The Mutational Spectrum of WT1 in Male Infertility

Author's Accepted Manuscript The mutational spectrum of WT1 in male infertility Catarina M. Seabra , Sofia Quental , Ana C. Lima , Filipa Carvalho , João Gonçalves , Susana Fernandes , Iris Pereira , Júlia Silva , Patrícia I. Marques , Mário Sousa , Alberto Barros , Susana Seixas , António Amorim , Alexandra M. Lopes

PII: DOI: Reference:

S0022-5347(14)04832-0 10.1016/j.juro.2014.11.004 JURO 11954

To appear in: The Journal of Urology Accepted Date: 3 November 2014 Please cite this article as: Seabra CM, Quental S, Lima AC, Carvalho F, Gonçalves J, Fernandes S, Pereira I, Silva J, Marques PI, Sousa M, Barros A, Seixas S, Amorim A, Lopes AM, The mutational spectrum of WT1® in male infertility, The Journal of Urology (2014), doi: 10.1016/j.juro.2014.11.004. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.

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TITLE The mutational spectrum of WT1 in male infertility AUTHORS

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Catarina M Seabra1,2, Sofia Quental1, Ana C Lima1,3,4,5, Filipa Carvalho6, João Gonçalves7, Susana Fernandes6, Iris Pereira7, Júlia Silva7, Patrícia I Marques1,4, Mário Sousa8, Alberto Barros6, Susana Seixas1, António Amorim1,9, Alexandra M Lopes1 1Institute

of Molecular Pathology and Immunology of the University of Porto, Portugal; 2Health Sciences

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Autonomous Section of the University of Aveiro, Portugal; 3Graduate Program in Areas of Basic and Applied Biology (GABBA); 4Abel Salazar Institute of Biomedical Sciences, University of Porto, Portugal; 5Department of

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Genetics, Washington University School of Medicine, St. Louis, MO, USA; 6Department of Genetics, Faculty of Medicine of the University of Porto, Portugal; 7Department of Human Genetics - National Institute of Health Dr. Ricardo Jorge, Lisboa, Portugal; 8Laboratory of Cell Biology, UMIB, ICBAS, University of Porto, Porto, Portugal; of Sciences of the University of Porto, Portugal.

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9Faculty

Running head: WT1 mutations in male infertility Key words: WT1 mutations; severe spermatogenic failure; rare variants Manuscript word count: 2994

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ABSTRACT Purpose: To evaluate the impact of WT1 mutations in isolated severe spermatogenic impairment in a population of European ancestry. WT1 was first identified as the gene responsible for Wilms’ Tumor and later has been associated with a plethora of clinical phenotypes often accompanied by urogenital defects and male infertility. The recent

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finding of WT1 missense mutations in Chinese azoospermic males without major gonadal malformations has broadened the phenotypic spectrum of WT1 defects and motivated this study.

Materials and methods: We have analyzed the WT1 coding region in a cohort of

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Portuguese non-obstructive azoospermic (NOA; n=194) and severe oligozoospermic (n=188) patients, with increased depth for the exons encoding the regulatory region of the protein. A group of 31 infertile males with clinical history of uni- or bi-lateral

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cryptorchidism and one patient with anorchia were also analyzed.

Results: We found two WT1 missense substitutions at higher frequency in patients than in controls: (i) a novel variant in exon 1 (p.Pro130Leu) disrupting a mammalian-specific polyproline stretch within the self-association domain was more frequent in azoospermia (0.27% vs 0.13%; p=0.549); (ii) a rare variant in a conserved residue in close proximity

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to the first zinc finger (pCys350Arg) was more frequent in severe oligozoospermia (0.80% vs 0.13%; p=0.113).

Conclusions: These results suggest a role for rare WT1 damaging variants in severe spermatogenic failure in populations of European ancestry and claim for large multicenter

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studies in order to fully assess the contribution of WT1 genetic alterations to male

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infertility in the absence of other disease phenotypes.

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INTRODUCTION The Wilms’ tumor 1 (WT1) gene, located at 11p13, encodes a protein with a C-terminal zinc-finger (ZF) domain involved in DNA- and RNA-binding. WT1 acts as a transcription factor through the interaction of WT1 activation and repression domains with its targets [1,2]. This activity is thought to be modified by dimerization of WT1 with other proteins

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(heterodimerization) or with itself (homodimerization), at the N-terminal self-association domain. Genetic defects in WT1 typically result in one of three congenital syndromes Wilms’ tumor, aniridia, genitourinary alteration and mental retardation (WAGR), DenysDrash (DDS) and Frasier syndromes characterized by malformations of the gonadal ridge (gonadal dysgenesis, hypospadias, cryptorchidism) and kidneys (horseshoe kidney, renal

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hypoplasia) [3]. This association concurs with the preponderant role of WT1 in the development and differentiation of the urogenital system.

