Clinical and Genetic Aspects of Mayer–Rokitansky–Küster– Hauser Syndrome

Übersichten medgen Susanne Ledig · Peter Wieacker https://doi.org/10.1007/s11825-018-0173-7 Institute of Human Genetics, Westfälische Wilhelms-Universität, Münster, Germany © The Author(s) 2018. This article is an open access publication. Clinical and genetic aspects of Mayer–Rokitansky–Küster– Hauser syndrome

Clinical aspects The MRKH syndrome can occur as tion of the coelomic epithelium into the an isolated or type I MRKH or in asso- mesonephros, and 3) elongation of the The Mayer–Rokitansky–Küster–Hauser ciation with extragenital malformations MD. (MRKH) syndrome [MIM 277000] is as type II MRKH. Upper urinary tract First, cells of the coelomic epithelium characterised by the congenital absence malformations are observed in about (Müllerian plaque) at the upper end of of the uterus and the upper two thirds 40%, including unilateral renal agenesis, the urogenital ridge are specified to be- of the vagina in 46,XX females with ectopia of one or both kidneys, renal come MD cells. Subsequently, within mostly normal ovarian function and hypoplasia, horseshoe kidneys and hy- the mesonephros, MD precursors invagi- therefore normal breast and pubic hair dronephrosis. The most frequent skeletal nate into the underlying mesenchyme development. The exact description anomalies include malformations of the and migrate caudally along the length of the genital manifestation of MRKH spine in 30–40% such as Klippel–Feil of the WDs extending posteriorly, cross is “uterus bipartitus solidus rudimen- anomaly or scoliosis. Müllerian hy- the WDs until the caudal tip of the MDs tarius cum vagina solida” and in fact poplasia, renal agenesis, cervicothoracic reaches the urogenital sinus, which is of endometrium islands can be detected in somite dysplasia (MURCS) association endodermal origin. It was suggested that a proportion of MRKH patients, leading is the most severe form of MRKH II thecellsformingtheMDswereofWD to complications in some cases. Occa- characterised by MD aplasia, renal dys- origin, but Orvis and Behringer were able sionally, the Fallopian tubes can also be plasia and cervical somite dysplasia. to show that the elongation of the MDs is affected, but the lower part of the vagina Less frequently, MRKH can be asso- accomplished predominantly by a small is usually unaffected. This is in good ciated with hearing defects including group of cells proliferating at the tip of the agreement with the hypothesis that the conduction defects such as stapes fixa- MDs. These cells are tightly associated lowerpartofthevaginamightdevelop tion or sensorineural deafness. Rarely, with the WDs and are guided by them from the urogenital sinus and might not cardiac (atrial septum defect, conotrun- [31]. The first phase of MD development be a derivative of the Müllerian ducts cal defects) and digital anomalies such as (specification and invagination) occurs (MDs). syndactyly, polydactyly or ectrodactyly independently from the WDs, but in the The first clinical feature is generally can occur. Occasionally, associations next phase of elongation it is dependent primary amenorrhea. Clinical examina- with situs inversus, Dandy–Walker mal- on the presence of the WDs and further- tion typically reveals a normal female formation, Meckel–Gruber syndrome, more on the expression of an elongation phenotype with breast development, ax- Bardet–Biedl syndrome, Holt–Oram signal (Wnt9b, wingless-type MMTV in- illar and pubic hair and normal exter- syndrome or McKusick–Kaufman syn- tegration site family, member 9b). nal genitalia. Differential diagnosis in- drome have been reported, leading to Although the MDs and WDs are of cludes isolated vaginal atresia, androgen the assumption that—in at least some different origin, they coexist during em- insensitivity syndrome caused by muta- cases—MRKHcanbeseenasaciliopathy. bryogenesisinbothsexesuntilgeneticsex tions of the androgen receptor gene (AR) triggers the differentiation of the indiffer- in XY individuals and WNT4 defects Embryogenetic aspects entgonadintoovaryortestisrespectively. characterised by MRKH and hyperan- In females, the MDs give rise by fusion drogenism. The mammalian female and male repro- to the utero-vaginal duct, which differ- Diagnosticsinclude differentmethods ductive tracts derive from the parameso- entiates into the uterus and the upper such as transabdominal ultrasound, MRI nephric or MDs and mesonephric or part of the vagina, whereas the unfused and pelviscopy. Wolffian ducts (WDs) respectively. Ac- part of the MDs develops into the Fallop- The incidence of MRKH is about cordingtoanalysesofmicemodels, ian tubes. In males, the testicular Sertoli 1:4,500 newborn girls. MD development includes three phases: cells secrete a glycoprotein, the anti-Mül- 1) initiation, 2) cranio-caudal invagina- lerianhormone(AMH),whichcausesthe

medizinische genetik Übersichten

