Williams et al. Molecular Cytogenetics (2016) 9:57 DOI 10.1186/s13039-016-0264-6 RESEARCH Open Access A balanced chromosomal translocation involving chromosomes 3 and 16 in a patient with Mayer-Rokitansky-Kuster- Hauser syndrome reveals new candidate genes at 3p22.3 and 16p13.3 Lacey S. Williams1, Hyung-Goo Kim1, Vera M. Kalscheuer2, J. Matthew Tuck1, Lynn P. Chorich1, Megan E. Sullivan1, Allison Falkenstrom1, Richard H. Reindollar3 and Lawrence C. Layman1,4,5* Abstract Background: Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, or the congenital absence of uterus and vagina, is the most severe anomaly of the female reproductive tract. It affects 1 in 5,000 females, and is the second most common cause of primary amenorrhea. The etiology remains unknown in most patients, although four single gene defects and some repetitive copy number variants (CNVs) have been identified. Translocations in MRKH patients are very rare, and reported only in three patients previously without breakpoint mapping. We have identified the fourth MRKH translocation patient and are the first to characterize the breakpoints mapped by molecular methods. Results: The proband is a 17- year old white female with agenesis of the uterus and vagina who had a peripheral blood karyotype revealing a de novo balanced translocation 46,XX,t(3;16)(p22.3;p13.3)dn. There were no known related anomalies present in the proband or her family. No CNVs were found by chromosomal microarray analysis, and no genes were directly disrupted by the translocation. DNA sequencing of six nearby candidate genes—TRIM71, CNOT10, ZNF200, OR1F1, ZNF205,andZNF213—did not reveal any mutations. RT-qPCR of proband lymphoblast RNA for 20 genes near the breakpoints of 3p22.3 and 16p13.3 showed significantly altered expression levels for four genes in the proband compared to three white female controls, after correction for multiple comparisons. Reduced expression was seen for CMTM7 and CCR4 on 3p22.3, while increased expression was observed for IL32 and MEFV on 16p13.3. Conclusion: We have mapped the breakpoints of our t(3;16)(p22.3;p13.3) translocation patient using molecular methods to within 13.6 kb at 3p22.3 and within 1.9 kb for 16p13.3 and have suggested 10 nearby genes that become plausible candidate genes for future study. * Correspondence: [email protected] 1Section of Reproductive Endocrinology, Infertility, & Genetics, Department of Obstetrics & Gynecology, Medical College of Georgia, Augusta University, Augusta, GA, USA 4Department of Neuroscience & Regenerative Medicine, Augusta University, Augusta, GA 30912, USA Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Williams et al. Molecular Cytogenetics (2016) 9:57 Page 2 of 7 Background has not been performed for any of them [21, 22]. In this Abnormal development of the uterus and vagina affects study we present an MRKH patient with a de novo 7-10 % of women, comprising a significant cause of balanced translocation of 46,XX,t(3;16)(p22.3;p13.3)dn impaired reproductive function [1]. Mayer-Rokitansky- with the purpose to: 1) identify the molecular breakpoints Kuster-Hauser (MRKH) syndrome, also known as con- of 3p22 and 16p13; 2) propose candidate genes for genital absence of the uterus and vagina or mullerian MRKH; and 3) compare the proband to other MRKH aplasia, is the most severe anomaly of the female repro- patients with balanced translocations presented in the ductive tract in which the uterus and vagina are absent literature. from birth [1]. MRKH (the name patients prefer) affects approximately 1 in 5,000 females, and is the second Case presentation most common cause of primary amenorrhea [1]. These The proband is a 17-year-old white female with agenesis women are 46,XX females that lack the vagina and most of the uterus and vagina who had a peripheral blood of the uterus, although fallopian tubes may be present karyotype revealing a de novo balanced translocation [2]. Ovaries are present with normal function, thus pa- 46,XX,t(3;16)(p22.3;p13.3). She has no associated renal, tients undergo spontaneous puberty. skeletal, or hearing anomalies. She has two unaffected MRKH is commonly classified with regard to the pres- sisters and two brothers (Fig. 1). Both parents and her ence or absence of additional anomalies [2, 3]. Isolated unaffected sister II-5 have normal karyotypes, and all agenesis of the uterus and vagina occurs in about two- three nieces (III-1, III-2, and III-3) have no known mul- thirds of MRKH patients, classified as Type I. The lerian, renal, or skeletal defects. remaining one-third of MRKH patients have one or more associated anomalies, and are classified as Type II. More