):515–519 20. Sanggaard KM, Rendtorff ND, Kjaer KW et al (2007) Branchiooto-renal syndrome: detection of EYA1 and SIX1 mutations in
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five out of six Danish families by combining linkage, MLPA and sequencing analyses. Eur J Hum Genet 15(11):1121–1131 Ozaki H, Watanabe Y, Ikeda K, Kawakami K (2002) Impaired interactions between mouse Eya1 harboring mutations found in patients with branchio-oto-renal syndrome and Six, Dach, and G proteins. J Hum Genet 47(3):107–116 Patrick AN, Schiemann BJ, Yang K, Zhao R, Ford HL (2009) Biochemical and functional characterization of six SIX1 branchiooto-renal syndrome mutations. J Biol Chem 284(31):20781–20790 Vivante A, Mark-Danieli M, Davidovits M et al (2013) Renal hypodysplasia associates with a WNT4 variant that causes aberrant canonical WNT signaling. J Am Soc Nephrol 24(4): 550–558 Saisawat P, Tasic V, Vega-Warner V et al (2012) Identification of two novel CAKUT-causing genes by massively parallel exon resequencing of candidate genes in patients with unilateral renal agenesis. Kidney Int 81(2):196–200 Saisawat P, Kohl S, Hilger AC et al (2013) Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association. Kidney Int. doi:10. 1038/ki.2013.417 Sanna-Cherchi S, Sampogna RV, Papeta N et al (2013) Mutations in DSTYK and dominant urinary tract malformations. N Engl J Med 369(7):621–629 Weber S, Moriniere V, Knu¨ppel T et al (2006) Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study. J Am Soc Nephrol 17(10):2864–2870 Hwang DY, Dworschak GC, Kohl S et al (2014) Mutations in 12 known dominant disease-causing genes clarify many congenital anomalies of the kidney and urinary tract. Kidney Int. doi:10. 1038/ki.2013.508 Wu W, Ren Z, Liu H et al (2013) Core promoter analysis of porcine Six1 gene and its regulation of the promoter activity by CpG methylation. Gene 529(2):238–244 de Vooght KM, van Wijk R, van Solinge WW (2009) Management of gene promoter mutations in molecular diagnostics. Clin Chem 55(4):698–708. doi:10.1373/clinchem.2008.120931
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ORIGINAL ARTICLE
SIX1 gene: absence of mutations in children with isolated congenital anomalies of kidney and urinary tract Susanna Negrisolo • Sonia Centi • Elisa Benetti Giulia Ghirardo • Manuela Della Vella • Luisa Murer • Lina Artifoni
•
Received: 8 January 2014 / Accepted: 13 May 2014 Ó Italian Society of Nephrology 2014
Abstract Background Mutations in human SIX1 gene cause branchiootorenal or branchiootic syndrome. Six1 deficient mice exhibit uni- or bilateral renal hypoplasia or kidney agenesis. Furthermore a lack of Six1 gene in the ureter leads to hydroureter and hydronephrosis. These murine malformations resemble human kidney and urinary tract congenital anomalies (CAKUT), a group of diseases with a diverse anatomical spectrum which includes duplex collecting system as much as urethra kidney and ureteropelvic anomalies. Our study focuses on whether mutations or deletion of this gene may be associated with nonsyndromic CAKUT. Methods Fifty unrelated patients (13–21 years) with nonsyndromic CAKUT were retrospectively recruited for SIX1 sequence variations analysis, and compared to three subjects without malformative nephrouropathies (controls). SIX1 coding sequence was screened by high resolution
melt analysis (HRMA) and by Sanger direct sequencing. A quantitative comparative real-time polymerase chain reaction (PCR) was later performed in order to detect the presence of SIX1 gene deletion. Results We did not find significant differences in the HRMA melting curves for each of the SIX1 coding exons between patients and controls, as also confirmed by Sanger direct sequencing. Moreover quantitative comparative realtime PCR for SIX1 and data normalization excluded total SIX1 gene deletion in our patients. Conclusions We did not find sequence variations in SIX1 coding regions or complete gene deletion in our CAKUT population. These results suggest that alterations in these sequences are unlikely to be a major cause of nonsyndromic CAKUT. Nevertheless, further studies are necessary to understand if altered SIX1 expression may play a role in human development of kidney and urinary tract congenital anomalies.
