Biallelic MAD2L1BP (p31comet) mutation is associated with mosaic aneuploidy and juvenile granulosa cell tumors

Abstract

MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.

Keywords: Cell cycle; Genetic diseases; Genetic instability; Genetics; Oncology.

Figure 1. Clinical phenotype of family 1 patients.

(A–J) Patient 1a. (A and B) Mild facial dysmorphism (4 years): long face, upward slanting sparse outer eyebrows, low columella, pointed chin, and microcephaly. (C) Fundus: retinal pigmentation defects. (D) Cranial MRI (cMRI) (24 months): underdeveloped brain with dysplastic frontal lobe, interhemispheric cyst, and polymicrogyria on the right near the cyst. Incomplete opercularization and left parietal cyst. (E–J) Histology of ovarian GCT. (E–G) H&E staining shows the faint preserved outlines of the macrofollicular architecture. Regressive calcification is visible in C. (H and I) Reticulin stains the preserved fibers, highlighting the typical and unique architecture for JGCT; E, at higher magnification. (J) Periodic acid–Schiff (PAS) staining shows the typical cytology of the JGCT with monomorphic, round to ovoid cells. (K–U) Patient 1b. (K and L) Face with mild dysmorphism (2 years of age). (M) Ultrasound image of left testicular mass. (N) Thin corpus callosum (cMRI at the age of 18 months). (O–U) Histology of testicular GCT. (O) Testis shows a macrofollicular pattern and contains multiple cysts replacing preexisting prepubertal testicular tissue. (P) Multilayered tumor cells surrounded by spindle cell stroma, reminiscent of ovarian follicles. (Q) Cysts contain pink mucoid material. The cells of the inner layer are pale and have luteinized cytoplasm. (R) Tumor cells show progesterone receptor (PR) expression. (S) Tumor cells with diffuse S100 expression. (T) Faint expression of estrogen receptor (ER). (U) Patchy expression of inhibin. (E, F, H, and O) Original magnification, 2×; scale bar: 500 μm. (G, I, S, and T) Original magnification, 4×; scale bar: 300 μm. (J, P, and Q) Original magnification, 10×, scale bar: 100 μm. (R and U) Original magnification, 40×, scale bar: 50 μm.

Figure 2. MAD2L1BP mutation and MVA in family 1.

(A) Pedigree. Filled symbols: Homozygous MAD2L1BP nonsense variant, c.757C>T [p.(R253*)]; affected. M, mutant allele; WT, wild-type allele. Electropherograms: homozygosity (patient 1a, patient 1b) and heterozygous carriership (healthy sisters, parents). (B) Regions with runs of homozygosity identified by genome-wide linkage analysis. Arrow, chromosome 6 region comprising MAD2L1BP. (C) Scheme of major MAD2L1BP isoforms. (D) Schematic representation of mapped sequencing reads (forward strand) visualized with the Integrative Genomics Viewer. In the patients, the c.757C>T mutation was present in all respective reads. (E) High-grade aneuploidy of cultivated lymphocytes from peripheral blood in patient 1a and (F) patient 1b. (G and H) Representative examples of aberrant karyotypes for both patients, including a 65,XXY constellation (bottom, right). Red asterisks in the karyograms indicate supernumerary or missing chromosomes.

Figure 3. Clinical and genetic findings in patient 2.

(A) Pedigree. (B) Subtle dysmorphism: large ears, microcephaly. (C–H) cMRI. (C and D) Diffuse T2 hyperintensity of the white matter and polymicrogyria particularly in the frontal lobes. (E–H) Generalized brain atrophy, including the cerebellum, the brainstem, and the cervical spinal cord. Thin corpus callosum. (I) Results from karyotyping of nontransformed lymphocytes. (J) Localization of both homozygous nonsense variants, M1 from patients 1a and 1b and M2 from patient 2, in the MAD2L1BP gene and observed disease expression.

Figure 4. MAD2L1BP c.757C>T (R253*) of patients 1a and 1b results in partial NMD and C-terminally truncated p31^comet protein.

