Discussion
The DMRT1 gene encodes a DNA-binding DM domain transcription factor and is widely conserved across animals. The DMRT1 protein can binds and regulates genes which were known to play an important role in sexual development. It can active male sex–determining gene Sox9 and repress female sex–determining genes Wnt4 and Rspo1 (10). It has been demonstrated that the mutation or loss of DMRT1 gene can disturb the normal male sexual development in several non-mammalian vertebrates like zebrafish (20-22), medaka (23), chicken (24), largemouth bass (25). In mammals, the DMRT1 gene is not the male sex determinant, but overexpression of Dmrt1 gene in XX mice can cause female-to-male sex reversal, indicating that this gene can initiate male development (26). In human fetal testis, repressing the expression of DMRT1 gene can change the expression of key genes which were necessary in gonadal development, leading to a move away from a male phenotype to a more ovarian-like phenotype (27).
In humans, the deletions or mutations of DMRT1 gene has been reported to cause DSD or sex-reversal (4, 8, 11, 28). Through literature review, we found twelve different kinds of mutations in the DMRT1 gene and the phenotypes are variable (Table1, Fig. 2C) (7, 13-18, 28). All of twelve mutations were heterozygous and no homozygous mutation was reported except the homozygous mutation c.967G>A detected in our study. Among these twelve mutations, seven mutations were related to sex determination. Four of these seven mutations (c.240G>C, c.251A>G, c.331A>G, c.332G>T) were located in the first exon of DMRT1 gene, and the other three mutations were mapped in exon 2 (c.416T>A), exon 4 (c.884C>T) and 3’UTR (3’UTR+11insT). The DMRT1 protein contains a highly conserved human DNA-binding domain (DM domain) located in the amino acid sequence 67 to 136 of the DMRT1 protein (DMRT167–136) (28). The four mutations c.240G>C, c.251A>G, c.331A>G, c.332G>T mapped in the DMRT167–136 caused DSD. The functional study of mutant protein (c.331A>G, R111G) showed that the mutant protein had strongly reduced DNA affinity and it also can interfere with the binding stoichiometry of wild-type DMRT1, suggesting that the mutation c.331A>G may combine haploinsufficiency with a dominant negative to cause 46,XY sex reversal (28). Deletions involving the DMRT1 gene have been reported in many individuals with GD, but most large deletions contain not only the entire DMRT1 gene but also other nearby genes. Review of literature found five intragenic deletions in the DMRT1 gene (Table 1, Fig. 3C). One case with a 103Kb deletion affecting exons 1 and 2 caused 46,XY GD (11); one case with a 35Kb deletion affecting exons 3 and 4 caused 46,XY ovotesticular disorder of sexual development (12); the other three cases including a 141Kb deletion affecting exons 3-5, a 150Kb deletion affecting exons 3-5 and a 55Kb deletion affecting exons 3 and 4, are all caused azoospermia (19). These cases demonstrated that intragenic deletions affecting exons 1 and 2 of the DM domain of the DMRT1 gene would disrupt the mRNA expression and exhibit haploinsufficiency for 46,XY GD, while the deletions affecting exons 3-5 might produce truncated proteins with reduced activity for a specific phenotype of azoospemia. The genotype-phenotype correlations for the DMRT1 gene from reported cases indicated that missense mutations and intragenic deletions affecting the DM domain would cause more severe phenotype of 46,XY GD to CGD. The variable phenotypes from azoospermia to sex reversal could be resulted from deletions and missense mutations involving exons 3, 4 and 5 of the DMRT1 gene.
In this study, we reported a homozygous mutation (c.967G>A, Val323Ile) of DMRT1 gene in a Chinese patient with 46,XY CGD. Her mother and old sister have a heterozygous c.967G>A mutation with a normal phenotype. Her healthy father refused to provide samples for testing, but he is an obligate heterozygous carrier of this mutation theoretically. The c.967G>A mutation was a missense variant with very low frequency in GnomAD (1/246186) and ExAC (1/121394 ) database and not reported in ClinVar. The homozygotes of this variant were not found in normal populations or DSD patients. According to ACMG variant interpretation guidelines, this variant was categorized as uncertain significance (29). This mutation was located outside of the DMN domain at the last base of the fourth exon of DMRT1 gene, and the minigene test showed that the mutation has no effect on mRNA splicing. It is reasoned that this c.967G>A is most likely hypomorphic mutation with reduced level of activity and minimal impact on sex development in heterozygous. When this mutation presented in a homozygous state, the reduced activity from both alleles probably reach the sensitive threshold of haploinsufficiency and caused 46,XY CGD in an autosomal recessive pattern.
In conclusion, we found a homozygous c.967G>A mutation of DMRT1 gene in a patient with 46,XY CGD. This unique case suggested the presence of hypomorphic mutations in the DMRT1 gene which could be inherited in an autosomal recessive pattern and exhibit haploinsufficiency of the DMRT1 gene at homozygous state to cause disorders of sex development. This finding enriched the mutational spectrum of the DMRT1 gene and highlighted the importance of accurate genotype-phenotype correlations for the interpretation of genetic defects for 46,XY GD. Further investigation should be performed to clarify the molecular mechanisms causing disorders of sex development by deletion or mutation of DMRT1 gene.