SNP array genotyping with the GeneChip Mapping 10K array and analysis with IBDfinder identified three areas of homozygosity common to 5 affected patients, two on chromosome 8 totaling 17.5 Mb (8p12–8q11.22 and 8q12.2–q12.3) and one of 4 Mb on chromosome 4q22.3–23 (Figure B). Genotyping of microsatellite markers confirmed homozygosity in the pericentric region on chromosome 8 in affected family members (Figure B). Exon capture and high-throughput sequencing of all three regions was carried out in one patient, identifying 680 variants from the 2009 human reference sequence (GRCh37/hg19). We reduced the number of variants to 3 by evaluating non-synonymous coding variants, splice variants, and indels and by excluding variants that were heterozygous or annotated in SNP databases (Ensembl SNP database, release 54). These three variants were then sequenced in other affected patients from the Irish Traveler community, their parents, and unaffected controls. Only one variant, c.71-1insG in minichromosome maintenance–deficient 4 (MCM4
), was homozygous in all 8 affected patients and segregated with the disease. (Figure , A and E). The two other variants in ankyrin 1 (ANK1
) and ADAM metallopeptidase domain 5, pseudogene (ADAM5P
) were subsequently recognized as SNPs (rs117614251 and rs73612404, respectively). In kindreds A and C, parents were heterozygous for the MCM4
variant, and all siblings tested had either heterozygous or homozygous wild-type sequences (parental and sibling samples were not available for kindred B). This variant (maximum lod score of 8.4) was not present in SNP databases and was not seen in 1000 Genomes (4
), the NHLBI Exome Sequencing Project (>10,700 alleles sequenced), or on screening of 300 control chromosomes of individuals of European descent.
This variant, a splice site mutation, altered the consensus sequence and would be predicted to shift the splice acceptor site upstream by one nucleotide and lead to a frameshift and a foreshortened open reading frame encoding a prematurely terminated translation product (p.Pro24ArgfsX4). The splice site shift was confirmed by performing RT-PCR on total RNA from whole blood isolated from two patients, a heterozygous relative, and an unaffected control (Figure , D and E).
Cell lysates from peripheral blood lymphocytes from unaffected human controls were immunoblotted for MCM4. This identified two major proteins migrating at approximately 96 kDa and 85 kDa. Patient samples showed only one major MCM4 species at 85 kDa (Figure A), suggesting an alternative transcription or translation start site may be present to explain the 85-kDa form. To our knowledge, no alternative splice variant has been reported that would explain this smaller MCM4 species.
Cell lysates from both control and patient lymphocytes show evidence of MCM4 protein.
cDNA encoding full-length MCM4 with a C-terminal HA tag was generated by PCR and cloned into an expression vector, and the initiating methionine was mutated using site-directed mutagenesis (M1X). Wild-type and/or M1X constructs were transiently expressed in HEK293 cells, and cell lysates were immunoblotted with HA and MCM4 antibody. M1X expression resulted in a smaller MCM4 species of approximately 85 kDa that corresponded in size to that seen in patient samples (Figure B).
cDNAs encoding proteins beginning at the first, second, and third in-frame ATGs (Figure C) were generated by PCR, then cloned, transfected into HEK293 cells, and immunoblotted. The first and third in-frame ATGs produced proteins corresponding in size to those seen in vivo, suggesting that the faster-migrating protein observed in patients may result from internal initiation from the third in-frame ATG (Figure C). This protein was also seen in a number of human cell lines (Figure D) but was not detected by immunoblotting with an N-terminal antibody for MCM4, suggesting this smaller MCM4 protein lacks the N terminus (Figure D). In patient samples and human cell lysates, the smaller band appeared to be a doublet. This may be due to phosphorylation, as there is evidence for multiple phosphorylation sites on MCM4 (4
Adrenal glands from affected patients were not available for study. In mice, homozygosity for a disrupted Mcm4
) caused pre-implantation lethality. A hypomorphic allele of Mcm4
, chromosome aberrations occurring spontaneously (Chaos3
), leads to genomic instability and cancer (6
), but Mcm4Chaos3/–
embryos died late in gestation. However, the near 100% lethality of Mcm4Chaos3/–
is rescued by Mcm3
), and these animals provide the closest viable animal model to our patient cohort, in that MCM4 levels were reduced to the lowest levels compatible with life. Adrenals from 3.5-month-old Mcm4Chaos3/–Mcm3+/–
mice were of normal size, but H&E staining revealed an abnormal morphology characterized by small, tightly packed, intensely stained spindle-shaped cells in the cortex just beneath the capsule that appeared to be migrating into the cortex (Figure , E and F). By 12 months of age, these cells were abundant and in some areas spanned the cortex from the capsule to the medulla (Figure , G and H). Such changes were not observed in wild-type littermate controls (Figure B), in Mcm4Chaos3/+Mcm3+/–
adrenals (Figure , C and D), or in other organs of the Mcm4Chaos3/–Mcm3+/–
). The adrenal capsule adjacent to these abnormal cells appeared to be much thinner, which is particularly obvious in the higher-power images (compare Figure , I and J). Analysis of the expression of steroidogenic enzymes indicated many cells in the mutants were negative for CYP11A1 (P450 side chain cleavage [SCC]) (Figure L), in contrast to the wild-type (Figure K). Co-staining with the zona fasciculata marker CYP11B1 (P450 11β-hydroxylase) showed that these CYP11A1-negative cells were also CYP11B1 negative (Figure P), implying they were not capable of producing glucocorticoid. These cells were also GATA-4 positive (Figure R); GATA-4 is a transcription factor that is not usually expressed in the adult adrenal (Figure Q and ref. 8
). The juxtaposition of the thinning capsule and the non-steroidogenic cells in the mutant adrenals suggested that these cells might be capsular in origin. Expression of the mesenchymal capsule marker Gli1 was analyzed by in situ hybridization, which indicated that the abnormal cells were Gli1 positive (Figure U). Taken together, these data indicate that the adrenal morphology in this MCM4 depletion mouse model is grossly abnormal, with steroidogenic cells displaced by non-steroidogenic, GATA-4– and Gli1-positive cells that are likely to compromise steroidogenic output.
Analysis of adrenals from MCM4 mutant mouse models.