(A) Inhibition of NMD in patients’ fibroblasts: increased mRNA expression compared with control cells (ratios of transcript expression in anisomycin-treated/-untreated cells; effect of anisomycin verified by expression change of NMD-sensitive (ENST00000452355.7) compared with NMD-nonsensitive SRSF2 transcript (ENST00000392485.2). Bar plot and scatterplot of 2/3 independent experiments analyzed in duplicates by digital droplet PCR (ddPCR). (B) Western blot of fibroblasts showing decreased p31^comet protein expression in both patients compared with 2 control samples with p31^comet WT. The blot is representative for 3 experiments. Right panel: Intensities of p31^comet bands in relation to β-tubulin standard. Quantification was done on 3 Western blots and the lower bands of p31^comet-R253* were used. All values: means ± SE (error bars). (C) p31^comet immunofluorescence staining in control and patients’ fibroblasts. Blue, Hoechst staining of DNA; green, p31^comet. Scale bars, 10 μm. (D) p31^comet staining of testicular JGCT in patient 1b (above) and in a reference case (below). There is faint nuclear p31^comet expression in <5% of tumor cells in patient 1b, compared with approximately 50% in the reference tumor. Original magnification, 40× (in both images); scale bar: 50 μm.

Figure 5. Mutated p31^comet is sensitive to proteosomal degradation causing slowed proliferation and decreased sensitivity to chemotherapeutic drugs in patients’ fibroblasts.

(A) Analysis of p31^comet protein half-life using cycloheximide. Fibroblasts were treated with cycloheximide at indicated time points, and p31^comet was analyzed by immunoblotting (β-tubulin as internal control). The blots are representative of 3 experiments. (B) Densitometric analysis of the immunoblots. Intensities of the p31^comet bands in relation to β-tubulin standard. Expression levels were normalized to untreated cells. The dashed line indicates t_1/2. The blots are representative for 3 repeated experiments with consistent results. (C) Inhibition of proteasomal degradation by MG132 increases p31^comet levels. Fibroblasts were treated with 10 μM MG132 for 2 and 4 hours, and p31^comet protein accumulation was analyzed by Western blot (β-actin as internal control). The blots are representative for 3 experiments. (D) Growth curves of fibroblasts from 2 controls and both patients. Data were analyzed by a 2-tailed, unpaired Student’s t test at each time point. Error bars represent the SD (n = 4). (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.) Each experiment was repeated 3 times. (E) Dose response to paclitaxel. Cell viability of fibroblasts from controls and patients were treated for 72 hours with the indicated concentrations of paclitaxel normalized to DMSO vehicle. Error bars represent the SD (n = 4). Data were analyzed by 2-tailed, unpaired Student’s t test (****P < 0.0001; ***P < 0.001; **P < 0.01). Each experiment was repeated 3 times.

Figure 6. Comparison of p31^comet-WT and -ΔC in isogenic cell lines.

(A) C-terminally truncated p31^comet cannot associate with Trip13 but retains weak Mad2 binding. Transiently transfected HEK293T cells expressing the indicated FLAG-tagged p31^comet variants were Taxol arrested and then subjected to anti-FLAG immunoprecipitation, followed by Western analysis using the indicated antibodies. WT, wild-type (isoform 2, NP_055443.1); PK, Trip13 binding-deficient p31-P228A,K229A; ΔC, amino acids 253–274 deleted. (B) Replacing endogenous p31^comet by p31^comet-ΔC greatly delays exit from G₂/M. HeLaK cells transfected to replace endogenous p31^comet by the indicated FLAG₃-Tev₂–tagged variants were synchronously released from an RO-336–mediated G₂ arrest and analyzed by time-resolved immunoblotting (Supplemental Figure 5B) and flow cytometry. (C) p31^comet-ΔC is unable to support Trip13-dependent disassembly of Mad2-containing complexes. HeLaK cells containing the indicated siRNAs were transfected to express FLAG₃-Tev₂–tagged p31^comet-WT or -ΔC or left untreated. Following their release from a G₂ arrest, cells were subjected to time-resolved immunoblotting either directly (upper panels, “input”) or following Mad2-IP (lower panels). Note that Trip13 association with Mad2 is delayed but still occurs in the absence of p31^comet; this association is reduced in the presence of p31^comet-ΔC. (D) p31^comet-ΔC is compromised in Rev7 and tp53 binding. HeLaK cells transiently transfected to express Myc₆-Rev7 and FLAG₃-Tev₂–tagged p31^comet-WT or -ΔC were treated for 2 hours with doxorubicin (DRB) or carrier solvent (-) and then subjected to IP-Western analyses using the indicated antibodies. Note that the interaction among p31^comet, Rev7, and tp53 is strongly induced by infliction of DSBs. Note also that Rev7 interacts with Mad2 in undamaged cells, which is consistent with a previous report (65). γH2AX, S139-phosphorylated histone H2AX (marker for DSBs).