Mutations in WNT9B are associated with Mayer Rokitansky Küster Hauser syndrome

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1 Clin Genet 2016: 89: Printed in Singapore. All rights reserved Short Report 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Mutations in WNT9B are associated with MayerRokitanskyKüsterHauser syndrome Waschk D.E.J., Tewes A.-C., Römer T., Hucke J., Kapczuk K., Schippert C., Hillemanns P., Wieacker P., Ledig S. Mutations in WNT9B are associated with Mayer Rokitansky Küster Hauser syndrome. Clin Genet 2016: 89: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2015 MayerRokitanskyKüsterHauser syndrome (MRKHS) is a well-known malformation pattern of the Müllerian ducts (MDs) characterized by congenital absence of the uterus and vagina. To date, most cases remain unexplained at molecular level. As female Wnt9b-/- mice show a MRKHS-like phenotype, WNT9B has emerged as a promising candidate gene for this disease. We performed retrospective sequence analyses of WNT9B in 226 female patients with disorders of the MDs, including 109 patients with MRKHS, as well as in 135 controls. One nonsense mutation and five likely pathogenic missense mutations were detected in WNT9B.Five of these mutations were found in cases with MRKHS accounting for 4.6% of the patients with this phenotype. No pathogenic mutations were detected in the control group (p = 0.017). Interestingly, all of the MRKHS patients with a WNT9B mutation were classified as MRKHS type 1, representing 8.5% of the cases from this subgroup. In previous studies, two of the patients with a WNT9B mutation were found to carry either an additional deletion of LHX1 or a missense mutation in TBX6. We conclude that mutations in WNT9B were frequently associated with MRKHS in our cohort and some cases may be explained by a digenic disease model. Conflict of interest The authors declare no competing interests. D.E.J. Waschk a,,a.-c. Tewes a,,t.römer b, J. Hucke c, K. Kapczuk d, C. Schippert e, P. Hillemanns e, P. Wieacker a and S. Ledig a a Institute of Human Genetics, University Hospital of Münster, Münster, Germany, b Department of Gynecology and Obstetrics, Evangelisches Krankenhaus Köln-Weyertal, Köln, Germany, c Department of Gynecology and Obstetrics, Agaplesion Bethesda Krankenhaus, Wuppertal, Germany, d Division of Gynecology, Poznan University of Medical Sciences, Poznan, Poland, and e Department of Gynecology and Obstetrics, University Hospital of Hannover, Hannover, Germany These authors contributed equally to this study. Key words: MayerRokitanskyKüsterHauser syndrome MRKHS Müllerian duct anomalies mutation WNT9B Corresponding author: Susanne Ledig, PhD, Institute of Human Genetics, University Hospital of Münster, Vesaliusweg 12-14, D Münster, Germany. Tel.: ; fax: ; sledig@uni-muenster.de Received 15 September 2015, revised and accepted for publication 2 November 2015 The mammalian female and male reproductive tracts develop from the Müllerian ducts (MDs) and Wolffian ducts (WDs), respectively. In the absence of testicular differentiation, the WDs regress and the MDs give rise to the fallopian tubes, uterus, cervix and upper part of the vagina. Disorders of normal MDs development can manifest as fusion anomalies (bicornuate uterus; uterus didelphys), formation defects (unicornuate uterus) and abnormal absorption of the midline septum (septate uterus), or more complex malformations like Mayer Rokitansky Küster Hauser syndrome (MRKHS; OMIM 27700). The incidence of MRKHS is estimated at about 1 out of 4500 female births (1). MRKHS type 1 is characterized by congenital absence of the uterus and vagina in women with a normal female karyotype and mostly regular ovarian 590

2 WNT9B mutations in MRKHS Fig. 1. (a) Schematic view of the localization of the detected mutations within WNT9B. (b) Electropherograms of the detected WNT9B mutations. (c) Mutation in TBX6 in the patient who also carries the WNT9B mutation c.919c>t. function. In MRKHS type 2, uterine and vaginal aplasia are associated with other malformations, e.g. of the kidneys and the skeletal system, or less frequently with auditory and cardiac defects. To date, the cause for most cases of MRKHS remains unknown. This might be due in part to the presumably heterogenic etiology of this disorder, which usually occurs sporadically. Nevertheless, familial clustering has been described, indicating a genetic cause of the disease (2). Familial cases may be explained by polygenic or autosomal dominant inheritance with reduced penetrance. By comparative genomic hybridization (array-cgh), several critical chromosomal regions have been detected in MRKHS cases (3, 4). Mutations in LHX1 (LIM homeobox gene 1, LIM1; OMIM ) and TBX6 (T-BOX 6; OMIM ) have been reported previously as a monogenic cause for MRKHS (3 6). Furthermore, mutations in WNT4 (wingless-type mouse mammary tumor virus integration site family member 4; OMIM ) have been described in a few cases with anomalies of the MDs and hyperandrogenism (7, 8). However, mutations in these genes account only for a minority of MRKHS cases. In search of further genes involved in MRKHS, WNT9B (wingless-type MMTV integration site family member 9B; OMIM ) has emerged as a promising candidate gene. The WNT gene family is composed of highly conserved developmental control genes which encode secreted 591

3 Waschk et al. glycoproteins that are involved in short-range signaling during embryonic patterning (9). Carroll et al. (2005) found that Wnt9b is expressed throughout the WDs epithelium and that it is essential for the caudal extension of the MDs in mice. They also showed that WNT9B acts upstream of WNT4 in the developing urogenital system. Wnt9b-/- female mice lack the uterus and upper vagina but have normal ovaries, which is comparable with the human MRKHS phenotype (10). This raises the question whether mutations in WNT9B may be associated with disorders of the MDs in women as well. The average probability to carry a likely pathogenic WNT9B mutation is in the order of 0.5% (Table S1, Supporting information) in both the European and African-American cohort of the NHLBI Exome Sequencing Project (ESP6500). It is important to note that these cohorts were not selected for gender or MD abnormalities. Therefore, studies of selected patient groups are needed to determine the connection between MRKHS and mutations in WNT9B. In this context, two novel WNT9B mutations have been reported in a Chinese woman with MRKHS, previously (11). However, another study in the Chinese population found no association between MD anomalies and mutations in WNT9B (12). To investigate whether mutations in WNT9B are causative for MD anomalies in the Caucasian population, we now performed retrospective sequence analyses of WNT9B in our study group. For cases with signs of hyperandrogenism, sequence analyses of WNT4 were carried out additionally. Materials and methods Our study cohort comprised a total of 226 Caucasian women with clinically characterized disorders of MDs. Of these patients, 109 were diagnosed with MRKHS (59 MRKHS type 1; 50 MRKHS type 2). The remaining 117 patients showed other malformations such as septate uterus (n = 58), bicornuate uterus (n = 23), unicornuate uterus (n = 9) and uterus didelphys (n = 17). Ten cases had Herlyn Werner Wunderlich syndrome, which is characterized by uterus didelphys, obstructed hemivagina and ipsilateral renal agenesis. A total of 135 Caucasian women who gave birth to at least one child were analyzed as controls. Fourteen patients of our cohort who showed clinical symptoms or laboratory signs of hyperandrogenism were additionally screened for WNT4 mutations. In our cohort, sequence analyses of LHX1 and TBX6, as well as array-cgh have been performed as previously reported elsewhere (3, 5, 6). Genomic DNA was extracted from peripheral blood leukocytes using standard methods. All samples were whole-genome amplified with an illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare Life Sciences, Chalfont St. Giles, UK) according to manufacturer s description. Polymerase chain reaction (PCR) was performed with 200 ng DNA in a volume of 20 μl and primers specific for coding regions of WNT9B and WNT4 (Tables S2 and S3). After treating the PCR products with ExoSAP-IT (USB Corporation, Cleveland, OH), sequencing was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA). Sequencing reactions were analyzed on a 3730 DNA Analyzer (Applied Biosystems). Sequence traces were analyzed with CodonCode Aligner (CodonCode Corporation, Dedham, MA). All detected mutations (according GenBank NM_ for WNT9B) were confirmed by sequence analyses of the original DNA samples. The online programs PolyPhen-2 ( SIFT ( sift.jcvi.org/www/sift_aligned_seqs_submit.html) and MutationTaster ( index.html), which are integrated in the biosoftware Alamut for interpretation