Table 1 Phenotypes of MRKH patients with imbalances in recurrently affected regions 1q21.1, 16q11.2, 17q12 and 22q11.21 Locus Copy number/ Causative Phenotype Reference size genes(s) 1q21.1 Dup/2.7 Mb RBM8A Complete uterine + vaginal agenesis, fused Cheroki et al. (2008; [11]) external labia, ovaries undetectable; inherited by unaffected mother 1q21.1 Del/0.378Mb RBM8A Müllerian aplasia type II, TAR syndrome Ledig et al. (2011; [20]) 16q11.2 Del/0.55Mb TBX6 MURCS, hypoplasia of the wrist, disturbed Nik-Zainal et al. (2011; [29]) psychomotor development, epilepsy, bilateral hearing loss 16q11.2 Del/0.6 Mb TBX6 Müllerian aplasia, short stature Nik-Zainal et al. (2011; [29]) 16q11.2 Del/0.55Mb TBX6 MURCS, long uterus horns, scoliosis, left Nik-Zainal et al. (2011; [29]) atrophic kidney 16q11.2 Del/0.55Mb TBX6 Müllerian aplasia Nik-Zainal et al. (2011; [29]) 16q11.2 Del/0.53Mb TBX6 Müllerian aplasia Sandbacka et al. (2013; [38]) 16q11.2 Del/0.53Mb TBX6 Müllerian aplasia Sandbacka et al. (2013; [38]) 16q11.2 Del/0.53Mb TBX6 Müllerian aplasia Sandbacka et al. (2013; [38]) 16q11.2 Del/0.53Mb TBX6 Müllerian aplasia Sandbacka et al. (2013; [38]) 16q11.2 Del/0.53Mb TBX6 Müllerian aplasia Sandbacka et al. (2013; [38]) 17q12 Del/1.2 Mb LHX1, HNF1B Müllerian aplasia, mild dysmorphic fea- Cheroki et al. (2008; [11]) tures, onychodystrophy, mental impairment, seizures 17q12 Del/1.5 Mb LHX1, HNF1B Müllerian aplasia, mild dysmorphic features Bernardini et al. (2009; [3]) 17q12 Del/1.5 Mb LHX1, HNF1B Müllerian malformations with right unicor- Bernardini et al. (2009; [3]) nuate uterus, no cavitating rudimentary left horn, right haematosalpinx and surgically corrected agenesis of the upper and middle thirds of the vagina, bilaterally multicystic kidneys 17q12 Del/1.8 Mb LHX1, HNF1B Müllerian aplasia Ledig et al. (2011; [20]) 17q12 Del/1.4 Mb LHX1, HNF1B Müllerian aplasia, unilateral kidney agenesis Ledig et al. (2011; [20]) 17q12 Del/1.4 Mb LHX1, HNF1B Müllerian aplasia Nik-Zainal et al. (2011; [29]) 17q12 Del/1.4 Mb LHX1, HNF1B MURCS, left kidney agenesis with absent Nik-Zainal et al. (2011; [29]) ureter, pelvic misalignment, type II diabetes 17q12 Del/1.4 Mb LHX1, HNF1B MURCS, absent right kidney, left pelvic kid- Nik-Zainal et al. (2011; [29]) ney, right convex kyphoscoliosis 17q12 Del/1.4 Mb LHX1, HNF1B MURCS, mild scoliosis Nik-Zainal et al. (2011; [29]) 17q12 Del/1.7 Mb LHX1, HNF1B Müllerian aplasia Sandbacka et al. (2013; [38]) 22q11.21 Del/2.6 Mb ? Müllerian malformation with vaginal age- Cheroki et al. (2006; [10]); Cheroki et al. nesis and rudimentary uterus, right kidney (2008; [11]) agenesis, scoliosis, mild to moderate learning disabilities, mild dysmorphic features 22q11.21 Del/0.39Mb ? Müllerian aplasia Ledig et al. (2011; [20]) 22q11.2 Del/0.39Mb ? MURCS, 1-cm blind ending vagina, rudimen- Nik-Zainal et al. (2011; [29]) tary uterus, normal right ovary, streak left ovary, fused pelvic kidney, fused vertebrae, scoliosis, hypoplastic first ribs, flattened sacrum, dysplastic auricles, right-sided cleft lip, cleft palate, atrial septal defect, persistent left superior vena cava, unroofed coronary sinus, patent ductus arteriosus, multiple nevi, left hypoplastic thumb, hypoplastic middle phalanx, absent distal phalanx second digit, absent middle and distal phalanges, fifth digit 22q11.21–q11.23 Dup/3.5 Mb ? Müllerian aplasia Ledig et al. (2011; [20]) TAR thrombocytopaenia/absent radius, MRKH Mayer–Rokitansky–Küster–Hauser syndrome, MURCS Müllerian hypoplasia, renal agenesis, cervicothoracic somite dysplasia medizinische genetik Abstract · Zusammenfassung regression of the MD. Targeted mutage- medgen https://doi.org/10.1007/s11825-018-0173-7 nesis in the mouse has identified several © The Author(s) 2018. This article is an open access publication. genes that are essential for proper development and differentiation of the female S. Ledig · P.Wieacker reproductive tract. Clinical and genetic aspects of Mayer–Rokitansky–Küster–Hauser The mice genes required for female syndrome reproductive tract development include paired-box-gene 2 (Pax2), LIM home- Abstract obox 1 (Lhx1), wingless-type MMTV in- The Mayer–Rokitansky–Küster–Hauser analysis revealed recurrent aberrations in different chromosomal regions such as TAR tegration site family, member 4 (Wnt4), (MRKH) syndrome [MIM 277000] is characterised by the absence of a uterus and vagina susceptibility locus in 1q21.1, chromosomal wingless-type MMTV integration site in otherwise phenotypically normal women regions 16p11.2, and 17q12 and 22q11.21 family, member 7a (Wnt7a), empty spir- with karyotype 46,XX. Clinically, the MRKH can microduplication and -deletion regions in acles homeobox 2 (Emx2), hepatocyte be subdivided into two subtypes: an isolated patients with MRKH. Sequential analysis of nuclear factor 1-beta (Hnf1b), wingless- or type I form can be delineated from a type II the genes LHX1, TBX6 and RBM8A,which are located in chromosomal regions 17q12, type MMTV integration site family, form, which is characterised by extragenital malformations. The so-called Müllerian 16p11.2 and 1q21.1, yielded in the detection member 5a (Wnt5a), dachshund ho- hypoplasia, renal agenesis, cervicothoracic of MRKH-associated mutations. In a subgroup molog 1 (Dach1), dachshund homolog 2 somite dysplasia (MURCS) association can be of patients with signs of hyperandrogenaemia (Dach2), wingless-type MMTV integra- seen as the most severe phenotypic outcome. mutations of WNT4 have been found to tion site family, member 9b (Wnt9b), and The MRKH syndrome affects at least 1 in be causative. Analysis of another member of the WNT family, WNT9B,resultedinthe genes of the abdominal BHoxacluster. 