frequent associated anomalies involve the kidneys Results with renal agenesis (32 %) and the skeletal system (12 %) Flow sorting of both derivative chromosomes 3 and 16 [3]. Less commonly, women with MRKH may also followed by comparative genomic hybridization (CGH) present with deafness, inguinal hernia, or abnormalities showed no deletions or duplications, and the breakpoints of the cardiac or nervous systems [3]. were localized (Fig. 2). The breakpoint of der(3) was nar- While the etiology of MRKH is not well understood, dis- rowed to within 13.6 kb at 3p22.3; and to within 1.9 kb on ease clustering in >67 families clearly indicates a genetic 16p13.3. In neither derivative chromosome was a gene dir- component [4]. A number of candidate genes including ectly disrupted, but nearby genes become candidate genes AMH, AMHR, WT1, WNT7A, CFTR, GALT, HOXA7, for MRKH (Fig. 2). HOXA13, PBX1, HOXA10, RARG, RXRA, CTNNB1, PAX2, Six genes near the breakpoints were prioritized as rea- LAMC1, DLGH1,andSHOX have been screened for muta- sonable candidate genes based upon either expression in tions in small numbers of MRKH patients, but no muta- appropriate tissues and/or proposed gene function. DNA tions were found [5–9]. Genomic regions 16p11 and 17q12 sequencing was performed on genomic DNA from 51 most commonly have been found to have copy number MRKH patients for the protein coding exons and splice variants (CNVs) identified by chromosomal microarray junctions for the two closest candidate genes near the analysis implicated in MRKH, but causation is difficult to breakpoint at 3p22.3—TRIM71 centromeric and CNOT10 prove [10, 11]. It is currently not clear if MRKH is a telomeric. In addition, ZNF200, OR1F1, ZNF213,and genomic disorder or if a single gene or several genes within ZNF205 at 16p13.3 were sequenced in 27 MRKH patients. these CNVs could be etiologic [5]. Single gene defects have No likely causative (nonsense, frameshift, or splice site) or uncommonly been identified–only a few patients have WNT4 [12–15], LHX1 [16], HNF1B [17], or TBX6 [18] gene mutations. The molecular basis for MRKH remains 1 2 unknown in the vast majority of patients [5]. I Patients with balanced translocations provide a unique and valuable opportunity to identify genes involved in 1 2 3 4 5 human genetic disorders [19]. The derivative chromosome breakpoint may disrupt or dysregulate genes, suggesting a II genomic region of etiologic candidate genes [20]. This MRKH method has been successful in identifying candidate genes 1 2 3 in other disorders, and may be valuable to elucidating the III molecular mechanisms of MRKH [20]. Only three MRKH patients with balanced translocations have been reported Fig. 1 The pedigree is shown for the proband with a de novo t(3;16) translocation in the literature, but fine mapping by molecular methods Williams et al. Molecular Cytogenetics (2016) 9:57 Page 3 of 7 32.24 Mb 33.15 Mb 909 kb TEL 3p.22.3 CEN DYNC1LI1 CMTM7 TRIM71 GLB1 CNOT10 TMPPE CMTM6 CCR4 CMTM8 CRTAP 13.6 kb breakpoint region 3.07 Mb 3.32 Mb 253 kb TEL 16p.13.3 CEN CASP16P OR1F1 ZNF200 LINC00921 ZSCAN10 ZNF263 OR1F2P IL32 ZNF75A ZNF205 ZNF213 MEFV 1.9 kb breakpoint region TIGD7 Fig. 2 Shown are the breakpoints with nearby candidate genes for chromosome 3p22.3 (top) and 16p13.3 (bottom) potentially causative (nonsynonymous missense changes) 16p13.3—IL32 with 7.3 fold and MEFV with 1.6 fold in- variants were identified for any of these six genes. creases in expression (Fig. 3). The altered expression of Since the translocation could disrupt regulatory ele- these four genes reached significance by a Z-test with a ments, we performed RT-qPCR from lymphoblastoid P < 0.00001 (P < 0.0025 was considered significant after RNA from the proband for 20 genes near the break- Bonferroni correction). points to see which ones, if any, had altered expression. This could provide additional supportive evidence for a Discussion and conclusions candidate gene(s). Using the CT method [23], four The etiology of MRKH remains largely unknown [5, 24], genes in the proband had significantly different expres- although when families are examined, autosomal domin- sion from the mean of three white female controls after ant or multifactorial/polygenic inheritance seems most correction for multiple comparisons. These included likely [4]. Several potentially causative repetitive CNVs two genes on chromosome 3p22.3—CMTM7 with 0.22 have been described—most commonly deletions in 17q12 fold (78 % reduction) and CCR4 with a 0.64 fold (36 % (n > 9) and 16p11 (n >5) [5], but it is not clear if they play reduction) change—and two genes on chromosome a pathogenic role [5, 24].