1, Part I: Gene Pathogenic Female Reproductive Tract Abnormalities In Patients 2, Part II: Clinical Phenotype, Family History Research 98 Cases Of Chinese MRKH Syndrome And Psychological Functions

Background:The complex topic of female genital tract malformations should include malformations that affect the development and morphology of the Fallopian tubes, uterus, vagina and vulva, with or without associated ovarian, urinary, skeletal, auditory or other organ malformations. Despite of growing interests and research in this field during the past20years, the etiology of female genital tract malformations is still unclear.Among the relevant research, an increasing number of studies have aimed at investigating the genetic basis of MRKH (Mayer-Rokitansky-Kuster-Hauser) syndrome. However, genetic investigations focused on the genes of anti-Mullerian hormone (AMH) and its receptor, as well as on LHX1, WNT and HOX genes, have been unproductive. Only the Wnt4gene has been clearly implicated in MRKH syndrome and found to be associated with clinical and/or biological signs of hyperandrogenism in three different reports. In the past years, Array-CGH technique has offered new opportunities to discover cryptic chromosome imbalances causative of congenital malformations. Four studies including a wide clinical spectrum of MRKH patients disclosed a similar deletion of genomic DNA at chromosome17q12by array-CGH analysis.17q12deletion appears to be pathogenetically related with a subset of individuals affected by MRKH syndrome, with or without renal disease. However, no CNVs (Copy Number Variatons) analysis was performed in Chinese MRKH patients till now.Methods:1. Copy number assays on the Affymetrix SNP6.0genotyping platform were conducted in10Chinese Type Ⅱ MRKH patients that recruited in the clinics of Peking Union Medical College Hospital from.2. A consanguineous family with a multiple malformations (including genitourinary system malformations) offspring was recruited in the clinics of Peking Union Medical College Hospital. Copy number assays on the Affymetrix SNP6.0genotyping platform were performed in the proband. Whole exome sequencing was performed in the proband and her parents.3^PCR amplification and Sanger sequencing were used to verify the mutational sites revealed by analysis of the whole exome sequencing.4mutational sites in4different genes that have destructive effects on the proteins encoded by these genes were verified both in the proband and other members of the consanguineous family. 4. To verify the rarity of these mutations, these4damaging mutational sites were screened in200Chinese Han female unrelated population using PCR amplification, Sanger sequencing and restriction enzyme digestion test.5. Function of proteins that encoded by these4genes was analyzed to determine whether these4mutational sites were associated with the development of the female genital tract.Results:1. Copy number assays in10Chinese Type ⅡMRKH patients did not reveal any CNVs presented in earlier studies or novel significant CNVs.2. No significant CNVs were found in the proband.3. De novo sequencing data analysis of whole exome sequencing revealed no significant mutations. Genome-wide homozygosity mapping analysis identified4damaging mutational sites in the proband, of which3were in Chr11and1in Chr9.4. Co-segregation analysis was performed to verify these4mutational sites in the consanguineous family. The first3variations were not found in other members of the family. Variation of HSD17B3c.749A>G (p.Y299H) was also found in the proband’s father. As the absence of female genital tract development in the proband’s father, the possibility that variation of HSD17B3c.749A>G (p.Y299H) may play a role in female genital tract malformations can not be ruled out.5. No heterozygous carrier was found for any of these4variants in200cases of normal Chinese female population.6. Functional analysis of these4mutations indicate that variations ofATG2A c.3313G>A (PI105S) and ZFPL1c.749G>A (S250N) are more likely to be causative gene mutations for female genital tract malformations, while variations of MYEOVc.59G>A (R20H) and HSD17B3c.749A>G (p.Y299H) are less possible to be causative mutations.Conclusions:1.10cases of type ⅡMRKH patients and a consanguineous family were studied to detect the causative genes of female genital tract malformation.2. An advanced genetic technique-whole genomic CNVs analysis on Affematrix SNP6.0genotyping platform was performed in10MRKH patients and the proband of the consanguineous family.3. As a pioneering study, the negative findings of copy number assays in Chinese MRKH patients might be attributed to the racial differences between Europeans and Asians.4. An advanced genetic technique-whole exome sequencing was used for the analysis of exome sequencing in the proband and her parents. 5. Functional analysis of these4mutations indicate that ATG2A c.3313G>A (P1105S) and ZFPL1c.749G>A (S250N) are more likely to be causative gene mutations for female genital tract malformations.6. Further animal and biological experiments are needed to verify the roles of ATG2A and ZFPL1played in the development of female genital tract.7. The study of causative genes in female genital tract malformation will provide new ideas and ways for molecular mechanism, genetics, embryologic screening and prevention of this complex disease. Background:The Mayer-Rokitansky-Kuester-Hauser (MRKH) syndrome is also called "Congenital absence of uterine and vagina" in China. Clinically, this syndrome presents as a rudimentary solid bipartite uterus with solid vagina. The extent of MRKH syndrome is variable, and it is associated with various additional malformations. This