4000 to 5000 female new-borns. Although most of the cases are sporadic, familial detection of some causative mutations in The homeodomain transcription fac- clustering has also been described, indicating MRKH patients. tor encoding gene Pax2 is expressed in a genetic cause of the disease. However, the the developing kidney and in the epithe- mode of inheritance is autosomal-dominant Keywords lium of the MD and the WD. According inheritance with reduced penetrance. MRKH · LHX1 · TBX6 · WNT9B to its expression mice deficient in Pax2 High-resolution array-CGH and MLPA lack kidneys and genital ducts in both sexes [18]. MD development is initi- Klinische und genetische Aspekte des Mayer-Rokitansky-Küster- ated by the expression of Pax2 together Hauser Syndroms with homeodomain coding gene Lhx1 in the coelomic epithelial cells and specifies Zusammenfassung them for a Müllerian fate. Furthermore, Das Mayer-Rokitansky-Küster-Hauser (MRKH) zur Identifizierung rekurrierender Aberra- Pax2 is also essential for the next steps Syndrom [MIM 277000] ist durch einen tionen in verschiedenen chromosomalen in MD development, the elongation and fehlenden Uterus und eine fehlende Vagina Regionen wie dem TAR-Suszeptibilitätslokus bei phänotypisch unauffälligen Frauen mit in 1q21.1, den Regionen 16p11.2 und maintenance of the MD. Therefore, in dem Karyotyp 46,XX gekennzeichnet. Klinisch 17q12 und der Mikroduplikations und Pax2-deficient mice the anterior portion werden beim MRKH 2 Subtypen unterteilt: –deletionsregion 22q11.21. of the MD initially forms, but then de- die isolierte oder Typ I Form wird von der In den Genen LHX1, TBX6 und RBM8A, generates, while the urogenital sinus still Typ II Form, bei der zusätzlich extragenitale die in den chromosomalen Regionen gives rise to the bladder and urethra [18]. Malformationen auftreten, unterschieden. 17q12, 16p11.2, bzw. 1q21.1 lokalisiert Hierbei kann die sog. MURCS-Assoziation sind, wurden zudem MRKH-assoziierte Wnt4 and Lhx1 areexpressedintheem- (MURCS: „Müllerian hypoplasia, renal Mutationen nachgewiesen. Hingegen werden bryonic mesonephric mesenchyme sur- agenesis, cervicothoracic somite dysplasia“) Mutationen des Gens WNT4 nur bei einer rounding the newly formed MD and both als schwerste phänotypische Ausprägung Subgruppe von Patientinnen mit zusätzlichen are required for embryonic MD develop- verstanden werden. Androgenisierungserscheinungen detektiert. ment. InWnt4-/- femalemice, theabsence Das MRKH tritt bei ca. einem von 4000 bis Interessanterweise konnten bei der Analyse 5000 weiblichen Neugeborenen auf; die eines weiteren Mitglieds der WNT-Familie, of MD formation and in contrast stabili- meisten Fälle kommen sporadisch vor. Das WNT9B, ebenfalls ursächliche Mutationen bei sation of WD suggest an essential role of Auftreten einiger familiärer Fälle weist auf MRKH-Patientinnen gefunden werden. Wnt4 in repressing male development in eine genetische Ursache mit autosomal- the XX gonad [45]. Furthermore, Jeays- dominanter Vererbung und einer reduzierten Schlüsselwörter LHX1 TBX6 WNT9B Ward et al. showed that the masculinised Penetranz hin. MRKH · · · Die Analyse von MRKH-Patientinnen mittels phenotype of Wnt4-/- female mice orig- hochauflösender Array-CGH und MLPA führte inates because of the disturbance of endothelial and steroidogenic cell migration into the developing XX gonad, pro- voking the formation of a male-specific coelomic blood vessel and production of steroids in the female gonad [16]. Possi- bly, Wnt4 is acting downstream of Lhx1

medizinische genetik Übersichten andWnt9b andinitiatestheMDinvagina- in the progenitor of the lower uterine seg- TAR syndrome a deletion affecting the tion [25, 45]. Wnt4 induces expression of ment and cervix and Hoxa13 in the cervix TAR susceptibility locus has been iden- Wnt7a [45]. Female mice lacking Wnt7a and upper vagina [47]. Mutations of ei- tified [20]. In a second patient, a gross are infertile owing to abnormal differ- ther Hoxa10 or Hoxa11 result in uterine duplication of approximately 2.7Mb, also entiation of the uterus and oviduct [33]. factor infertility in mice due to an im- overlapping the common TAR deletion The LIM domain-expressing gene, Lhx1 plantation defect in the uterus [39]. interval has been described [11]. Most is essential for the development of the TARpatientscarrydeletionsofdiffer- epithelial cells of MDs and WDs. There- Genetic aspects ent sizes, but always affecting a 200- fore, female Lhx1 knockout mice lack the kb gross common deletion interval, the uterus and the upper part of the vagina, Currently,thegeneticsofMRKHremains TAR susceptibility locus. Rarely, malfor- whereas male mice are deficient in the elusive. There is a risk of recurrence in mations of the genitourinary anomalies WD derivatives [18]. relatives,butmostcasesofMRKHare have been observed in patients with TAR Emx2 is another homeodomain tran- sporadic. Familial cases can be explained syndrome including horseshoe kidney, scription factor coding gene that is ex- by autosomal dominant inheritance with hypoplasia of the uterus and vagina, and pressed in the epithelial cells of the uro- reduced