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.