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Indeed WT1 is a crucial factor in male sex determination, and performs an essential role in the male gonadal differentiation pathway, as supported by the fact that Wt1-/- mice fail to develop gonads [4]. Moreover, several lines of evidence have demonstrated its importance in different stages of testicular and germ cell development. Chang et al. reported that WT1dependent suppression of WNT/β-catenin signaling in Sertoli cells is essential for the normal development of primordial germ cells [5] and studies where Wt1 was specifically

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inactivated in Sertoli cells at different developmental stages have demonstrated its importance in testicular differentiation [6] and in the maintenance of Sertoli cells polarity and spermatogenesis in adulthood [7]. Interestingly, the phenotype of germ cell loss observed in Wt1 conditional knock-out mice resembles that observed in Sertoli cell only

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syndrome and, concordantly, several missense mutations in the WT1 gene were recently described in a cohort of Chinese patients with non-obstructive azoospermia (NOA) [7].

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The phenotypic expression of WT1 defects is variable and dependent on the protein domains that are affected. Patients with C-terminal missense or nonsense mutations typically display severe gonadal dysgenesis and/or nephropathy, resulting from a dominant negative action of heterozygous WT1 missense mutations or from haploinsuficiency [8-10]. Most of the mutations reported within the first exons of this gene result in truncated proteins and have been found in patients with renal tumor and genitourinary abnormalities. In fact, missense mutations affecting only the N-terminus of the WT1 protein are expected to have a milder impact on its physiological function and to result in milder gonadal malformations, since the DNA-binding domain should remain intact [11]. Accordingly, most of the mutations as yet found in infertile patients that do not

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ACCEPTED MANUSCRIPT present major disturbances of testicular development are located in the N-terminus of the WT1 protein [7]. Here we have resequenced the WT1 coding region in a cohort of Portuguese patients, focusing on the first 6 exons, which encode the regulatory domain. To grasp the impact of the two missense WT1 variants found we: (i) reassessed the protein conservation

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across vertebrates; (ii) reviewed the spectrum of WT1 mutations affecting male fertility and compared them to those found in control populations from large genome sequencing projects.

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MATERIALS AND METHODS Patients and Control Populations

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DNA samples extracted from peripheral blood leukocytes of 194 NOA and 188 severe oligozoospermic male individuals (<1 million sperm/mL) with idiopathic spermatogenic failure were collected at the Human Genetics Department from INSA-IP and at the Genetics Department from the Faculty of Medicine, University of Porto (where routine molecular diagnosis for male infertility is established). A group of 31 infertile males with clinical history of uni- or bi-lateral cryptorchidism and one patient with anorchia were also

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selected following physical examination, hormonal testing (FSH, testosterone) and standard clinical genetic screening for karyotypic anomalies and Y chromosome microdeletions. Patients with known causes of infertility were excluded from this study. Molecular studies were performed after informed consents had been obtained and in

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coded DNA samples.

As controls, we obtained DNA from peripheral blood from 373 Portuguese men: 72 normozoospermic (normal sperm parameters) and 301 males who fathered at least one

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child. The study was included in the project ‘Copy number variation in infertile men genomic

regions:

screening

in

the

Portuguese

population’

(PTDC/SAU-

GMG/101229/2008), approved by the INSA Ethics Committee (Lisbon, Portugal on 6 November 2007).

Analysis of WT1 coding sequence A table containing the WT1 primers used in this study is available in the supplementary data (Supplementary Table 1). For the analysis of NR5A1 sequence we have used the primers described in [12] DNA fragments were amplified and sequenced and all putative variants were individually confirmed.

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ACCEPTED MANUSCRIPT Restriction Fragment Length Polymorphism (RFLP) Analysis This technique was used to screen the Portuguese controls for the presence of the c.1048T>C variant in WT1 exon 6. The restriction endonuclease Cfr42I was used to cleave a 1020 bp amplicon into two fragments of 832 bp e 188 bp, resolved by electrophoresis in

In silico analysis

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polyacrylamide gels.