penetrance and variable mani- renal pelvis dilatation. Furthermore, it genital tract. Consequently, Emx2-mu- festation. However, oligogenic or poly- is known from analysis of TAR patients tant mice lack kidneys, ureters, gonads genic inheritance has also been discussed that about 75% have inherited the dele- and genital tracts and die soon after birth [20]. tion from an unaffected parent [17]. [26]. However, in these mice, WDs ini- There are some reports of monozy- Therefore, the authors supposed that in tial develop, but then degenerate. The gous twins discordant for MRKH, which addition to the rare deletion, a second POU domain-containing Hnf1ß is essen- may be explained by mosaicism or im- frequent change, possibly a frequent tial for general epithelial differentiation printing effects. Recently, insights into variant, is needed for the phenotypic and is expressed in very early urogenital genetics and the pathogenesis of MRKH manifestation of TAR. However, muta- tract formation, continuing into adult- have come from genetic techniques such tional analysis of 10 genes, located in the hood [13]. Wnt5a is expressed in mes- as array CGH. minimal deletion interval, in 3 patients enchymal cells of the uterus, cervix and Until recently, WNT4 deficiency was revealed in a first approach no second vagina and is required for the growth the only known monogenetic cause for causative mutation [17]. However, re- of the female reproductive tract [25]. MRKH, but different groups identified by cently in all patients analysed, one of two Therefore, in mice deficient in Wnt5a, multiplex ligation-dependent probe am- rare intronic regulatory polymorphisms the cervix and the whole vagina are ab- plification (MLPA) and genome-wide ar- in the RBM8A gene, which is located in sent [25]. Female mice double mutant ray comparative genomic hybridisation the minimal deletion interval of the TAR for the putative transcriptional cofactors (CGH) microimbalances affecting new susceptibility locus, have been found on Dach1/Dach2show a severe disruption of genes that play a role in the pathogenesis the second allele [2]. Furthermore, in MD development [14]. The adequate de- of this condition. So far, different recur- the case of patients with clinical signs of velopment, fusion and resorption of the rentlyaffectedchromosomalregionshave TAR and the causative polymorphism separating wall between the MDs seem been identified with the following fre- in the regulatory region of RBM8A,but to be induced by the WDs [1]. Thus, it quencies: ~1%in1q21.1, ~1%in16p11.2, without the deletion in 1q21.1, nonsense is known, that the WD secretes Wnt9b, ~6% in 17q12 and ~4% in 22q11.21 [20, mutations of RBM8A in a compound whichservesasacanonicalWntsignal 27]. . Table 1 summarises the pheno- heterozygous manner have been found essential for caudal MD extension [9]. types of MRKH patients with imbalances [2]. RBM8A encodes the Y14 protein, In Wnt9b -deficient mice, the MDs start in the above-mentioned recurrently af- which is one of four core components to invaginate, but there is no elongation fected regions. of the exon junction complex (EJC). In caudally. Interestingly, the WDs are un- Drosophila melanogaster,theY14protein affected [9]. Different genes of the Hox 1q21.1 is necessary for oocyte differentiation family play a major role in body pat- and determination of primordial germ terning and organogenesis and are ex- Imbalances in 1q21.1 affecting the so- cells [32]. pressed during the development of the called common thrombocytopaenia/ Allofthesefindingssuggestedastrong female genital tract in different areas of absent radius (TAR; MIM27400) sus- associationbetweenRBM8AandMRKH. the MD. The expression of the different ceptibility locus have been identified in Therefore, by performing sequence anal- Hox genes divides the homogeneous MD patients with or without signs of the ysis of RBM8A in a group of 116 MRKH into segments along the anterior–pos- TAR syndrome (hypomegakaryocytic patients, one of the two TAR-associated terior axis with each segment develop- thrombocytopaenia, bilateral absence variants and a second undescribed in- ing into different structures according to of the radius in the presence of both tronicvariantwerefoundwithhigherfre- their 3’–5’order in the Hox cluster: Hoxa9 thumbs) in addition to Müllerian mal- quencies in the patient group in contrast is expressed in the subsequent oviduct, formations (. Table 1;[11, 20]). In to the general population [43]. Interest- Hoxa10 in the developing uterus, Hoxa11 an MRKH type II patient with signs of ingly, one patient carried both RBM8A

