A Novel Missense Mutation 224G>T (R75M) in SRY Coding Region Interferes with Nuclear Import and Results in 46, XY Complete Gonadal Dysgenesis

Abstract

SRY-mutation-caused sex reversal is a rare disease and mostly associated with a de novo mutation since the patients with defective SRY is infertile. There are many reports about SRY-mutation associated 46, XY ovarian disorder of sex development (DSD), but few described their molecular mechanism. Here we report a de novo mutation 224G>T (R75M) in SRY associated with a phenotypic female, 46, XY karyotype and dysgerminoma. The wild and mutated SRY were cloned into recombinant plasmid and expressed in cells in vitro, the result showed the mutated SRY is greatly accumulated in cytoplasm while the wild type SRY is mostly localized in nucleus. To make sure no other genes were involved, we performed the trio-based whole exome sequencing using the DNA samples from the proband and the parents, and no mutations were identified especially in DHH, NR0B1, NR5A1, SOX9 and MAP3K1, indicating the de novo mutation in SRY is the single defect responsible for the female sex reversal. We also used bioinformatics simulation analysis to predict impact of the mutation on SRY function, and find the R75 in wild type SRY can form a hydrogen bond with serine at 91 (S91) that make the SRY protein well fit into the minor groove of target DNA, while the M75 in the mutated SRY can't. Finally, we reviewed SRY mutations based on the available references and analyzed the mutation distribution patterns according to density and continuity, which may be useful for further study of the SRY structure, function, and its relatedness with DSD.

Fig 1. Karyotype of the proband.

The Y chromosome is marked by a red arrow.

Fig 2. Gel Electrophoresis of PCR Product with SRY-Specific Primers.

Lane-1 represents the normal male; Lane-2 the proband; and Lane-3 the negative control (everything except template DNA).

Fig 3. DNA Sequencing to identify Point Mutation.

A. The mutation 224G/T is identified in proband's SRY; B. No sequence change is identified in proband’s father.

Fig 4. The Point Mutations Distribution in SRY.

A. Mutations occurred in SRY from 1^st to 56^th amino acids (AAs) upstream the HMG domain. A total of 9 mutations were out of 56 AAs (16.07%) including 5 missense mutations (5/9, 55.6%) and 4 nonsense mutations (4/9, 44.4%); B. Mutations occurred in SRY from 57 to 136 AAs covering the whole HMG domain. A total of 66 mutations were out of 80 AAs (82.5%), including 54 missense mutations (54/66, 81.8%), 12 nonsense mutations (12/66, 18.2%); the novel mutation described in this paper is highlighted by red color and arrow; C. The mutations occurred in SRY downstream of 3’ HMG box from 137 to 163 AAs. A total of 5 mutations were out of 27AAs (18.5%); including 2 missense mutations (2/5, 40%), 1 nonsense mutation (1/5, 20%), and 2 frame-shift mutations (2/5, 40%). D. No mutations were reported in SRY from 164 to 204 AAs. Note: the first row of AAs in each figure represents amino acid sequences of the normal SRY protein, the AAs beneath the first row represent the mutated amino acids; the pound sign# represents frame shift, and asterisk* represents nonsense mutations. Y-axis indicates mutation frequencies, and X-axis marks AAs sequence in SRY protein.

Fig 5. Impact of R75M Mutation on SRY Nuclear Import.

MGC-803 cells were transiently transfected to express GFP and GFP-SRY fusion proteins. Photos were taken 24h later by laser scanning confocal microscope (40 x magnifications). A. Laser confocal microscope observation of pZsGreen1-N1, showing the GFP is distributed mainly in cytoplasm; B. Laser confocal microscope observation of pZsGreen1-N1-SRY, showing the overwhelming accumulation of WT-SRY in nucleus; C. Laser confocal microscope observation of pZsGreen1-N1-R75M, showing both nuclear and cytoplasm accumulation of mutant SRY protein as granules or clumpy spots.

Fig 6. Simulation Analysis of R75M Point Mutation that Causes Impaired DNA-Binding Capability.

A. The arginine at position 75 (R75) can form a hydrogen bond with serine at position 91 (S91) which fits well with the minor grove of target DNA. B. Showing how the R75 form a hydrogen bond with S91 in detail. C. When arginine (R75) is substituted by methionine (M75), the hydrogen bond with S91 is destroyed.