WT1 protein sequences of several species of mammals and chicken were retrieved from the Ensembl database, manually curated and aligned using the ClustalW algorithm in

Geneious

v.5.5.8.

PolyPhen-2

[13]

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available

(http://genetics.bwh.harvard.edu/pph2/) and SIFT (http://sift.jcvi.org/) were used to predict the impact of non-synonymous substitutions in WT1 patients and controls,

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using as reference the Ensembl WT1 Protein - ENSP00000331327 (transcript sequence ENST00000332351). This transcript comprises all 10 WT1 exons (3122 bp) and initiates with the upstream CUG codon. We retrieved variant data from the 1000 genomes Project [14], NHLBI GO Exome Sequencing Project (ESP; http://evs.gs.washington.edu/EVS/), and

RESULTS

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CLINSEQ projects (Supplementary Table 2).

Mutation screening in patients

The mutation screening was conducted in two stages: initially the whole WT1 coding

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region (Figure 1) and flanking intronic regions were sequenced in 92 patients with NOA and one new missense variant was found within the first exon of the gene c.389C>T

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(p.Pro130Leu; ENST00000332351). This finding was in agreement with our hypothesis that this cohort would be enriched for rare damaging variants in the N-terminal region of the protein and therefore in the second stage we increased the coverage of the first exons (1 to 6) of the WT1 gene that were sequenced in a larger number of patients. In total this region was analysed in 169 patients with NOA and two missense variants were found: one in exon 1, resulting in a proline to leucine substitution (p.Pro130Leu; c.389C>T; ENST00000332351); and one variant in exon 6, resulting in a cysteine to arginine substitution (p.Cys350Arg; c.1048T>C; ENST00000332351). The patient carrying the p.Cys350Arg variant had been referred to the fertility clinic with a primary diagnosis of NOA, but spermatozoa were recovered from the ejaculate on a second attempt and a final diagnosis of severe oligozoospermia was established. We have also screened 31 patients 5

ACCEPTED MANUSCRIPT with uni- or bi-lateral cryptorchidism and one patient with anorchia for WT1 coding mutations and detected the rare p.Cys350Arg in the anorchia patient but found no additional alterations. The patients harbouring WT1 coding variants (one azoospermic, one severe oligozoospermic and the patient with anorchia) were also screened for exonic as well as proximal flanking intronic mutations in NR5A1, which has been associated with severe spermatogenic impairment [12,15] , but no

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alterations were detected.

Overall WT1 exon 1 was sequenced in 183 azoospermic patients and 373 Portuguese control individuals (301 fertile and 72 normozoospermic) and the p.Pro130Leu variant was found in heterozygosity in one patient and one fertile control with unknown sperm

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count; exon 6 was sequenced in 194 patients with severe spermatogenic impairment (193 NOA and 1 severe oligozoospermic) and the p.Cys350Arg variant was then tested by RFPL

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in a total of 188 severe oligozoospermic patients and in 371 controls (299 fertile and 72 normozoospermic) and was detected in heterozygosity in three patients and one fertile individual of unknown sperm count. The allele frequency of each missense variant was higher in patients than in controls: the p.Pro130Leu variant was twice more frequent in azoospermic patients than in controls (0.27% vs 0.13%), while the p.Cys350Arg variant showed an over 6-fold difference in frequency between severe oligozoospermic patients

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and controls (0.80% vs 0.13%), even though these differences did not reach statistical significance (Fisher exact test, p=0.549 and p=0.113, respectively). None of these variants was present in individuals with known sperm counts. The p.Cys350Arg substitution within exon 6 (rs142059681) had been previously detected in a large scale genome

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sequencing project (ESP – Exome Sequencing Project), at very low frequency both in European Americans (0.08%) and in African Americans (0.02%).

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Functional impact of coding variants detected in our patients Two missense variants were detected at a higher frequency in our group of patients with severe spermatogenic impairment than in control individuals from the same population. We have detected in three severe oligozoospermic men and in one patient with anorchia a rare variant within exon 6 (rs142059681) that results in a substitution predicted to be damaging by Polyphen-2 (score 0.999) and SIFT (score 0), where a conserved cysteine residue is replaced by an arginine (p.Cys350Arg). This substitution in close proximity to the first zinc finger may interfere with the stabilization of this important functional domain.