medizinische genetik variants mentioned above, whereas an- more, two known polymorphisms could LHX1 on chromosome 17 belongs to other carried a gross duplication, which be associated with Müllerian aplasia, as the LIM homeodomain family of tran- contains the Bardet–Biedl syndrome they were found at a higher frequency scriptionfactors, whichisimplicateddur- (BBS)-associated BBS9 gene [43]. Fur- in patients in contrast to the general ing embryogenesis in processes such as thermore, in a patient with MRKH I and population [38]. body axis determination, in addition to XX gonadal dysgenesis, a heterozygous tissue and regional specification. LIM RBM8A missense mutation was found, 17q12 homeodomain proteins contain two tan- making RBM8A an interesting candidate dem LIM domains followed by a cen- for MRKH syndrome associated with The most recurrently affected chromoso- tral homeodomain with DNA-binding ovarian dysgenesis too [43]. mal region in MRKH is 17q12. Different activity and a C-terminal transactiva- array-based studies identified deletions tion domain, which may be involved in 16p11.2 of 1.4–1.8Mb in size in chromosomal re- the transcriptional regulation of target gion17q12inpatientswithMRKHtypesI genes. The LIM domain is a cysteine- Lossesin16p11.2have primarilybeende- and II (. Table 1;[3, 11, 20, 29, 38]). rich double zinc finger motif that binds scribed in combination with autism spec- Associated malformations were bilater- zinc and functions as a protein adapter trum disorders, but also with epilepsy, ally multicystic kidneys, mild facial dys- module that can interact with different seizures, developmental delay and learn- morphisms [3], but also severe learning protein domains and regulate by this the ing disability, dysmorphism/congenital disability and seizures (. Table 1;[11]). function of different components in the anomalies (abnormal head size) and obe- Furthermore, deletions of 17q12 can also transcriptional complex. Female Lhx1- sity. Furthermore, deletions in 16p11.2 give rise to other phenotypes without any null mice lack a uterus and oviducts to- were also identified in unaffected per- impairment of MD. gether with a complete absence of both sons.However,inanarray-basedstudy Due to expression data and mouse the epithelium and the mesenchyme of of patients with isolated and syndromic models, different studies favoured LHX1 the female reproductive tract, while the Müllerian aplasia, in 4 of the 63 patients andHNF1Baspromisingcandidategenes ovaries are unaffected [18], a phenotype deletionsofthislocuswereidentified, forMRKH,butalsoforassociatedtraitsof strongly resembling MRKH syndrome in suggesting a strong association of this re- the 17q12 deletion. Both genes are dis- humans. Additionally, micelackingLhx1 gion with MRKH syndrome (. Table 1; cussed in the following. Furthermore, lack kidneys and are anencephalic [40]. [29]). Among the genes deleted in the the finding that the deletion size and Most embryos deficient of Lhx1 die at common deletion interval, TBX6 seems the breakpoints observed in patients with embryonic day E10 because of defects in to be a good candidate gene, as it encodes MRKH type I are similar to those in pa- allantois differentiation, and only a few a conserved transcription factor, playing tients with a more severe phenotype or are stillborn. an essential role in developmental pro- with malformations not affecting the MD Expression of Lhx1 in the epithelium cesses such as mesoderm formation and makes the involvement of other genes of the developing MDs in a mouse model specification. outside the deletion interval for this ex- is dynamic with onset of the expression at Sequence variants in TBX6 are known tended phenotype likely. An oligogenic embryonic day 11.5 (E11.5) in the most to cause congenital scoliosis in the Chi- mode of inheritance has also been sug- anterior region of the urogenital ridge nese Han population and spondylocostal gested for MRKH and would explain the and caudal extension at E13.5 in both dysostosis [15]. A mouse model, the ho- rare familial cases and the difficulty in sexes [18]. The Lhx1 expression in the mozygous Tbx6rv (rib-vertebrae), show identifying a single genetic cause. epithelium of the MDs becomes sexu- a hypomorphic phenotype, with an oc- ally dimorphic at E14.5, with persistent casionally unilateral absence of kidneys LHX1 strong expression until E16.5 in females and reduced female fertility [49]. The Of a total of 118 MRKH patients, we and a weaker expression in males than phenotype in this mouse model and the could detect in two of them heterozygos- in females consistent with the regression known association between TBX6 mu- ity for a frameshift mutation and a mis- of the MDs in males to this point of tations in humans and scoliosis strongly sense mutation in the LHX1 gene respec- time. Afterwards, the Lhx1 expression resembles the MURCS association in hu- tively [20, 21]. The frameshift causes also becomes downregulated in females, mans. a very early premature stop codon at but persists in the differentiating oviduct. Sequencing of the TBX6 gene in two amino acid position 33 and was detected These findings correspond to an essen- studies with MRKH patients resulted in a type II patient with unilateral kid- tial role of Lhx1 in the formation of the in the identification of one possible ney agenesis [21]. The patient, who car- MDs in the female and suggest a further pathogenic missense and one splice site ries the LHX1 missense mutation, has role in the development of the oviducts. mutation in a total of four patients [38, atypeIMRKHsyndrome[20]. Further- Furthermore, by using a chimera assay, 43]. Corresponding to the phenotype more, three pathogenic LHX1 mutations Kobayashi et al. showed that Lhx1 is re- seen in mice with homozygous Tbx6 mu- were found in 5 out of 112 Finnish pa- quired cell-autonomously for very early tations, two of these patients also show tients with aplasia of the Müllerian ducts MD epithelium formation and that its ex- skeletal malformations [43]. Further- [38]. pression in the Müllerian precursor cells

