MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans

Abstract

An interesting variant of familial glucocorticoid deficiency (FGD), an autosomal recessive form of adrenal failure, exists in a genetically isolated Irish population. In addition to hypocortisolemia, affected children show signs of growth failure, increased chromosomal breakage, and NK cell deficiency. Targeted exome sequencing in 8 patients identified a variant (c.71-1insG) in minichromosome maintenance-deficient 4 (MCM4) that was predicted to result in a severely truncated protein (p.Pro24ArgfsX4). Western blotting of patient samples revealed that the major 96-kDa isoform present in unaffected human controls was absent, while the presence of the minor 85-kDa isoform was preserved. Interestingly, histological studies with Mcm4-depleted mice showed grossly abnormal adrenal morphology that was characterized by non-steroidogenic GATA4- and Gli1-positive cells within the steroidogenic cortex, which reduced the number of steroidogenic cells in the zona fasciculata of the adrenal cortex. Since MCM4 is one part of a MCM2-7 complex recently confirmed as the replicative helicase essential for normal DNA replication and genome stability in all eukaryotes, it is possible that our patients may have an increased risk of neoplastic change. In summary, we have identified what we believe to be the first human mutation in MCM4 and have shown that it is associated with adrenal insufficiency, short stature, and NK cell deficiency.

Figure 1. Pedigree and linkage analysis of 3 affected kindreds and identification of a mutation in MCM4 leading to a truncated protein p.Pro24ArgfsX4.

(A) Pedigree of affected patients. (B) SNP and microsatellite genotyping of chromosome 8p12.2–q12.3 locus surrounding MCM4. Genotyping was not carried out on patients 1, 2, and 8. (C) Gene structure of MCM4 (top). The location of the mutation, c.71-1insG, prior to exon 2 (red arrow), which leads to a shift in the consensus splice site (nucleotides ag in red) and the introduction of an extra G into patient mRNA/cDNA (shown below), is indicated. (D) Protein structure of MCM4 indicating (top) the positions of the two cyclin-dependent kinase (CDK) binding domains, zinc finger, Walker A and B motifs, and an arginine ring finger; these motifs are conserved across all MCMs and are essential for MCM4 functionality. Also indicated are 4 in-frame methionines (M). The predicted consequence of the c.71-1insG mutation leading to premature truncation of the protein at amino acid 27 (p.Pro24ArgfsX4) is shown (bottom). (E) Partial sequence chromatograms showing (left) the intron 2–exon 2 junction in genomic DNA from control, parent, and patient (intronic bases in lowercase, exonic in uppercase). Arrows indicate the base change from A to G and (right) cDNA from control, parent, and patient indicating wild-type, mixed, and mutant cDNA sequences, respectively, the latter resulting in a premature stop codon.

Figure 2. Cell lysates from both control and patient lymphocytes show evidence of MCM4 protein.

(A) Lysates from patient and control human lymphocytes were immunoblotted with an anti- MCM4 antibody. While control subjects have two main protein species, of approximately 96 and 85 kDa, the predicted full-length 96-kDa product is missing in the patients. (B) HEK293 cells were transfected with full-length MCM4 or MCM4 with the first methionine mutated, both of which were tagged with a C-terminal HA tag. The lysates were then blotted with both MCM4 antibody (left panel) and HA antibody (right panel). Abolition of the initiating methionine of MCM4 leads to expression of a smaller MCM4 species, of approximately 85 kDa. (C) FLAG-tagged full-length MCM4 and constructs beginning at the second and third in-frame ATGs were expressed in HEK293 cells. Lysates from each were run individually (lanes 1–3), all together (lane 4), or in combination with the full-length and third ATG (lane 5) and immunoblotted using an anti-FLAG antibody. The relative mobility of the different species indicates that the smaller MCM4 species may be produced by translation from the third in-frame ATG. (D) Different cell lysates show evidence of both the major full-length and smaller MCM4 isoforms when immunoblotted with the MCM4 C-terminal antibody. However, when the same lysates are immunoblotted with the N-terminal antibody, the smaller MCM4 isoform is no longer seen, suggesting that the smaller isoform is missing the N terminus of MCM4.

Figure 3. Analysis of adrenals from MCM4 mutant mouse models.

(A–J) Adrenal cortices of Mcm4^Chaos3/–Mcm3^+/– mice have an abnormal morphology. H&E staining of wild-type (A, B, and I), Mcm4^Chaos3/+Mcm3^+/– (C and D), and Mcm4^Chaos3/–Mcm3^+/– adrenals at 3.5 months (E and F) and Mcm4^Chaos3/–Mcm3^+/– at 12 months (G, H, and J). Atypical spindle-shaped cells are observed within the cortex in the Mcm4^Chaos3/–Mcm3^+/– adrenals 3.5 months (arrows in F), with a more severe phenotype at 12 months (H). The normal capsule architecture in the wild-type (I) is lost adjacent to the abnormal cells (J). (K–V) Atypical cells in the cortex are not steroidogenic. 3.5-month-old wild-type (K, Q, and S) and Mcm4^Chaos3/–Mcm3^+/– (L, R, and U) adrenals were stained for P450 SCC (K, L, M, and N), CYP11B1 (B1) (O), double SCC-B1 (P), and GATA-4 (Q and R). The atypical cells populating the zona fasciculata in Mcm4^Chaos3/–Mcm3^+/– adrenals are negative for both SCC and CYP11B1 (arrows in N and P). GATA-4 immunoreactivity was rarely observed in the wild-type adrenals and always in cells close to the capsule (arrows in Q). In Mcm4^Chaos3/–Mcm3^+/– samples, the atypical cells are GATA-4 positive (arrows in R). In situ hybridization on 12-month-old adrenals showed that Gli1 expression is restricted to the capsule in the wild-type (S) but detected in the cortex in the atypical cells in Mcm4^Chaos3/–Mcm3^+/– mice (U). T and V are sense probe controls. Cap, capsule; Med, medulla; ZF, zona fasciculata; ZG, zona glomerulosa. Scale bars: 100 μm (A–H, K–V), 40 μm (I and J).