MCM complex members MCM3 and MCM7 are associated with a phenotypic spectrum from Meier-Gorlin syndrome to lipodystrophy and adrenal insufficiency

Abstract

The MCM2-7 helicase is a heterohexameric complex with essential roles as part of both the pre-replication and pre-initiation complexes in the early stages of DNA replication. Meier-Gorlin syndrome, a rare primordial dwarfism, is strongly associated with disruption to the pre-replication complex, including a single case described with variants in MCM5. Conversely, a biallelic pathogenic variant in MCM4 underlies immune deficiency with growth retardation, features also seen in individuals with pathogenic variants in other pre-initiation complex encoding genes such as GINS1, MCM10, and POLE. Through exome and chromium genome sequencing, supported by functional studies, we identify biallelic pathogenic variants in MCM7 and a strong candidate biallelic pathogenic variant in MCM3. We confirm variants in MCM7 are deleterious and through interfering with MCM complex formation, impact efficiency of S phase progression. The associated phenotypes are striking; one patient has typical Meier-Gorlin syndrome, whereas the second case has a multi-system disorder with neonatal progeroid appearance, lipodystrophy and adrenal insufficiency. We provide further insight into the developmental complexity of disrupted MCM function, highlighted by two patients with a similar variant profile in MCM7 but disparate clinical features. Our results build on other genetic findings linked to disruption of the pre-replication and pre-initiation complexes, and the replisome, and expand the complex clinical genetics landscape emerging due to disruption of DNA replication.

Fig. 1. Individuals with biallelic variants in MCM3 and MCM7.

A Schematic indicating amino acid positions of identified variants, numbering according to RefSeq NM_002388.4 (MCM3) and NM_005916.4 (MCM7). NTD, N-terminal domain; AAA + , AAA ATPase domain; WD, winged-helix domain; red bar, nuclear localization signal. B Clustal Omega alignment demonstrating strong conservation of substituted residues in MCM3 and MCM7. C Substitution of MCM3 Gln806 (yellow) with a Leu may impact upon binding of MCM3 (green) to the ORC-CDC6 complex, where interactions with ORC2 (red) and CDC6 (blue) would likely be impacted. Substitution of MCM7 Gly259 (yellow) with an Ala may distort the MCM7 protein core (blue), as the side-chain would clash with that of Glu135. Substitution of MCM7 Tyr539 (yellow) with a Cys may distort the alpha helix and surrounding structures of MCM7 (blue), thereby impacting MCM3 (green) and ATP binding. ASG (yellow) represents the likely binding mode of ATP. D Photographs of patients. P1 and P2 demonstrate classical features of Meier-Gorlin syndrome, with microtia, thick lower lip vermillion and a pointy jaw. P2 and P3 share similar features of narrow face, prominent nose with a high nasal bridge and prominent teeth. P3 displayed a progeroid appearance at age 4 months (top). Facial photographs at the age of 14 yrs are shown in the bottom panel.

Fig. 2. Consequences of MCM3 and MCM7 variants on transcript and protein.

Ai, A Quantification of MCM3 in cells from P1. (left) qRT-PCR of MCM3 from Control and P1 fibroblasts, quantified by 2^{(−Δ ΔCT)} method, normalized to RPS18. (Three primer sets located across the transcript were used, n = 3 independent experiments). (right) Representative blot and quantification of MCM3 in P1 vs. control fibroblasts, normalized to HDAC (student’s t-test, mean ± SD, n = 3 independent experiments). B Quantification of MCM7 in cells from P2 and P3. (left) qRT-PCR of MCM7 from Control, P2, and P3 fibroblasts, quantified by 2^{(−Δ ΔCT)} method, normalized to RPS18. (Three primer sets located across the transcript were used, n = 3 independent experiments). ****p < 0.0001, one-way ANOVA, treating each cell line as one group). (right) Representative blot and quantification of MCM7 in control fibroblasts compared to P2 and P3 fibroblasts, normalized to HDAC as a loading control (one-way ANOVA, P2 vs. control, **p = 0.0040; P3 vs. control, **p = 0.0028, mean ± SD, n = 2 independent experiments).

Fig. 3. Cellular consequences of variants in MCM3 and MCM7.

A A representative immunoblot resulting from a pull-down of the MCM2-7 helicase by MCM7-2×Strep-V5 (encoding either the canonical protein sequence or with patient substitutions) highlighting the effect of MCM7 patient variants. *MCM7 degradation. B Quantification of the MCM3-6 helicase complex pulled-down, and C quantification of a single subunit, MCM5, for each of the pull-downs from A. MCM5 was used as a proxy for the MCM3-MCM7 interaction. Pull-downs involved co-transfection of HEK293 cells with 3×FLAG-tagged MCM subunits for MCM2-6, and MCM7-2XStrep-V5 (encoding either the canonical protein sequence or with patient substitutions). Samples were immunoprecipitated using MCM7-2×Strep-V5. Band intensities were measured using Image Studio software. MCM bands were normalized to HDAC and then relative intensities to wildtype calculated. Differences were statistically analysed using a one way ANOVA with Dunnett’s multiple comparisons test (mean ± SEM). MCM3-6, *p = 0.0158 (n = 3 independent experiments); MCM5, *p = 0.0191 (n = 2 independent experiments). D Representative flow cytometry plots in control, P2 and P-1 over a 6 h time course. E Quantification of control vs. P1 (left) and P2, P3 (right) cells in early S-phase, relative to t = 0 time point. (mean ± SEM, n = 6 independent experiments, **p = 0.0047, *p = 0.0122, two-way ANOVA).