medizinische genetik Übersichten is independent of Wnt7a, Pax2 and Wnt4 out of Lhx1 in epiblast derivative that 22q11.21 [18]. Lhx1 is also expressed in the WD Lhx1 has no impact on the formation of in both sexes. In females, Lhx1 expres- PGCs, but influences their retention in 22q11.21 deletions are commonly as- sion is lost from the anterior gonadal the hindgut endoderm, resulting in the sociated with DiGeorge or velocar- region around E15.25, whereas Lhx1 ex- loss of PGCs subsequently [41]. These diofacial syndrome (DG/VCFS OMIM pression in males persists and becomes observations make LHX1 an interesting 188400/192430). DG/VCFS belongs to upregulated around E17.5. Interestingly, candidate for MRKH syndrome associ- a group of related dysmorphic syndromes the only Lhx1-null male neonate lacks ated with gonadal dysgenesis. However, with highly variable clinical phenotypes Wolffian derivatives, also suggesting that in two previously described patients with encompassing congenital heart defects, Lhx1 might play an essential role in the MRKH and gonadal dysgenesis, no mu- hypocalcaemia, immunodeficiency, typi- development of the male genital tract tation in LHX1 was found [20, 21], al- cal facial dysmorphism, learning, speech [18]. though one male patient with a 17q12 and behavioural disorders. Moreover, Furthermore, the specific knockout of duplication has a 46,XX sex reversal [24]. some publications showed an associ- Lhx1 in the WD epithelium causes the LHX1 is also essential for differentiation between 22q11.21 deletions and lack of the WDs, but also impairs the ation of the central nervous system [19, Müllerian aplasia. Therefore, MRKH further development of the MDs, con- 34] and there are rare reports of MRKH syndrome has been considered to be part firming the importance of WDs for the patients with mild mental retardation or of the spectrum of clinical features of the elongation of the MDs [19]. learning disabilities [11]. Both patients DG/VCFS. Deletions and duplications In addition to the malformations of with mental retardation in our collec- of the DiGeorge syndrome-associated the MDs, Lhx1-minus mice lack, as men- tive harbour no LHX1 mutation, but they region 22q11.21 have also been found in tioned above, any kidneys and fail to both have a heterozygous deletion of ap- MRKH patients (. Table 1;[11, 20, 29]). form normal anterior head structures proximately 1.4–1.8Mb in 17q12 encom- Cheroki detected a gross deletion in a pa- [18, 40]. Renal malformations such as passing the LHX1 gene [21]. tient with uterus agenesis and further unilateral agenesis, ectopia of kidneys features present in DG/VCFS (. Table 1; or horseshoe kidneys are quite often in HNF1B [10, 11]). The deletion was disrupted MRKH, but even bilateral renal agene- Heterozygous mutations and whole gene by a short unaffected region containing sis has been reported (Potter sequence). deletions of the tissue-specific home- the TBX1 gene, which is responsible for Interestingly the patient who carries the odomain transcription factor HNF1B some of the major clinical features of LHX1 frameshift mutation shows uni- gene are typically associated with re- DGS/VCFS. The authors suggest that the lateral renal agenesis. This phenotypic nal cysts and diabetes (OMIM 137920). non-deletion of the TBX1 gene might outcome is similar to observations from However, other phenotypic characteris- be causative of the milder phenotype. aconditionalknockoutofLhx1innephric tics have also been described in associa- A smaller deletion, also non-affecting the epithelium after nephric duct develop- tion with HNF1B alterations. These are TBX1 gene, has been identified in a pa- ment, which led to hypoplastic kidneys, in good agreement with the tissues that tient with type I MRKH syndrome [20]. hydronephrosis and unilateral agenesis express HNF1B such as kidney, pancreas, Furthermore, an adjacent duplication of [19]. liver and the uterus. Expression of Hnf1b approximately 3.4Mb has been found in Lhx1 is expressed in the gonads and has been shown in MDs in the mouse another type I MRKH patient, overlap- complete loss of Lhx1 has first been de- embryo and in the inner epithelial layer ping with the distal part of the 22q11.21 scribed to cause defective head struc- in the adult mouse [13]. Therefore, in microdeletion–microduplication region. tures and a lack of kidneys in addition very few cases, HNF1B mutations have Thesefindingssuggestthatgenesother to a loss of gonads in mice [40]. How- also been reported to cause, in asso- than the TBX1 gene in the 22q11.21 dele- ever, such agonadism was not found in ciation with renal tract malformations, tion syndrome might play a role in uter- neonate Lhx1minus mice with mixed ge- abnormalities of the MDs in females [7]. ine malformations. Interestingly, analy- netic background [18]. Gonadal dysge- However, studies analysing the HNF1B sis of MRKH patients with a commer- nesis and agenesis are also rarely associ- gene in patients with MRKH failed to cially available DGS and DGS-like MLPA ated