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ACCEPTED MANUSCRIPT The second, here reported for the first time, is located within exon 1 and alters the encoded amino acid from proline to leucine (p.Pro130Leu c.389C>T; ENST00000332351), disrupting a polyproline stretch within the WT1 self-association domain. In silico prediction of the functional impact of this substitution using PolyPhen-2 tool (http://genetics.bwh.harvard.edu/pph2/) and the protein isoform starting at the major initiation site classified this variant as ‘possibly damaging’ (score 0.770). It should be

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noted that in the absence of a tridimensional structure of this region of the protein PolyPhen only takes into account the phylogenetic conservation of this residue and the mutations annotated in this position known to cause Mendelian disease. The score obtained in SIFT also supports a deleterious impact on the protein, even though the

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confidence is low (score 0).

In an attempt to better evaluate the functional impact of the variants found in our patients

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we have performed an analysis of WT1 conservation, by retrieving and aligning all high quality sequences from different mammalian species available, an avian and a fish sequence

(Figure 2). Although the C-terminus of WT1 (harbouring the four

Kruppel-like zinc fingers) is more highly conserved across species than the transregulatory domain, as previously noted [16,17], the conservation of other regions in the N-terminus of the protein is also remarkable. Indeed, most of the

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activation domain, a block of 19 aminoacids in the self-association domain and the Cterminal part of exon 6 juxtaposed to the zinc-finger domain are completely conserved in mammals and also in amniotes. Some mammalian specific features also standout in the alignment, such as the existence of a polyglycine and a polyproline stretch in the N-

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terminus, as well as the alternatively spliced exon 5 [18]. The polyproline stretch is highly conserved in mammals (placental and marsupials), with a minimum of six consecutive

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proline residues present in all species of this group.

WT1 variants and male fertility – pathological mutations and coding polymorphisms

In order to grasp the potential impact of coding variants within the protein we compared the spectrum of missense WT1 variants found in controls of large sequencing projects to that of substitutions found in patients with impaired fertility [3,11,19,20] (Table 1). Apart from three well-defined syndromes (Table 2), WT1 mutations have also been identified in 46,XY male patients with genital abnormalities (typically gonadal dysgenesis, hypospadias and/or cryptorchidism) who may develop Wilms’ tumor but do not present nephropathy typical of DDS or Frasier syndromes [9,11,20-23]. The mutations found in 7

ACCEPTED MANUSCRIPT these patients cluster in the initial exons of the gene and are either missense or nonsense, in the latter case leading to very prematurely truncated proteins. Two missense mutations have been previously reported in patients with isolated genital and gonadal malformations such as hypospadias and cryptorchidism and considered as pathogenic [11,22,23]: one in the self-association domain (p.Ala199Thr) and a second in the activation domain (p.Pro249Ser). Surprisingly these variants are present in unphenotyped

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controls of European ancestry, with the p.Ala199Thr attaining a not so negligible frequency for a deleterious variant (rs9332973; 1.3% in CEU). The functional predictions for this variant are different depending on the algorithm used: according to PolyPhen-2 it is benign (0.172) but the score obtained with SIFT is within the range expected for a

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deleterious substitution (0.02). The p.Pro249Ser substitution, which had also been found in a patient with XY ambiguous genitalia [20], is rare in controls reaching only 0.06% in European-Americans of the ESP (rs2234584) and is classified as non-damaging by both

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PolyPhen-2 and SIFT (0.041 and 0.16, respectively). Even though we cannot exclude that some of these controls may present genital or gonadal malformations, the relatively high frequency of the p.Ala199Thr allied to the bioinformatics predictions suggest it is more likely a risk factor for the observed phenotype rather than a highly penetrant pathogenic variant.

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The missense variants recently associated with NOA in Chinese patients are equally distributed throughout the activation domain, exon 6 and the zinc-finger region. The two substitutions within the zinc-fingers (p.Arg430Gln and p.Lys454Arg) are predicted to be damaging (PolyPhen-2 scores of 0.994 and 0.807, respectively), affect amino acids

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conserved in amniotes and have been shown in vitro to interfere with the function of the protein [7]. Of the five missense variants within the zinc-fingers found in controls all are rare (minor allele frequency <0.1%) and only one is benign, reflecting the strong