S. Negrisolo and S. Centi have contributed equally to this work.
Keywords CAKUT Children Kidney and urinary tract congenital anomalies Renal transplant recipients SIX1 gene
Electronic supplementary material The online version of this article (doi:10.1007/s40620-014-0112-x) contains supplementary material, which is available to authorized users. S. Negrisolo S. Centi M. Della Vella L. Artifoni (&) Laboratory of Immunopathology and Molecular Biology of the Kidney, Women’s and Children’s Health Department, University of Padua, Via Giustiniani 3, 35128 Padua, Italy e-mail: [email protected]
E. Benetti G. Ghirardo L. Murer Pediatric Nephrology, Dialysis and Transplant Unit, Women’s and Children’s Health Department, University of Padua, Via Giustiniani 3, 35128 Padua, Italy e-mail: [email protected]
S. Negrisolo e-mail: [email protected]
G. Ghirardo e-mail: [email protected]
S. Centi e-mail: [email protected]
L. Murer e-mail: [email protected]
M. Della Vella e-mail: [email protected]
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Introduction Human SIX1 gene, a member of the mammalian homeobox family (SIX1–SIX6), maps to chromosome 14q23.1 and consists of two exons, coding for a transcription factor of 284 amino acids that is widely expressed in many tissues in mammalian organogenesis (e.g. ear, branchial arch and kidney development) [1, 2]. SIX1 is a master regulatory protein which is required for the activation of multiple sets of genes during the earliest phase of kidney development. It is expressed in the metanephric mesenchyme differentiation (MM) before ureteric bud (UB) branching morphogenesis. Ureteric budding during MM differentiation represents a crucial step which is regulated by a complex network of genes that, if altered, may lead to impaired ureterorenal development and renal anomalies [3–5]. In Six1-/- mutant mice, UB grows out normally, elongates to differentiate into ureter, but it fails to form the collecting system [6–8]. Despite these observations, the mechanism by which Six1 induces epithelial branching to form the collecting duct system remains unclear. Kidney and urinary tract congenital anomalies (CAKUT) may vary phenotypically and may affect kidney(s) alone or the lower urinary tract. The spectrum of anomalies ranges from more common anomalies like vesicoureteral reflux (VUR) to more severe malformations such as uni- or bilateral renal hypo/dysplasia and renal agenesis. CAKUT are common in humans, occurring approximately in 1 out of 500 newborns [9]. Congenital renal anomalies can be considered a major cause of chronic renal failure in infants and young children, but little is known about their molecular pathogenesis. In humans, SIX1 mutations cause branchiootorenal (BOR) or branchiootic (BO) syndromes [10, 11]. BOR (MIM# 113650) is a clinically heterogeneous autosomal dominant form of syndromic hearing loss which is characterized by variable hearing impairment, pinnae malformations, branchial arch remnants and renal anomalies (e.g. collecting system duplication, renal hypoplasia, dysplasia and agenesis, hydroureter or megaureter, and hydronephrosis) [12]. BO (MIM# 608389) without renal anomalies is thought to be a variant of the same disorder. As Six1 deficient mice exhibit malformations that resemble human CAKUT (i.e. uni- or bilateral renal hypoplasia, kidney agenesis [13], hydroureter and hydronephrosis) [8], we evaluated whether SIX1 gene deletion or point mutations may be associated with nonsyndromic kidney and urinary tract congenital anomalies.