with MRKH [8]. Complete gonadal identify mutations in HNF1B [3, 20, kit revealed imbalances in 22q11.2 and dysgenesis with only fibrogenous tissue 21]. So far, only one case of a heterozy- in the DGS-like phenotype associated re- and a lack of germ cells can be distin- gous HNF1B missense mutation and gions 4q34-qter, 8p23.1 and 10p14 [28]. guished from a partial form with residues an isolated bicornuate uterus has been In addition to recurrent aberrations, ofhormonal active tissues and some germ described [7]. Therefore, HNF1B muta- array CGH-based studies also identified cells. A premature loss of germ cells in tions are associated as a cardinal feature various interesting private losses and the female causes gonadal dysgenesis. In- with malformations of the renal tract gains such as 2p24.1–24.3, 7p14.3 and terestingly, mouse embryos lacking Lhx1 and are a very rare cause of Müllerian Xq21.31 [11, 20, 29]. activity are deficient of primordial germ disorders. cells (PGCs; [44]). Recently, it could be shown by performing conditional knock-

medizinische genetik Members of the WNT family WNT9B could be detected in 11 MRKH mutational analysis of HOXA10 and patients [37]. HOXA11 in a small group of patients WNT4 Despite these findings, WNT9B with malformations of the female genital WNT4, which maps to human chromo- seemed to be a good candidate, as tract revealed only one missense variant some 1, isa memberofthe WNTfamilyof Wnt9b-/- female mice have no uterus of unknown pathogenicity, which was structurally related and highly conserved and upper part of the vagina, but have also present in the patients’ unaffected genes that encode secreted signalling fac- normal ovaries, which is comparable to mother [23]. tors that regulate a broad range of de- the MRKH phenotype in women [9]. Finally, it should be considered that velopmental processes, but has also been Furthermore, Carroll et al. showed in exogenous factors such as diethylstilbe- implicated in carcinogenesis. WNT4 is the same work that Wnt9b acts up- strol (DES), which functions as a strong known to be essential for the develop- stream of Wnt4 in the development of oestrogen, may be involved in the patho- mentofthefemalereproductivetract, the urogenital tract and is essential for genesis of MRKH syndrome. whereas it has been shown to play in the the development of mesonephric and female gonad a double role, on the one metanephric tubules and caudal exten- Conclusion handbycontrollingthefemaledevelop- sion of the Müllerian ducts in mice. mentandontheotherhandbypreventing A first association between WNT9B Mayer–Rokitansky–Küster–Hauser syn- testes formation. andMRKHwasfoundinaChinesestudy drome is a phenotypically and genetically Heterozygous mutations in the WNT4 with 42 patients, in which two possible very heterogeneous disorder and has an gene have been associated with MRKH pathogenic WNT9B mutations were de- incidence of 1:4,500 newborn females. in humans [4, 5, 35, 36]. The 4 patients tected in one patient [46]. Although it Most of the cases are sporadic, but described so far displayed an agenesis or was unknown if these two mutations had analyses of the few reported familial hypoplasia of the Müllerian derivatives, been in cis or trans, the authors suggested cases suggest an autosomal-dominant but also clinical or biochemical signs of a synergistic effect. In contrast, another inheritance with reduced penetrance. hyperandrogenism (hirsutism, acne, ele- Chinese study found no association be- As array CGH analyses in MRKH pa- vated plasma testosterone levels). These tween anomalies of the Müllerian ducts tients identified recurrent aberrations in findings are in good agreement with the and mutations in WNT9B [42]. chromosomal regions 1q21.1, 16p11.2, phenotype found in Wnt4-deficient mice, However, by analysing WNT9B in 17q12 and 22q11.21 respectively, array which fail to develop MD and are mas- a group consisting of 59 MRKH and CGH analyses should be performed in culinised. However, unilateral renal age- 50 MRKH II patients, Waschk et al. women with the suspected diagnosis nesis has been identified in females with identified in five of the MRKH I pa- of MRKH syndrome. These recurrent heterozygous WNT4 mutations. Func- tients potential pathogenic mutations aberrations are associated with highly tional analysis of the WNT4 mutations (one nonsense and four missense muta- variable clinical phenotypes and can also revealed failure of post-transcriptional tions) [48], but no WNT9B mutation was cause further disorders, e.g. in the case lipid modification, misfolding and for- detected in MRKH II patients. Interest- of 22q11.21 deletion heart defects or mation of intractable aggregates, defects ingly, previous studies showed that two 17q12 deletion maturity-onset diabetes in receptor-binding and partial dereg- of the patients with a WNT9B mutation of the young (MODY) due to deletions ulation of enzymes involved in ovarian carried either an additional deletion of of HNF1B. androgen biosynthesis [6, 36]. LHX1 or a missense mutation in TBX6 Furthermore, the clinical overlap Studies involving classical MRKH pa- [20, 43], suggesting digenic inheritance of MRKH syndrome with different cil- tients failed to identify WNT4 mutations