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functional constraints in this region of the protein. Within exon 6, both the p.Arg363Ser substitution found by [7] in one azoospermic man and the p.Cys350Arg, more frequent in Portuguese oligozoospermic patients than in fertile controls, affect positions that are highly conserved in vertebrates and are classified by PolyPhen-2 as probably damaging, while the p.Gly338Ala substitution also described in azoospermia is conserved across amniotes and is predicted to be benign. Only two rare missense variants (minor allele frequency of 0.01%) have been found in this exon in controls, apart from the p.Cys350Arg, and their functional impact is very different: Ser336Asn (rs371021920) in the first amino acid of exon 6 is predicted not to affect the protein function and it is a fairly variable position even in mammals, while Phe362Cys (rs150194429) in the C-terminal end of exon 6 is predicted to be damaging. The different conservation level and predicted functional 8

ACCEPTED MANUSCRIPT impact of variants in the N- and C-terminal of exon 6 strongly suggest that the latter may be crucial for protein function, likely due to its proximity to the first zinc-finger.

DISCUSSION Most WT1 substitutions reported to date are associated with severe phenotypes, such as

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cryptorchidism, hypospadias, ambiguous genitalia, syndromic complications and renal tumor [9,10,24,25] and are located within the zinc finger region of the WT1 gene. More recently WT1 missense substitutions were reported in Chinese infertile males without syndromic manifestations or other major gonadal and/or urogenital abnormalities [7].

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The majority of the latter variants (4 out of 6) are clustered in the N-terminal region of the protein, suggesting that missense substitutions in this region of the protein result in a milder impairment of gonadal function. We have screened the WT1 coding region and

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flanking intronic sequence for mutations in a cohort of Portuguese males with NOA previously analyzed for genome-wide copy number variation [26] and oligozoospermic patients, focusing on the N-terminal region of the gene and including the first exon, which had not been efficiently captured in a previous mutation screen due to its high GC content [7].

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We have identified two missense variants at higher frequencies in our infertile patients when compared to geographically matched controls, which were absent from men with known sperm counts. Within exon 1 the p.Pro130Leu, here reported for the first time, disrupts a polyproline stretch in the self-association domain, which is involved in

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transrepression of WT1 target genes [27]. All the mutations previously reported in this exon lead to truncated, hence, dysfunctional WT1 proteins associated with severe phenotypes [8,22]. This substitution was found in one azoospermic patient and was also

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present in one Portuguese fertile control with unknown sperm count. This variant appears to be population specific and its pathogenic potential should be evaluated through the analysis of a larger cohort of European azoospermic patients and matched normozoospermic controls. The second substitution was identified in severe oligozoospermic patients (p.Cys350Arg; rs142059681) and displayed an over 6-fold difference in frequency compared to controls. This variant was also present in a patient with anorchia, who did not harbour any NR5A1 coding mutations. The p.Cys350Arg has also been detected in control individuals of European and African ancestry and therefore it is either a recurrent mutation or a relatively old mildly deleterious allele that likely represents a risk factor for spermatogenic failure. By surveying the available data on the

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ACCEPTED MANUSCRIPT WT1 missense variants present in control populations we have concluded that the rare variants with higher probability of damaging the protein are located in the C-terminus of exon 6 and in the zinc-fingers, in agreement with the existence of strong functional constraints in these regions. The analysis of WT1 orthologs confirmed the high conservation of the C-terminal region of exon 6 in vertebrates and the close proximity to the first zinc-finger suggests that this region may have a role in stabilizing this crucial

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DNA-binding element.

The WT1 variants associated with azoospermia in Chinese patients [7], as well as those reported here are clustered in the N-terminus of the protein. Two other substitutions in the WT1 N-terminus previously reported as pathogenic, one in the self-association domain

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(p.Ala199Thr) and other in the activation domain (p.Pro249Ser) are also found in control individuals, with the former reaching a not so negligible frequency (1.3%). The

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pathogenic potential of these variants must be further evaluated considering that rather than having a strong effect in heterozygosity these may instead confer risk to spermatogenic impairment. Conclusions

The available evidence indicates that WT1 N-terminal missense variants result in milder

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impairment of gonadal and kidney development, manifested by isolated spermatogenic failure in otherwise healthy men or in increased risk for gonadal or genital malformations, such as hypospadias or cryptorchidism. These results claim for large multicenter studies in order to fully assess the contribution of WT1 genetic alterations to male

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infertility, establish clear genotype-phenotype correlations and determine the

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usefulness of WT1 mutation screening in patients of European ancestry.