is reflected in the classification, which is subdivided into type I (typical) and type II (atypical) depending on each additional malformation that is present. The clinical spectrum of MRKH syndrome has been expanded with the advances in its research. However, there is still no research of large samples in the spectrum of genital and associated malformations in women with Mayer-Rokitansky-Kuster-Hauser syndrome in China. Although the increased familial frequency of the syndrome reported, most MRKH patients are sporadic. Also with the difficulty in having their own genetic children, the etiology of MRKH syndrome is still unclear and continues to be a matter of controversy. With a lack of comprehensive family tree analyses for MRKH patients, screening of the relatives of these patients is important. Treatment of MRKH patients should not only meet the need of sexual life anatomically, but also improve their psychological status and quality of life. There have been dozens of studies about the sexuality of MRKH patients after different treatments these years. However, few studies have been conducted in the psychological status of MRKH patients.Methods:From2012March to2013March,106cases of MRKH patients were recruited in the gynecology clinic of Peking Union Medical College Hospital.6ml of venous blood samples were collected from each patient and some patients’first-degree relatives to establish the blood specimen bank of MRKH patients. General information and family medical history were collected from each patient. Comprehensive laboratory examination was conducted in each patient. Specific psychological function questionnaires were surveyed in these patients. For patients who had sexual life within1month of our survey, Chinese version of Female Sexual Function Index was dispatched. All patients and some patients’ first-degree relatives have singed informed consent in the clinic.Results:1.98MRKH patients accepted comprehensive laboratory examination in our study. Of them,56(57.1%) cases were type Ⅰ,42cases (42.9%) were type Ⅱ. Among type Ⅱ MRKH patients,34cases(34.7%,34/98) had various kinds of renal malformations,26 cases(26.5,26/98) had various kinds of skeletal malformations,18patiens (18.4%,18/98) had both renal and skeletal malformations.2. A Survey of these98patients’ family medical history revealed that first degree relatives of8MRKH patients had renal and (or) skeletal malformations. A consanguineous family with a MRKH patient offspring was reported for the first time in our study. Also we reported two cases of heterozygotic discordant twins, among whom one twin had MRKH syndrome while the other had severe skoliosis for the first time.3. A survey of the psychological function of these98MRKH patients revealed that only11.2%of patients could accept the diagnosis at the first time. After mental adjustment,68.4%of patients could accept the diagnosis at the time of our survey. Also we find that60.2%of patients had a sense of embarrassment,90.8%of patients feared that others would know. A survey of reactions of56patients’partners revealed that41.1%(23/56) partners were ignorant of their disease,57.1%(32/56) were keen on having offsprings.4. A survey of the postoperative situation in these56MRKH patients revealed that the time of mould wearing in the biological mesh group is shorter than in the peritoneum group. The time interval between the end of treatment and the time of having sexual life in the biological mesh group is also shorter than that in the peritoneum group. Ratios of patients in biological mesh group who reported had improved body evaluation and quality of life were both higher than the other two groups. Survey of FSFI in these56MRKH patients revealed that sexual desire and sexual arousal in the biological mesh group were better than those in the peritoneum group. No statistically significant differences in the other aspects and total score of FSFI were found between these two groups.Conclusions:1. Our study revealed that about1/3of MRKH patients had renal malformations and almost1/3of MRKH patients had skeletal malformations. We suggest that for MRKH patients, medical history of kidney and skeletal systems should be routinely asked. Five laboratory examinations including chromosome karyotype analysis, pelvic ultra-sonography, kidney ultrasonography, spinal radiographs and assessment of female sexual hormones should all be conducted in MRKH patients.2. According to the findings in the family medical history of these patients, we suggest that all first (or second) degree relatives of MRKH patients should be screened for renal and (or) skeletal malformations. Besides, the monozygotic discordant twins and consanguineous family that first repoted in our study would be helpful in the etiological study of MRKH syndrome.3. According to our study in the psychological status of MRKH patients, most of them had a sense of embarrassment and fear. After mental adjustment, the acceptance of their diagnosis improved significantly. This further suggests the importance of psychological counseling and therapy in the treatment of MRKH syndrome. The survey of partners’ reactions revealed that the way for patients to communicate with their partners, to maintain and improve the relationship with their partners is a critical and difficult problem for clinical workers.4. With the shorter time of mould wearing and time interval between the end of treatment and the time of having sexual life, the higher ratios of patients who reported that had improved body evaluation and quality of life and better sexual desire and sexual arousal in FSFI, we recommend that vaginoplasty with biological mesh should be extended for the treatment of MRKH patients.