in MRKH. Interestingly, it was shown iopathies suggests that MRKH syndrome [10, 12]. Therefore, it has been sug- that the expression of Wnt9b in Lhx1- might also be a ciliopathy. gested that MRKH syndrome with signs deficient mice is markedly altered [34]. By analysing candidate genes, being of androgenisation due to heterozygous All of these findings suggest a com- located in these aberrations, mutations in WNT4 mutations is a distinct clinical en- mon pathway in MRKH syndrome with genes such as LHX1, RBM8A and TBX6 tity that can be delineated from typical or WNT9B acting upstream of WNT4 and have been identified as being causative of classic MRKH. Moreover, because of its LHX1. MRKH syndrome. However, in WNT4, role in gonadal development, folliculo- Furthermore, in the past, the possible which is associated with a distinct clini- genesis can also be disturbed in affected involvement of other genes in the patho- cal entity of MRKH syndrome and signs women [35]. genesis of MRKH has been tested. of hyperandrogenism, and in its fam- Mutations affecting AMH, which ini- ily member WNT9B, causative mutations WNT9B tiates regression of MDs during male have also been detected. Furthermore, in However, alterations in other members embryonal development, anti-Mülle- some patients with fusion anomalies of of the complex WNT signalling pathway rian hormone receptor (AMHR)and the MDs such as uterus didelphis, the have been suggested as being causative, various homeobox (HOX) genes, have same causes as for MRKH syndrome, butnomutationinWNT5A, WNT7Aand been excluded as causative factors for e.g. deletions in 17q12, duplications of MRKH syndrome [30]. Furthermore, chromosomal region 22q11.21, and vari-

medizinische genetik Übersichten ants in WNT9B, TBX6 and RBM8A have 4 Various surgical procedures are 6. Biason-Lauber A, Konrad D (2008) WNT4 and sex development. SexDev2:210–218 been described, suggesting that Mülle- available for building a neovagina. In 7. Bingham C, Ellard S, Cole TR, Jones KE, Allen LI, rian fusion anomalies and MRKH syn- a few cases, uterus transplantation has Goodship JA, Goodship TH, Bakalinova-Pugh D, drome might have a partially common been performed to enable pregnancy. Russell GI, Woolf AS, Nicholls AJ, Hattersley AT (2002) Solitary functioning kidney and diverse aetiology [22, 43, 48]. genital tract malformations associated with New insights can be expected from hepatocyte nuclear factor-1ß mutations. Kidney studies on large cohorts of well-char- Corresponding address Int61:1243–1251 8. Bousfiha N, Errarhay S, Saadi H, Ouldim K, acterised patients in combination with Dr.rer.nat.S.Ledig Bouchikhi C, Banani A (2010) Gonadal dysgenesis 46,XX associated with Mayer-Rokitansky-Kuster- technologies such as next-generation se- Institute of Human Genetics, Westfälische quencing. Hausersyndrome: onecasereport. ObstetGynecol Wilhelms-Universität Int. https://doi.org/10.1155/2010/847370 Vesaliusweg 12–14, 48149 Münster, Germany 9. Carroll TJ, Park JS, Hayashi S, Majumdar A, Aspects of genetic counselling [email protected] McMahon AP (2005) Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis 4 MRKH syndrome is a rare, hetero- of the mammalian urogenital system. Dev Cell geneous disease characterised by the Compliance with ethical 9:283–292 absence of a uterus and the upper two 10. CherokiC,Krepischi-SantosAC,RosenbergC,Jehee guidelines FS,Mingroni-NettoRC,PavanelloFilhoI,Zanforlin thirds of the vagina in 46,XX females. FilhoS,KimCA,BagnoliVR,MendoncaBB,Szuhai 4 Most cases are sporadic, but familial Conflict of interest. S. Ledig and P.Wieacker declare K, Otto PA(2006) Report of a del 22q11 in a patient occurrence is well documented and that they have no competing interests. with Mayer-Rokitansky-Kuster-Hauser (MRKH) anomaly and exclusion of WNT-4, RAR-gamma, indicates autosomal-dominant inher- and RXR-alpha as major genes determining MRKH This article does not contain any studies with human anomaly in a study of 25 affected women. Am J itance with variable manifestation. participants or animals performed by any of the au- MedGenetA140:1339–1342 4 MRKH syndrome is frequently asso- thors. 11. Cheroki C, Krepischi-Santos AC, Szuhai K, Brenner ciated with malformations, especially V, Kim CA, Otto PA, Rosenberg C (2008) Genomic OpenAccess. Thisarticleisdistributedundertheterms imbalances associated with mullerian aplasia. of the kidneys (unilateral renal agen- of the Creative Commons Attribution 4.0 International JMedGenet45:228–232 esis 30%), skeleton (10–15%), cardiac License (http://creativecommons.org/licenses/by/ 12. Clement-Ziza M, Khen N, Gonzales J, Cretolle- 4.0/), which permits unrestricted use, distribution, anomalies (2–3%) and deafness VastelC,PicardJY,Tullio-PeletA,BesmondC, and reproduction in any medium, provided you give Munnich A, Lyonnet S, Nihoul-Fekete C (2005) (2–3%). appropriate credit to the original author(s) and the ExclusionofWNT4asamajorgeneinRokitansky- 4 Siblings of MRKH patients can also source, providealinktotheCreativeCommonslicense, Küster-Hauser anomaly. Am J Med Genet A and indicate if changes were made. show, for example, malformations of 137:98–99 the MDs or associated anomalies. 13. 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