of genomic variations ( interactive-biosoftware.com/), were used to predict the pathogenicity of nucleotide changes. Missense mutations were considered as pathogenic when at least two programs reported them to be disease causing. An association between WNT9B mutations and MRKHS was evaluated by means of Fisher s exact test (with p values of <0.05 considered to indicate statistical significance). Results We identified the heterozygous missense mutations c.472c>g (p.gln158glu), c.665g>a (p.arg222his), c.722g>a (p.arg241his), c.919c>t (p.arg307trp), c.974g>a (p.arg325his) and the heterozygous nonsense mutation c.1029c>a (p.cys343x) in WNT9B in six different patients (Fig. 1). Five of these showed a MRKHS type 1 phenotype and one patient had bicornuate uterus (Table 1). The mutation c.1029c>a has been seen once in the European-American cohort of ESP6500, which includes about 4300 probands. In the same cohort, the mutations c.665g>a and c.722g>a have been detected 14 and 4 times, leading to minor allele frequencies (MAFs) of and , respectively. The mutation c.919c>t is not listed in ESP6500 but in the database of Single Nucleotide Polymorphisms (dbsnp; with a MAF of To our knowledge, the mutations c.472c>g and c.974g>a are neither listed in ESP6500 nor in other genome databases. Additionally found variants were considered as benign and are listed in Table 1. No pathogenic mutations were detected in the control group. No WNT4 mutations were detected in cases with hyperandrogenism. Discussion To date, the majority of MRKHS cases remain unexplained at molecular level. Wang et al. (2014) detected two possibly pathogenic WNT9B mutations in 1 out of 42 women in a Chinese cohort of MRKHS patients (11). The authors suggested that these mutations might function through a synergistic effect in this patient. However, it is unclear whether the mutations were found in trans or in cis. Tang et al. (2014) found the two novel WNT9B variants c.566g>a (p.arg189gln) and 592

4 WNT9B mutations in MRKHS Table 1. Results of WNT9B sequence analysis. Pathogenicity analyses of amino acid substitutions were performed with the online programs PolyPhen-2, MutationTaster and SIFT. In silico-scores range from 0 (predicted benign by all programs) to 3 (predicted pathogenic by all programs). Missense mutations with a score of 2 were considered as pathogenic. Minor allele frequencies (MAF) and allele counts are given from the European-American cohort of the NHLBI Exome Sequencing Project (ESP6500), except for c.919c>t which is not listed in ESP6500 but in the database of single nucleotide polymorphisms (dbsnp). WNT9B variants considered as pathogenic Alleles Variant State Location AAC up to Phenotype MAF Allele-count RefSNP-ID In silico-score Cases (n = 226) Controls (n = 135) c.472c>g p.gln158glu c.665g>a a p.arg222his c.722g>a p.arg241his c.919c>t b p.arg307trp c.974g>a p.arg325his c.1029c>a p.cys343x Exon 3 Fruitfly Tetraodon Tetraodon Frog Frog MRKHS type 1 Not listed MRKHS type /8600 rs MRKHS type /8600 rs Bicornuate uterus (dbsnp) rs MRKHS type 1 Not listed MRKHS type /8600 rs WNT9B variants considered as benign Alleles Variant State Location AAC up to MAF Allele-count RefSNP-ID In silico-score Cases (n = 226) Controls (n = 135) c.140a>g p.gln47arg c.252c>t p.leu84leu Exon 2 Moderately conserved Exon /8588 rs Not listed

5 Waschk et al. Table 1. continued WNT9B variants considered as benign Alleles Variant State Location AAC up to MAF Allele-count RefSNP-ID In silico-score Cases (n = 226) Controls (n = 135) c.317 T>C p.met106thr c.399g>t p.arg133arg c.454g>a p.glu152lys c.627g>a p.thr209thr c.766 T>C p.leu256leu c.846g>c p.val282val /hom. Exon 2 Weakly conserved Exon3 Exon 3 Frog T = C = rs /8598 rs /8600 rs /8600 rs /8600 rs /8584 rs T/C: 95 C/C: T/C: 40 C/C: 85 AAC, amino acid conservation;, heterozygous; hom., homozygous; MRKHS, MayerRokitanskyKüsterHauser syndrome. a This patient additionally has a deletion in 17q12 including LHX1. b This patient additionally has a likely pathogenic missense mutation in TBX6. 594

