Cell Research | 2019
Joint utilization of genetic analysis and semi-cloning technology reveals a digenic etiology of Müllerian anomalies
Abstract
Dear Editor, Identifying pathogenic gene mutations and their combination is critical but challenging in dissecting the etiology of complex diseases when more than one gene is involved. The digenic/ oligogenic/omnigenic models, holding that more than one gene could act synergistically, appeal to a wide range of genes responsible for complex phenotypes. These genetic models advanced our understanding of genetic factors underlying complex phenotypes, yet an accordingly rapid and efficient experimental assay for identifying pathogenic combinations of genetic variants at animal model level is lacking and urgently needed. Recapitulating multiple human genetic variants in mice allows them to be examined in a fixed genetic background, which is especially powerful for establishing oligogenicity. In a newly published study, a combination of triple-compound heterozygous variants was identified in a family affected by a heart disease, and was further verified in a mouse model by zygotic injection of CRISPR-Cas9 system followed by multiple generations of intercrossing. These findings revealed the joint contributions of gene variants to the etiology of complex diseases. However, sequential intercrossing for generations to obtain mice harboring multiple genetic variants is time-consuming. Additionally, founders generated from zygotic injection of CRISPR-Cas9 system are usually genetically mosaic, making phenotyping of founders extremely difficult. Our previous work successfully derived androgenetic haploid embryonic stem cells (AG-haESCs), which can maintain haploidy via periodic cell sorting, from sperm-originated haploid embryos. Importantly, AG-haESCs can serve as a “sperm replacement” to deliver multiple genetic modifications into descendants via intracytoplasmic AG-haESC injection (ICAHCI), enabling rapid phenotyping of uniform founders without mosaicism in one generation. Consequently, this AG-haESC-mediated semi-cloning technology may be a rapid and efficient experimental assay for identifying pathogenic combinations of genetic variants in complex diseases. Müllerian anomalies (MA) include a wide variety of anatomic malformations in the uterus, cervix, fallopian tubes, or vagina, stemming from the variable aberrances during the development of Müllerian ducts. MA brings with women not only adverse reproduction capacity, but also psychological distress. Regarding the etiology of MA, genetic risk factors are deemed to harbor a strong influence; however, the wide phenotypic and genotypic heterogeneity across MA individuals makes it extremely difficult to determine the underlying genetic risk factors. For years, no major genes have been found to account for human MA in the monogenic inheritance, implying the genetic complexity of MA and an urgency to interrogate the potential involvement of pathogenic variant combinations in MA and other analogous complex diseases. So far, several previous studies have implicated the involvement of genomic copy number variants (CNVs, especially genomic deletions) in the complex etiology of MA. To explore the etiology of MA, we thus first employed comparative genomic hybridization (CGH) microarrays to analyze genomic CNVs in 25 women with MA. We found 7/25 (28%) of the subjects carried rare and large (>100 kb) CNVs (Supplementary information, Tables S1, S2 and Data S1). Changes in gene dosage caused by CNVs are critical for the pathogenesis of human developmental disorders. We continued to identify the CNV-affected genes that may play crucial roles in MA. We focused on genes involved in MA or related to female reproductive tract and examined whether they are deleted in these 7 subjects. Three genes, namely GEN1, TBX6, and LHX1 are supposed to fit the bill. Thus the newly identified GEN1/2p24.2 deletion (carried by subject M45; 1/25), along with two previously reported TBX6/16p11.2 deletion (carried by subject B03; 1/25) and LHX1/17q12 deletion (carried by subject B07; 1/ 25), are potential risk CNVs for MA (Fig. 1a; Supplementary information, Tables S2, S3). Herein, the frequency of rare MAassociated CNVs is at least 12% (3/25) in our discovery cohort of MA. Subsequently, we continued to explore whether these rare CNVs (GEN1/2p24.2, TBX6/16p11.2 and LHX1/17q12) are recurrent in a larger MA population. For this, we enrolled another 100 women with MA, and conducted targeting qPCR analysis to preliminarily screen for CNVs in the above regions. We identified another 6 cases carrying potential deletions in TBX6/16p11.2 (4/ 100) or LHX1/17q12 (2/100). To verify these potential CNVs identified by qPCR, we further conducted CGH microarray analysis in these 6 cases, and found that all of them are indeed carriers of the corresponding TBX6/16p11.2 or LHX1/17q12 deletion CNVs (Fig. 1a; Supplementary information, Table S3). In total, we identified 9 cases carrying potential pathogenic CNVs in the 125 patient cohort (9/125) and demonstrated that two of these CNVs are recurrent in Chinese MA cases (Fig. 1a; Supplementary information, Tables S1, S3). CNVs may contribute to human diseases with variable clinical manifestations. As shown in our cohort and previous studies, TBX6/16p11.2 deletion and LHX1/17q12 deletion are recurrent in human subjects with MA. However, deletion CNVs of 16p11.2 or 17q12 can also lead to other diseases without MA phenotypes. As for the GEN1/2p24.2 deletion, the heterozygous null mutant of mouse Gen1 showed no obvious MA phenotypes in females. Based on these facts of incomplete penetrance, we raise our hypothesis that single genetic variant might be insufficient for MA manifestation, and other genetic factors could contribute synergistically to the pathogenesis or increase the penetrance, resembling the digenic/oligogenic models (Fig. 1b). Deleterious genetic variants, such as deletion CNVs and single nucleotide variants (SNVs), can destroy gene function. CGH microarray analysis can efficiently identify genetic variants involving CNVs, but is not applicable to SNVs. For this reason, to gain a comprehensive insight into genetic etiology of MA, we detected SNVs using whole exome sequencing (WES) in the 9 CNV carriers. The genes with hints for contributions to MA or involvements in the female reproductive tract development in