Pathogenic Mechanism Of BMP4 And BMP7 Genes In The Etiology Of Mayer-Rokitansky-Küster-Hauser Syndrome

Part ⅠPathogenic Mechanism of BMP4 in the Etiology of Mayer-Rokitansky-Kiister-Hauser syndromeBackground:MRKH（Mayer-Rokitansky-Kuster-Hauser）syndrome is caused by abnormal development of the Mullerian duct.Although the disease was first reported in the early 19th century,until now,the etiology remains unclear.In recent years,with the development and application of genome sequencing technology,several candidate pathogenic genes of MRKH syndrome have been discovered.BMP4（Bone morphogenetic protein 4）plays an important role in the development and differentiation of embryos.It has been confirmed that BMP4 mutation can lead to the deformity of bone,teeth,kidneys,and other organs.However,there is no research on the pathogenicity of BMP4 gene mutation in MRKH syndrome.Objects:To investigate the pathogenesis of BMP4 gene mutation in MRKH syndrome at the cellular and animal model levels based on sequencing data from a large cohort of MRKH syndrome patients.Methods:Whole exon sequencing was performed in 442 patients with MRKH syndrome to screen out likely pathogenic mutations of the BMP4 gene.Sanger sequencing was used to verify the authenticity of the variants.Then the effects of the variants on the transcription,translation,and function of the BMP4 gene were evaluated in vitro.Bmp4flox/flox mice were constructed and bred with Cre mice to achieve conditional knockout of the Bmp4 gene in different parts of the Mullerian duct to observe its effect on murine Mullerian duct development.Results:1.Total 12 likely pathogenic missense variants of BMP4 gene were screened out（c.305C>T、c.326G>T、c.338G>A、c₄50C>G、c.619C>G、c.712G>A、c.751C>T、c.874C>T、c.918G>T、c.925C>T、c.979G>A、c.1216G>A）.2.Cell experiments showed that the variant c.305C>T reduced the total protein expression of BMP4.Variants c.338G>A,c.450C>G,c.712G>A,c.751C>T,c.925C>T,and c.979G>A led to a decrease in the amount of BMP4 protein secreted to the extracellular area.However,Variants c.326G>T and c.918G>T resulted in a significant increase in the extracellular secretion of BMP4 protein.Protein localization experiments showed that variants c.326G>T and c.918G>T caused the abnormal localization of BMP4 protein in the intracellular;the BMP4 protein became granular and could not enter the endoplasmic reticulum.3.In animal experiments,both Bmp4flox/flox:Amhr2-Cre female mice and Bmp4flox/flox:Wnt7a-Cre female mice showed phenotypes similar to those of patients with MRKH syndrome,including abnormal uterine development and vaginal atresia.Conclusions:This study,for the first time,found and confirmed that the mutation of the BMP4 gene is the pathogenic cause of MRKH syndrome,and expanded the pathogenic gene spectrum of MRKH syndrome.Part ⅡPathogenic Mechanism of BMP7 in the Etiology of Mayer-Rokitansky-Kiister-Hauser syndrome.Background:Congenital female genital tract malformation is a congenital disorder caused by abnormalities in the differentiation and development of the Mullerian duct,including MRKH（Mayer-Rokitansky-Kuster-Hauser）syndrome,Oblique vaginal septum syndrome,and vaginal atresia.At present,the specific pathogenesis of female genital tract malformation is unclear.More research is needed to uncover the genes responsible for the disease.Studies have confirmed that the gene BMP7（Bone Morphogenetic protein 7）plays an indispensable role in the growth,differentiation,and formation of various tissues and organs.BMP7 mutations can cause malformations in several organs,including bones,kidneys,and eyes.However,there is no research on the pathogenicity of BMP7 gene mutation in genital tract malformation.Objects:To study the pathogenicity of BMP7 gene mutation in genital tract malformation at the cellular and animal levels based on a large cohort of patients with genital tract malformations.Methods:Whole exon sequencing was performed in 492 patients with reproductive tract malformation to identify the possible pathogenic BMP7 variants.Sanger sequencing was used to verify the authenticity of the variants.Then the effects of the variants on the transcription,translation,and function of the BMP7 gene were evaluated by cell experiments.Then Bmp7flox/flox mice were constructed and bred with Cre mice to achieve conditional knockout of the Bmp7 gene in different parts of the Mullerian duct to observe its effect on murine Mullerian duct development.Results:1.Total 10 likely pathogenic missense variants of BMP7 gene were screened out（c.52T>G、c.461G>A、c.496G>A、c.713A>G、c.865C>T、c.866G>A、c.941G>A、c.1126C>G、c.1244T>A、c.1277G>A）.2.It was found that the variants c.52T>G、c.713A>G,and c.865C>T reduced the total protein expression of the BMP7 gene.Variants c.52T>G and c,713A>G resulted in decreased mRNA expression.Variants c.52T>G、c.461G>A、c.496G>A、c.713A>G、c.865C>T,and c.1244T>A resulted in decreased extracellular secretion of BMP7 protein.Variant c.866G>A increased the extracellular secretion of BMP7 protein.Protein localization experiments showed that Variant c.52T>G resulted in abnormal intracellular localization of BMP7 protein,and the protein became granular without obvious overlap with the distribution of the endoplasmic reticulum.3.In animal experiments,the reproductive tract of Bmp7flox/flox:Amhr2-Cre and Bmp7flox/flox:Wnt7a-Cre female mice showed no abnormal phenotype.Conclusions:Through sequencing and analysis of a large sample of clinical cases of reproductive tract malformation,the possible pathogenic mutations of the BMP7 gene were screened.Then the functional verification of the mutations proved the pathogenicity of some mutations of the BMP7 gene.It provides evidence for clinical molecular diagnostic detection.