Subjects and methods Fifty unrelated patients (35 males and 15 females, aged 13–21 years) with nonsyndromic CAKUT (renal hypo/
123
dysplasia with or without VUR, oligomeganephronia, unior bilateral renal agenesis, ureteral anomalies with or without VUR, and hypoplasia of urethra) were retrospectively analyzed for SIX1 variations. All patients had been previously checked in terms of clinical signs and symptoms, as well as for family and personal medical history. They had also undergone diagnostic work-up for renal disorders through kidney and urinary tract ultrasound scan as well as contrast voiding cystourethrogram and diuretic renography. Renal dysplasia was defined as ‘poor cortical–medullary differentiation’ or ‘diffuse hyperechogenicity’ on sonography and renal hypoplasia was defined as ‘kidney length \ 2 standard deviations (SD) for age’ [14, 15]. Three subjects without malformative nephrouropathies (negative renal ultrasound nuclear scan and voiding cystourethrogram) were also analyzed for SIX1 encoding region by Sanger direct sequencing, and were employed as a control group. The study was approved by our Institutional Ethical Committee and informed consent for genetic screening was obtained from the patients themselves or their parents. Mutation and deletion screening Samples of frozen blood (200 ll) were used to isolate genomic DNA with a DNA extraction kit, according to manufacturer instructions. DNA concentration was determined by spectrophotometry (NanovueÒ). Exon 1 and the coding region of exon 2 (including exon–intron boundaries) of SIX1 gene were amplified using overlapping oligonucleotide primer pairs designed by the software Primer3 Input (http://frodo.wi.mit.edu/). The genomic reference sequences for SIX1 gene (GenBank: NC_000014.8 and cDNA GenBank: NM_005982.3) are available from The National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). SIX1 coding sequence was screened by high resolution melt analysis (HRMA) with RotorGeneTM 6000. Sanger direct sequencing of both strands was performed using a Big Dye Terminator v3.1 Cycle Sequencing Kit and analyzed on an ABI PRISMÒ 3100 Genetic Analyzer for all DNA samples. A quantitative comparative real-time polymerase chain reaction (PCR) using SIX1 coding exon 1 primers and MTR primers as reference gene [16] was later performed in order to detect the presence of SIX1 gene deletion. MTR codifies for methionine synthase, an enzyme that plays an important role in folate metabolism, since it catalyzes the reaction of homocysteine transmethylation to methionine. Deletion analysis was performed by RotorGeneTM 6000 using EvaGreenÒ DNA dye and experiments were done in triplicate for each data point.
J Nephrol
Results We retrospectively analyzed genomic DNA of 50 patients aged 13–21 years with CAKUT for SIX1 sequence variations. Mean age at examination was 14 years and males represented 70 % of the cohort. Clinical and instrumental analyses revealed the renal and urinary tract anomalies shown in Table 1. Twenty-four of the 50 patients had undergone renal transplantation for end-stage renal disease (ESRD) due to CAKUT at the Pediatric Nephrology, Dialysis and Transplantation Unit of the Women’s and Children’s Health Department of Padua (Italy): 6/50 Table 1 Congenital anomalies of kidney and urinary tract (CAKUT) observed in our study population CAKUT phenotype
Number patients/ total patients
Bilateral renal hypo/dysplasia
10/50
Bilateral renal hypo/dysplasia associated with primary vesicoureteral reflux
13/50
Unilateral renal agenesis and contralateral hypo/dysplasia
5/50
Bilateral renal agenesis
1/50
Bilateral oligomeganephronic renal hypoplasia Ureteropelvic junction obstruction
1/50 9/50
Megaureter
6/50
Ureteral duplicity
3/50
Hypoplasia of urethra
1/50
Hydronephrosis with bilateral IV grade vesicoureteral reflux
1/50
showed ESRD and 20/50 showed normal renal function. None had deafness, branchial anomalies or other extrarenal anomalies. No family history for renal anomalies or hearing loss was recorded. We did not find significant differences in the HRMA melting curves (Fig. 1) for each of the coding exons of SIX1 (including exon–intron boundaries) in our CAKUT cohort compared to controls. Sanger direct sequencing confirmed these results. None of the sequence variations reported in Ensembl Genome Browser (www.ensembl.org/) were present in our cohort, according to their very low frequencies. Quantitative comparative real-time PCR for SIX1 and relative data normalization to values obtained from our reference gene MTR also excluded total SIX1 gene deletion in our cohort.
