Results from pedigree and genetic analyses
The phenotypic female proband was the fifth child of a healthy woman. The pedigree of the family as shown in Figure 2A indicated that the parents (III-1/2) of the proband (IV-5) are first cousins. All elder sisters (IV-1/2/3) and the brother (IV-4) of the proband were phenotypically normal, the eldest sister (IV-1) had given birth to two female babies (V-1/2)(Fig. 2A). Informed consent for genetic analysis was obtained from the patient (IV-5), the eldest sister (IV-1), and the mother (III-2), but the other family numbers refused to accept the genetic analysis.
Routine chromosome analysis performed on the proband found a normal male karyotype of 46, XY. Genomic DNA was extracted from peripheral blood samples of the patient, the eldest sister and the mother, using QIAamp DNA Blood Mini Kit (Qiagen). The concentration and quality of genomic DNA was analyzed by Nano-drop ND-1000 (Thermo Scientific, Wilmington, DE, USA).
Chromosomal Microarray Analysis (CMA) was performed to detect the copy-number variant (CNV) and the region of homozygosity (ROH) using a CytoScan 750 array (Affymetrix, Santa Clara, CA, USA) by following the manufacturer’s instructions. No causative deletion or duplication was found, while 19 ROH regions amounting to 175Mb (6.31% of total autosomal length) were noticed on the proband, which was consistent with the consanguinity of the parents. Actually, the patient initially concealed the genetic relationship of her parents, until we reminded the indication of the CMA result.
To detect the causative gene mutation, next generation sequencing was performed on two gene panels, the first one including more than 200 genes for DSD was analyzed at BGI (Shenzhen, Guangdong, PRC), the second one including more 2800 genes by clinical whole-exome sequencing (WES) was performed at our laboratory. A homozygous mutation c.967G>A (p. Val323Ile) in the DMRT1 gene was detected in both panels. No other pathogenic variants were found in other DSD-related genes. Further analysis using Sanger sequencing confirmed this mutation in the patient and detected the mother and the eldest sister as heterozygous carrier for the c.967G>A mutation (Fig. 2B). Because the father refuse the test,the CMA test were done on the sister and mother. A haplotyping analysis on the SNP nearby the DMRT1 gene to dissect mother’s and father’s haplotype from the proband and her sister was done by using the CMA results of proband and her sister and mother to provide an indicated evidence the father should an obligate carrier of this mutation(Fig. 2A).
To rule out the polymorphism of this variant c.967G>A, we screened 64 normal fertile Chinese male individuals by Sanger sequencing, compared the conservatism of the amino acid among different species, and searched online databases including ExAC (http://exac.broadinstitute.org/) and ClinVar (https://www.ncbi.nlm.nih.gov/ClinVar). We did not find c.967G>A (p.Val323Ile) variant among 64 normal fertile male individuals. Val323 amino acid of AR protein is highly conserved in compared mammals. The variant was presents in ExAC (Exome Aggregation Consortium) database (Chr9:916907 G/A, rs746758951) as a singleton state (heterogeneous), so the frequency of the allele is extremely low (1 in total 121394 alleles, 8.238e-06), and the carrier was a European (Non-Finnish) individual, the gender and phenotype of the carrier was not mentioned.
Considering that the c.967G is the last nucleotide of DMRT1 exon 4, we performed Minigene assay to evaluate the effect of c.967G>A to the pre-mRNA splicing. PCAS2 plasmid was used as a vector for this Minigene assay following the protocol of Gaildrat P, et al [9]. The PCAS2 plasmid was generated through a collaboration with Zhang Xue’s Laboratory of Peking Union Medical College. The Minigene assay result showed that the c.967G>A variant had no effect on splicing in comparison with wild type.
To evaluate the genotype-phenotype correlations for the DMRT1 gene, a review of literature on PubMed and of cases on ClinVar database was performed. A total of 12 mutations and five intragenic deletions in the DMRT1 gene was identified from 11 reports (7, 10, 11, 12, 13-19). These mutations and deletions and their associated phenotypes were summarized in Table 1 and Fig 2C.