Primate genome gain and loss: a bone dysplasia, muscular dystrophy, and bone cancer syndrome resulting from mutated retroviral-derived MTAP transcripts

Abstract

Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal-dominant syndrome characterized by bone dysplasia, myopathy, and bone cancer. We previously mapped the DMS-MFH tumor-suppressing-gene locus to chromosomal region 9p21-22 but failed to identify mutations in known genes in this region. We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously uncharacterized terminal exons of the gene encoding methylthioadenosine phosphorylase, MTAP. Intriguingly, two of these MTAP exons arose from early and independent retroviral-integration events in primate genomes at least 40 million years ago, and since then, their genomic integration has gained a functional role. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine and methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Six distinct retroviral-sequence-containing MTAP isoforms, each of which can physically interact with archetype MTAP, have been identified. The disease-causing mutations occur within one of these retroviral-derived exons and result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Our results identify a gene involved in the development of bone sarcoma, provide evidence of the primate-specific evolution of certain parts of an existing gene, and demonstrate that mutations in parts of this gene can result in human disease despite its relatively recent origin.

Figure 1

Pedigrees of the Five DMS-MFH-Affected Families Families 1, 2, and 3 were originally described as the “American,” “Australian,” and “New York” families, respectively. Family 4 is a previously undescribed DMS-MFH-affected family from New York. Family 5 has been described as having autosomal-dominant bone fragility and limb-girdle myopathy. Family members classified as “suspected by history” were unavailable for radiological diagnosis but had a history of multiple (>2) pathological fractures and/or had children who were clinically diagnosed with DMS-MFH on the basis of plain-film X-rays and family history.

Figure 2

Identification of the Critical Region and the DMS-MFH-Associated Gene (A) The results of a combined multipoint parametric linkage analysis for families 2, 3, and 4. The maximal location score is 4.27 at marker D9SB3. (B) Haplotype analysis of informative individuals from families 2, 3, and 4 narrowed the DMS-MFH critical region (boxed). (C) Physical map of the DMS-MFH critical region, which spans 1.3 Mb between the flanking markers AL882 and D9S976. Arrows indicate transcriptional direction of candidate genes. The exon-intron structure of MTAP highlights the eight exons of MTAP (green) and the three terminal exons (blue). Maps are not drawn to scale. (D) Representative DNA-sequence chromatograms from selected affected and unaffected individuals from each family. (E) Tumor DNA sequence revealing homozygosity of the diseased allele and loss of the unaffected allele.

Figure 3

Histopathological Analysis of an Osteosarcoma from Patient F4 IV-2 (A) Histologic analysis revealed that 95% of the studied tumor specimen displayed the typical pattern of malignant spindle (fibroblastic) cells of bone MFH. (B) Malignant cells forming neoplastic bone. (C) Focal sheets of neoplastic bone within the tumor are shown to be entrapping pre-existing bone trabeculae, and the overtly malignant cells are shown to produce bone. All sections were stained with hematoxylin and eosin.

Figure 4

Patient-Derived MTAP Mutations Result in Exon Skipping (A) Sequence differences of the three minigene constructs, WT, c.813-2A>G, and c.885A>G, are highlighted. (B) Schematic representation of the major sequence-verified isoforms flanking the electrophoretic profile of each minigene construct. Arrowheads above exons depict the positions of translational stop codons. The WT construct expresses all alternative splice variants that are detectable with this combination of primers (exon 6 forward [Ex6F] and exon 11 reverse [Ex11R]) (left), whereas patient-derived mutant constructs, c.813-2A>G and c.885A>G, expressed relatively very low levels of exon-9S- and -9L-containing variants, v1/v4 and v2/v5, respectively. By comparison, the expression of isoforms v3 and v6 was markedly elevated in both mutant constructs. (C) qRT-PCR analysis of archetype MTAP and isoform expression in cells that express each of the minigene constructs. Expression analysis of the three minigene constructs demonstrated that the c.813-2A>G mutant construct resulted in significantly increased archetype MTAP expression levels, whereas both the c.813-2A>G and c.885A>G mutant constructs were associated with an absence of and/or significantly decreased levels of MTAP_v1, _v2, _v4, and _v5. Both mutant constructs resulted in significantly increased expression levels of MTAP_v3 and _v6. The following abbreviation is used: wtMTAP, archetype MTAP. The error bars represent the averages of three independent experiments.

Figure 5

Dysregulated MTAP Splicing Patterns in Patient-Derived Cell Lines MTAP expression patterns in patient-derived and control fibroblast (A) and lymphoblast (B) cell lines. Semiquantitative RT-PCR analysis is on the left, and qRT-PCR analysis of archetype MTAP is on the right. (A) C1, C2, C3, and C4 are controls. Fibroblast and tumor cell lines are from affected individual F4 III-3. (B) C1 and C2 are controls. Lymphoblast cell lines are from individuals F3 IV-5, F3 IV-6, F3 IV-7, F4 III-5, and F4 III-6. The following abbreviation is used: wtMTAP, archetype MTAP.

Figure 6

Molecular Modeling of MTAP Modeling is based on the coordinates of the trimeric enzyme complex including 5′-deoxy-5′-methylthioadenosine sulfate (PDB ID 1CG6) and the MTAP apoprotein (PDB ID 1CB0). Rendering was done with the program O v.13 (courtesy of Dr. Alwyn Jones, University of Uppsala, Uppsala, Sweden), and visualization was done with the PyMOL molecular graphics system v.1.3 (Delano Scientific). (A) Overview of the top of the MTAP trimer. The stick representation (arrow) indicates the MTA substrate. (B) View of an MTAP monomer. The insertion positions of exon 7 (salmon) and exon 6 (tan) are at opposite ends. (C) View of the bottom of the MTAP trimer. Subunits and the insertion positions of exon 7 (salmon) and exon 6 (tan; discontinuous electron density) are indicated. Displayed is the close proximity of one of these junctions: exon 7 in subunit 2 with exon 8 in subunit 3. Because of the three-fold symmetry, there is the possibility of three splice-variant insertion points in the trimer.

Figure 7

Evolution of MTAP Exon 9 (A) Schematic representation demonstrating that exons 9 and 10 arose from distinct families of retroviral integration events. (B) The PCR results of exon 9 in a primate genomic DNA panel are superimposed adjacent to a phylogenetic tree. The results demonstrate that exon 9 was integrated into the primate genome at some point in evolution between the divergence of the ring-tailed lemur and the common woolly monkey approximately 40 million years ago.