Discussion Studies on knockout mice and cell cultures have identified murine Six1 gene homolog of Drosophila sine oculis as an important factor in the early stages of renal development. This gene is expressed in the metanephric mesenchyme both before and after ureteric budding as well as during condensation. It is not expressed, however, in the Wolffian duct or in the ureteric bud epithelium. In mature kidney, Six1 expression is restricted to a subpopulation of collecting tubule epithelial cells [5, 17]. Mutations in human SIX1 gene are associated with BOR and BO syndromes [11, 18–20]. However, the frequency of SIX1 mutations is less than 4 % [18] and, in general, 40 %
Fig. 1 Example of the profiles obtained with HRMA: the melting curves of amplicons of 197 bp of exon 1 are shown. No differences were observed between 20 patients and 3 controls
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of patients with BOR/BO syndromes carry mutations in EYA1, the human homolog of the Drosophila eyes absent gene. Murine Eya1 gene and Six1 gene products are coexpressed during branchial arch system, otic, and kidney development. Six1 forms a complex with Eya1 and synergistically activates the target gene promoter through nuclear translocation of Eya1 by Six1 [21]. The experiments of Patrick et al. [22] demonstrated that the SIX1 BOR mutations contributed to the syndrome pathogenesis by abolishing the formation of the SIX1–EYA1 complex or by diminishing SIX1 ability to bind DNA. Moreover, Six1 is differentially expressed during ureter morphogenesis and is required for the maintenance and normal differentiation of smooth muscle progenitors. A lack of Six1 in the murine ureter leads to hydroureter and hydronephrosis. In the ureteral mesenchymal progenitors Six1 acts as a co-transcription factor for Tbx18 (an important T-box protein for smooth muscle cells differentiation) by forming a complex that regulates signaling pathways in order to control mesenchymal–epithelial interaction during ureter patterning [4]. This interaction was also confirmed in HEK293 cells in as much as the functional analysis of two missense mutations of SIX1 gene from BOR patients demonstrated a decrease or complete abolition of SIX1–TBX18 complex formation [4]. According to these findings, we included 19 patients with ureteral anomalies (i.e. hydroureter and hydronephrosis) in our study, but no variation of SIX1 sequence was found in these patients. More recently, however, high-throughput mutation analyses and next generation sequencing in nonsyndromic familial and isolated CAKUT [23–26] have identified new causative genes such as WNT4, FRAS1, FREM2, RET, BMP4, TRAP1 and DSTYK. These findings support a wide locus heterogeneity for CAKUT, but their frequency decreases from 10 % for FRAS1 mutations to 5 % for WNT4 mutations and even further down to 0.15 % for TRAP1 gene mutations. Only nine SIX1 causative mutations are reported in the Human Gene Mutation Database (HGMD) in patients with BOR/BO. Only one single patient showed both SIX1 missense substitution and PAX2 gene mutation. In the latter case, the authors suggested that SIX1 may act as a modifier gene in renal coloboma syndrome [27]. In our study no mutations and no gene deletion were identified in our patients with CAKUT, suggesting that alterations in the coding sequence of SIX1 gene is probably not a major cause of CAKUT. A lower frequency of mutations, however, cannot be excluded altogether. More recently, however, Hwang et al. [28] analyzed 17 dominant CAKUT-causing genes (BMP4, BMP7, CDC5L, CHD1L, EYA1, GATA3, HNF1B, PAX2, RET, ROBO2, SALL1, SIX1, SIX2, SIX4, SOX17, UMOD and UPK3A) by target exome sequencing in 749 individuals from 650
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families with CAKUT. The authors detected 37 heterozygous mutations in 12/17 genes but, in line with our findings, no causative mutations were identified in coding regions of SIX1. Recent animal studies in vivo and in vitro [29] identified porcine Six1 gene core promoter region and demonstrated that DNA methylation level of the region is critical for promoter activity. No studies, however, have to date been carried out to investigate transcriptional regulatory mechanisms of human SIX1. The gene promoter is a critical cis-regulatory element for transcription, and sequence variations in this region can be a putative cause of disturbed transcriptional regulation that leads to the disease. Depending on the genetic defect location and nature, a mutation in the promoter region of a gene may disrupt the normal process of gene activation and may decrease or increase the level of mRNA and thus of its protein [30]. Taken together, these data suggest that SIX1 coding sequence mutations are not responsible for the CAKUT phenotype in our cohort. Therefore, the frequency of SIX1 mutations in nonsyndromic CAKUT may be assumed to be very low and alterations of transcriptional regulatory mechanisms of human SIX1 gene may be decisive for the CAKUT phenotype. Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
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