ACKNOWLEDGMENTS

This work was partially funded by the Portuguese Foundation for Science and Technology FCT/MCTES (PIDDAC) and co-financed by European funds (FEDER) through the COMPETE program, research grant PTDC/SAU-GMG/101229/2008 to AML and through CIGMH and Pest-OE/SAU/UI00009/2011. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology, and Higher Education and is partially supported by FCT. AML, SQ, ACL, and PIM are funded by FCT fellowships SFRH/BPD/73366/2010,

SFRH/BPD/64025/2009,

SFRH/BD/51695/2011

and

SFRH/BD/68940/2010, respectively. We thank Dr. Ana Aguiar, Joaquim Nunes, Ana Paula 10

ACCEPTED MANUSCRIPT Soares and Carlos Calhaz-Jorge from Unidade de Medicina da Reprodução, Departamento de Obstetrícia, Ginecologia e Medicina da Reprodução, CHLN-Hospital de Santa Maria, Lisboa and Graça Pinto and Sónia Correia from Unidade de Medicina da Reprodução, Maternidade Dr. Alfredo da Costa for the clinical evaluation and sperm analysis of the

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patients. The authors declare no competing interests.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1 – Schematic representation of WT1 gene exons and corresponding functional domains of the encoded protein. The c.389C>T and c.1048C>T substitutions found in exons 1 and 6, respectively, are underlined in the electropherogram depicted above. The yellow triangles represent three alternative transcription start sites, where the middle one is the canonic. Exon 5 is alternatively spliced, as well as the KTS insertion between

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exons 9 and 10 (light blue).

Figure 2 - WT1 protein conservation and alignment of human WT1 sequences with

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mammalian orthologues, chicken and zebrafish – only the sequence downstream the main initiator AUG site has been retrieved. A high level of amino acid similarity is evident at the C-terminus domain across vertebrates. In the N-terminal region most of the activation

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domain, a block of 19 aminoacids in the self-association domain (blue box) and the Cterminal part of exon 6 juxtaposed to the zinc-finger domain (black box) are completely conserved in mammals and also in amniotes. Some mammalian specific features also standout in the alignment, such as the existence of a polyglycine and a polyproline stretch in the N-terminus (red boxes), as well as the alternatively spliced exon 5 (positions 265272). Marked under the human sequence are previously reported mutations implicated in

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non-syndromic male infertility (purple) and missense variants found in controls from large sequencing projects annotated in the Ensembl database (release 75, February 2014). Symbols are colored according to PolyPhen-2 predictions: red – probably damaging,

these variants).

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yellow – possibly damaging; green – benign (see Supplementary table 2 for details on

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Authors’ contributions

CMS, SQ and ACL performed the WT1 sequence analysis. FC, JG, SF, IP, JS, MS and AB performed the clinical and genetic characterization of the samples for the routine workup of male infertility. CMS drafted the manuscript. PIM collected the samples and extracted the DNA of the normozoospermic individuals. AML obtained financial support, conceived and designed the study, conducted the data analysis, interpreted the results and wrote the manuscript. CMS, SQ and ACL participated in the data analysis and interpretation of results. SS and AA contributed with a critical review of the manuscript.

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21. Evaluation of the azoospermic male. Fertil Steril 2008; 90: S74-77. 22. Köhler B, Biebermann H, Friedsam V, et al. Analysis of the Wilms' Tumor Suppressor Gene (WT1) in Patients 46,XY Disorders of Sex Development. Journal of Clinical Endocrinology & Metabolism 2011; 96: E1131-E1136. 23. Wang Y, Li Q, Xu J, et al. Mutation analysis of five candidate genes in Chinese patients with hypospadias. Eur J Hum Genet 2004; 12: 706-712. 24. Baird PN, Santos A, Groves N, et al. Constitutional mutations in the WT1 gene in patients with Denys-Drash syndrome. Human Molecular Genetics 1992; 1: 301305. 25. Melo KFS, Martin RM, Costa EMF, et al. An unusual phenotype of Frasier syndrome due to IVS9+4C > T mutation in the WT1 gene: Predominantly male ambiguous genitalia and absence of gonadal dysgenesis. Journal of Clinical Endocrinology & Metabolism 2002; 87: 2500-2505. 26. Lopes AM, Aston KI, Thompson E, et al. Human Spermatogenic Failure Purges Deleterious Mutation Load from the Autosomes and Both Sex Chromosomes, including the Gene DMRT1. PLoS Genet 2013; 9: e1003349. 27. Madden SL, Cook DM, Rauscher FJ, 3rd A structure-function analysis of transcriptional repression mediated by the WT1, Wilms' tumor suppressor protein. Oncogene 1993; 8: 1713-1720.

