Genome Sequencing and Transcriptome Profiling in Twins Discordant for Mayer-Rokitansky-Küster-Hauser Syndrome

Abstract

To identify potential genetic causes for Mayer-Rokitansky-Küster-Hauser syndrome (MRKH), we analyzed blood and rudimentary uterine tissue of 5 MRKH discordant monozygotic twin pairs. Assuming that a variant solely identified in the affected twin or affected tissue could cause the phenotype, we identified a mosaic variant in ACTR3B with high allele frequency in the affected tissue, low allele frequency in the blood of the affected twin, and almost absent in blood of the unaffected twin. Focusing on MRKH candidate genes, we detected a pathogenic variant in GREB1L in one twin pair and their unaffected mother showing a reduced phenotypic penetrance. Furthermore, two variants of unknown clinical significance in PAX8 and WNT9B were identified. In addition, we conducted transcriptome analysis of affected tissue and observed perturbations largely similar to those in sporadic cases. These shared transcriptional changes were enriched for terms associated with estrogen and its receptors pointing at a role of estrogen in MRKH pathology. Our genome sequencing approach of blood and uterine tissue of discordant twins is the most extensive study performed on twins discordant for MRKH so far. As no clear pathogenic differences were detected, research to evaluate other regulatory layers are required to better understand the complex etiology of MRKH.

Keywords: MRKH syndrome; Müllerian ducts; genome sequencing; monozygotic discordant twins; transcriptome analysis.

Figure 1

Mosaic missense variant in ACTR3B identified in blood and tissue of twin 2-1. IGV reads show that the variant c.1066G>A, p.Gly356Arg in ACTR3B has an allele frequency of about 39% in uterine tissue and an allele frequency of about 11% in blood of the affected twin 2-1, while blood of twin 2-2 only has 1 supporting read.

Figure 2

No significant change in genes expression observed for ACTR3B, GREB1L, PAX8, and WNT9B. Expression levels for the selected DEGs plotted as individual data points with mean ± SEM.

Figure 3

Endometrial transcriptomes of MRKH twins and sporadic cases show largely similar changes. (A) Schematic diagram of comparisons between MRKH monozygotic twins (MZT), MRKH sporadic cases (SP) and unaffected women (controls) indicating number of differentially expressed genes (DEGs). DEGs between sporadic cases and controls based on previous work [13]. Fold change and significance cut-offs below. (B) Venn diagram showing number of common DEGs MRKH twins and sporadic cases each compared to controls. (C) Expression profiles (log₂ expression change relative to Ctrl group) of 174 DEGs (common DEGs indicated in Figure 3B) across all samples. Rows hierarchically clustered by Euclidian distance and ward.D2 method. Cycle information (proliferative or secretory) and patient type (monozygotic twin, sporadic, or control) on top.

Figure 4

Gene expression changes in MRKH monozygotic twins point at regulators linked to estrogen receptor. (A) Predicted upstream regulators for common 174 DEGs (from Figure 3B) based on Ingenuity Pathway Analysis. Top five significant regulators shown. (B) Network of DEGs associated with upstream regulator fulvestrant. Interactions based on Ingenuity Pathway Analysis with line width indicating number of curated interactions. Genes color-coded by mean expression change observed in MZT/CTRL on top and SP/CTRL separately for type 1 and type 2 below. (C) Expression levels for the selected DEGs plotted as individual data points with mean ± SEM.