6 WNT9B mutations in MRKHS c.773g>a (p.arg258his) in a first stage analysis of 191 Chinese women with MD anomalies (12). In a second stage analysis, 351 additional cases and 563 controls were screened for the presence of these two WNT9B variants. Of all patients from stages I and II, 34 had uterine agenesis and 508 showed other defects of the MDs. The authors found no significant differences in the frequencies between the patients and the control group for the two variants and concluded that mutations in WNT9B were not causative for MD anomalies in their cohort. However, it is unclear whether the patients from the second stage analysis were tested for other mutations besides the two variants found in stage I and if any of the patients with uterine agenesis fulfilled the criteria for MRKHS. As the studies performed in the Chinese population have produced contradictory conclusions, it is of interest which results can be expected in a different ethnic background. In this context, we now detected six presumably pathogenic WNT9B mutations in our Caucasian cohort. The mutation c.1029c>a generates a premature stop codon at amino acid position 343. Premature stop codons usually lead to a truncated protein or nonsense-mediated mrna decay (13), which in both cases would interfere with the regular functioning of the affected allele. We therefore consider the mutation c.1029c>a as pathogenic. Each of the five detected missense mutations causes an exchange of a highly conserved amino acid at the respective position of the protein structure (Table 1). In silico analyses using PolyPhen-2, SIFT and MutationTaster consistently indicate that the mutations c.665g>a, c.722g>a and c.919c>t are pathogenic. Both mutations c.472c>g and c.974g>a are considered disease causing by two out of three programs, respectively. Unfortunately, DNA samples of the parents were not available to examine their genetic status. However, paternal inheritance obviously would not contradict causality in women, whereas in the theoretical case of maternal transmission, a reduced penetrance or a digenic disease model is thinkable. The WNT9B mutation c.919c>t was detected in 1 out of 23 women with bicornuate uterus. Because of the small sample size, statistical significance cannot be deduced for this group of patients. Nevertheless, five of the WNT9B mutations were found in cases with MRKHS accounting for 4.6% of the patients with this phenotype (5 out of 109). None of the mutations were detected in the control group (p= 0.017). Interestingly, all of the MRKHS patients with a WNT9B mutation were classified as type 1, representing 8.5% of the cases from this subgroup (5 out of 59). Although statistically not significant at this point (p= 0.061), further WNT9B analyses in larger cohorts may reveal that mutations in this gene are specific for type 1 MRKHS. In addition to the mutation c.665g>a in WNT9B, we previously found a deletion of megabases in the chromosomal region 17q12 including LHX1 in the same patient by array-cgh (Fig. S1). Recently, the authors and others showed that mutations in LHX1 are associated with MRKHS (3, 4). Female Lhx1-knockout mice have ovaries but lack derivatives of the MDs including uterus and the upper part of the vagina (14). Besides, Pedersen et al. showed that the expression of Wnt9b in Lhx1-deficient mice is markedly altered (15). This suggests a common pathway of MRKHS including LHX1 and WNT9B. Furthermore, we previously found the missense mutation c.815g>a of a highly conserved amino acid (up to Tetraodon) in TBX6 in the patient who also carries the WNT9B mutation c.919c>t (Fig. 1). The TBX6 mutation is listed in ESP6500 with three allele counts leading to a MAF of (rs ) and all three prediction programs consider it as pathogenic. TBX6 encodes a conserved transcription factor playing an important role in mesodermal development and has been described to be associated with anomalies of the MDs (4, 6, 16). The phenotypes of the latter patients may therefore be explained by a synergistic effect of a WNT9B mutation in co-occurrence with the deletion of LHX1 or the mutation in TBX6, respectively. In summary, we could show that mutations in WNT9B were frequently associated with MRKHS in our cohort and found evidence that some of the cases with MD anomalies may be explained by a digenic disease model. Supporting Information Additional supporting information may be found in the online version of this article at the publisher s web-site. Acknowledgements We thank all patients for participating in this study and Anne-Lena Bröcher, Steffi Burkhardt, Mandy Hoffmann and Silvia Roßkamp for great technical assistance. References 1. Folch M, Pigem I, Konje JC. Müllerian agenesis: etiology, diagnosis, and management. 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