14

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TABLES Table 1 - WT1 genetic defects detected in male patients with clinical presentation including isolated (non-syndromic) genital anomalies, morphological gonadal defects or

Phenotype

E1

Kohler (2011) J Clin Endocrinol Metab 96(7):E1131-E1136

PubMed 21508141

N-terminus

Hypospadias

Wang (2004) Eur J Hum Genet 12:706

PubMed 15266301

N-terminus

Hypospadias, unilateral cryptorchidism

Kohler (2011) J Clin Endocrinol Metab b 96(7):E1131-E1136

PubMed 21508141

Non-obstructive azoospermia

Wang et al (2103) PLoS Genet 9:e1003645

PubMed 23935527

Non-obstructive azoospermia

Wang et al (2103) PLoS Genet 9:e1003645

PubMed 23935527

Non-obstructive azoospermia

Wang et al (2103) PLoS Genet 9:e1003645

PubMed 23935527

Wang et al (2013) PLoS Genet 9:e1003645

PubMed 23935527

E1 E2

Pro249Ser E3

N-terminus

Ala282Pro E4

N-terminus

Asn307Ser E6

N-terminus

Gly338Ala E6

N-terminus

Non-obstructive azoospermia

Arg363Ser E8

C-terminus

Arg430Gln E8

Arg430Term

C-terminus

Non-obstructive azoospermia

Wang et al (2013) PLoS Genet 9:e1003645

PubMed 23935527

Hypospadias, bilateral cryptorchidism, Wilms tumor

Kohler (1999) Pediatr Res 45:187

PubMed 10022588

a

a

Functional Domain affected

PolyPhen2

Self-association domain

NA

NA

0.172

0.02

0.041

0.16

Activation domain

0.983

0

Activation domain

0.994

0

0.075

0.06

0.996

0.01

0.994

0.16

NA

NA

Self-association domain

M AN U

Hypospadias, unilateral cryptorchidism

TE D

Ala199Thr

N-terminus

EP

Val130Term

Reference

SC

Exon

AC C

Amino acid change

RI PT

spermatogenic failure.

Zinc-fingers (non DNAcontacting residue) Zinc-fingers

SIFT

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C-terminus

Non-obstructive azoospermia

Lys454Arg

Wang et al (2013) PLoS Genet 9:e1003645

0.807

Severe NA hypospadias, C-terminus bilateral Kohler (2001) J PubMed Arg458Term cryptorchidism, Pediatr 138(3):421 11241055 glomerulosclerosi s a PolyPhen and SIFT scores presented were retrieved from Ensembl and refer to the longest WT1 transcript (ENST00000332351) b

M AN U

SC

E9

PubMed 23935527

Zinc-fingers (non DNAcontacting residue) (non DNAcontacting residue) Zinc-fingers

RI PT

E9

also detected in a patient with XY Ambiguous genitalia, congenital heart disease [Kohler (2004) Eur J Endocrinol 150:825]

TE D

Table 2 - WT1-associated syndromes WAGR

Large 11p13 deletions:

Tumors usually carry intragenic somatic mutations in the remaining WT1 allele. Functional

Consequences

Heterozygous point mutations within the exons coding the ZF region of WT1.

Constitutional intronic mutations in the second donor splice site of intron 9, on one WT1 copy.

EP

Frasier

AC C

Genotype


WT1 gene: genitourinary features.
PAX6 gene: aniridia.

Denys-Drash

Individuals with a constitutional WT1 deletion present a high risk of

DDS mutant WT1 proteins fail to bind DNA and act in a dominant negative fashion by

Prevention of the production of the KTS-containing isoforms. There is a shift in the KTS

0.12

NA

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XY pseudohermaphroditism
Glomerulonephropathy
Usually do not develop renal tumor

EP

TE D

M AN U


Mesangial sclerosis (nephropathy)
Genital abnormalities (mild to XY pseudohermaphroditism)
Wilms’ tumor

isoform ratio, leading to an imbalance of WT1 isoform functions, rather than a formation of mutant protein.

RI PT

DDS triad:

AC C

Clinical Phenotype


Wilms’ tumor
Aniridia
Genitourinary anomalies
Mental retardation

forming homodimers with normal WT1 protein. Prevents WT1 physiological activity.

