Noninvasive Prenatal Diagnosis Of Disorders Of Sex Development By Maternal Plasma DNA Sequencing

【 Objective 】 Disorders of sex development(DSD)is defined as congenital conditions in which development of chromosomal,gonadal,or anatomic sex is atypical.However,a specific molecular diagnosis is identified in only 20% of the cases.As for 46,XYchildren with DSD,only50% of them will receive a definitive diagnosis.In the present study,we aimed to expand the spectrum of genetic findings for DSD,then introduced a new haplotype-based approach for the noninvasive prenatal testing(NIPT)of monogenic diseases associated with DSD.【Method】1.Sanger sequencing and multiplex ligation-dependent probe amplification were used to detect point mutations and large gene deletions in 95 Chinese suspected 21-hydroxylase deficiency patients,respectively.2.Whole-genome sequencing was used to identify the candidate genes in one family with testicular regression syndrome(TRG).3.The reconstructed plasmid was transducted into the COS-7 cells to detect subcellular localization of mutant human recombinant protein.4.Immunohistochemical method was performed to detect the expression of gene of interest in testis of adult male mice.5.The m RNA levels in testis of male mice during different development stage were measured with realtime PCR.6.Parental haplotypes were constructed using sequencing data of the parents and the proband.On the basis of the parental haplotypes,fetal haplotypes was recovered by a hidden Markov model through maternal plasma DNA sequencing.【 Result 】 1.35 patients were diagnosed according to clinical manifestations and 17-hydroxyprogesterone(17-OHP)levels and confirmed by genotyping.The most common mutation was c.293-13A/C>G(30.0%),followed by p.I173N(20.0%),large gene conversions(14.3%),large gene deletions(11.4%),and p.R484Pfs*58(4.3%).2.Remarkably,a novel p.F450 L mutation,in silico predicted to be associated with salt-wasting(SW)form,was identified.Two mutations including p.R409 C and p.R427 H,previously considered as conserved in specific ethnicities due to a founder effect,were detected in our cohort.Further,a rare p.H63L+p.V70 L mutation,hitherto only observed in the Chinese population,in trans with different mutations corresponding to SW form resulted in diverse phenotypes.3.A heterozygous c.923G>A variant in DHX37 gene was detected in the family with an autosomal dominant,sexlimited pattern of transmission.4.Wild-type protein was localized to nuclei in COS-7 cell lines,while mutant protein was localized to both the nuclei and cytoplasma.5.DHX37 gene was expressed in stertoli cells,Leydig cells,peritublar cells,and germ cells(present in spermatocytes and spermatids and absent in spermatozoa).6.The DHX37 m RNA level in testis of male mice was significantly increased at 10 days(P<0.02),then reached a peak at 20 days(P<0.0001).7.The fetuses were both predicted to be unaffected carriers,which were in accordance with the results of amniocentesis.【Conclusion】1.The sequence of CYP21A2 gene must be analyzed carefully in case rare or novel deleterious mutations exist.2.DHX37 may be required for the normal development of testis and play a key role in the regulation of spermatogenesis.3.The haplotype-based approach is feasible for NIPT of 17 hydroxylase deficiency and 5-alpha reductase deficiency.