SC

developing Wilms’ Tumor (>20%) and should be monitored.

AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

M AN U

SC

RI PT

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NOA – non-obstructive azoospermia DNA – desoxyribonucleic acid WT1 – Willms’ tumor 1

AC C

EP

TE D

M AN U

SC

RI PT

NR5A1 - nuclear receptor steroidogenic factor 1

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Primer

Sequence CAGCGCTGAACGTCTCCA

Exon 1A_R kohler (Kohler, Pienkowski et al. 2004)

GGGTGTCCTAGAGCGGAGAG

WT1_Exons 2+3_F

AGCCCCAGACAGATAACA

WT1_Exons 2+3_R

TTCCTCCCAGTAAAGACC

WT1_Exons 4+5_F

CTGGAAAATGTGGAGGCT

WT1_Exons 4+5_R

TGCTACCCTGATTACCCA

WT1_Exon 6_F

GCCTCATCTCATCTGGAAGT

WT1_Exon 6_R

GGTGTCCCTGATGTTAAAGG

WT1_Exon 7_F

CCTCAAGACCTACGTGAATGT

WT1_Exon 7_R

ACTTTCTCTCTACCACTCTGCTC

WT1_Exon 8_F

Length (bp)

GC %

Product Length 573 bp

18

66,72

61,11

20

65,14

65,00

18

58,08

50,00

18

56,47

50,00

18

60,21

50,00

18

59,00

50,00

20

60,81

50,00

20

60,78

50,00

21

60,59

47,62

23

59,72

47,83

CTAACAAGCTCCAGCGAAGT

20

61,13

50,00

WT1_Exon 8_R

TCATGCCTCACCCTTAGATT

20

61,03

45,00

WT1_Exon 9_F

TAGCAGTGGGCTGATGATAC

20

60,27

50,00

WT1_Exon 9_R

GTAGGGACCTGGCTTATCTCT

21

60,12

52,38

WT1_Exon 10_F

GTTAGCTCAGGGACAGAATGA

21

60,82

47,62

WT1_Exon 10_R

TGACCTCGGGAATGTTAGAC

20

61,49

50,00

AC C

EP

TE D

M AN U

SC

Exon 1A_F kohler (Kohler, Pienkowski et al. 2004)

Tm (°C)

RI PT

Supplementary table 1 - WT1 primers used in this study

1505 bp

1386 bp

1020 bp

458 bp

697 bp

734 bp

772 bp

Primers for amplification and sequencing were designed in Primer3 v.0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/) using the latest version of the human genome assembly (GRCh37).

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Exon

Variation ID

Source

193

E1

rs377072761

ESP

199

E1

rs9332973

215

E1

249

MAF (Global)

Alleles

Residues

0.0008

C/A

A, S

0.034

HapMap CEU

0.0130

C/T

A, T

0.496

rs373935628

ESP

0.0008

G/T

Q, K

SC

Residue

RI PT

Supplementary table 2 – WT1 missense variants found in large scale sequencing projects (Exome Sequencing Project – ESP; 1000Genomes – 1KG and HapMap) with global minor allele frequencies (estimated using allele frequencies across European Americans and African Americans – ESP or across all 1000G Phase I populations).

E2

rs2234584

ESP

0.0006

G/A

250

E2

rs142653301

ESP

0.0008

T/C

272

E3

rs138073760

ESP

0.0002

G/A

282

E3

rs368452754

ESP

0.0008

C/A

336

E6

rs371021920

ESP

0.0008

350

E6

rs142059681

ESP

0.0008

362

E6

rs150194429

ESP

380

E7

rs147241955

1KG

381

E7

rs142937387

413

E7

430 485

EP

A/G

0.479

M AN U

TE D C/T

Polyphen

P, S

0.034

M, V

0.020

T, I

0.133

A, S

0.277

S, N

0.057

C, R

0.987

A/C

F, C

0.994

0.0010

C/T

R, Q

0.945

1KG

<0.001

G/C

S, W

0.998

rs373176048

ESP

0.0008

C/A

R, M

0.999

E8

rs144788858

ESP

0.0008

C/T

R, Q

0.096

E10

rs139893274

<0.01

C/T

R, Q

0.684

AC C

0.0008

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1KG 0.0008

G/C

N, K

0.925

M AN U

SC

RI PT

ESP

TE D

rs369940913

EP

E8

AC C

504