Discussion
Here, we report that biallelic mutations in TOP3A cause prenatal growth restriction, microcephaly, and short stature, a clinical phenotype overlapping that seen in Bloom syndrome. Furthermore, we have identified a rare RMI1 truncating variant (allele frequency = 1.1 × 10−5) in two individuals. Given that no homozygous loss-of-function variants are reported in gnomAD, we expect this to be the cause of microcephalic dwarfism in individuals P11 and P12. However, it will be necessary to identify more individuals with other deleterious sequence variants to establish RMI1 as a disease-related gene.
Notably, individuals with TOP3A mutations exhibit elevated amounts of SCEs on cytogenetic testing. Although cell lines were not available from P11 and P12, we also predict that increased SCEs will occur in cells with the homozygous RMI1 p.Lys419Leufs∗5 variant given the important role of this protein in the BTRR complex.9, 10, 37 For many years, the diagnosis of Bloom syndrome rested on a demonstration of elevated SCEs, and this has continued to be employed in cases where molecular testing is inconclusive (see GeneReviews in Web Resources). Hence, analysis of other BTRR components is now warranted when elevated SCEs are detected. Indeed, given the clear phenotypic overlap between Bloom syndrome and individuals with TOP3A mutations, re-evaluation of individuals previously cytogenetically diagnosed with Bloom syndrome could be fruitful.
RMI2 is the fourth component of the BTRR complex. A single family in which two children were also found to have elevated SCEs was recently reported to have a homozygous deletion of RMI2; café-au lait macules were evident, but only one individual showed mild-growth impairment.38 Because RMI2 is not essential for BTR (BLM, TopIIIα, and RMI1) enzymatic function,12 a more subtle cellular and developmental phenotype might therefore result. It also remains possible that the BTR complex has functions independent of RMI2 given that BLM, TopIIIα, and RMI1 are conserved in all eukaryotes but RMI2 is absent in invertebrates and yeast.13
Bloom syndrome is associated with a predisposition to both solid tumors and hematological malignancies.3 However, cancers have not been reported in our individuals with TOP3A mutations. Nevertheless, all are still children, and because malignancy in Bloom syndrome typically manifests in early adulthood, TOP3A mutations could also confer an increased risk of neoplasia.
Distinct from Bloom syndrome, some of our individuals with TOP3A mutations have clinical features consistent with mitochondrial dysfunction, and several exhibit a dilated cardiomyopathy. Notably, an individual with chronic progressive external ophthalmoplegia and cerebellar ataxia, along with a novel functional role for TopIIIα in the decatenation of mitochondrial DNA after its replication, has been recently reported32 (denoted as subject MC1). The compound-heterozygous mutations c.[298A>G];[403C>T], p.[Met100Val];[Arg135∗] could have milder consequences than those reported in our cohort, and consistent with this, we found that MC1 does not exhibit short stature (Table 2) or elevated numbers of SCEs in a primary fibroblast line derived from this individual (Figure S2). Thus, it appears that the residual TopIIIα activity in MC1 is sufficient for the nuclear function of the BTRR complex in dHJ dissolution and for normal growth but could have become rate limiting for the role of TopIIIα in mitochondrial DNA replication.
Mechanistically, our cellular and biochemical studies suggest that the identified TOP3A mutations reported here are severe hypomorphs, consistent with the established essential cellular and developmental functions of TopIIIα.34 Given that marked growth restriction is common in individuals with TOP3A and BLM mutations, it seems likely that they have a shared pathogenic basis arising from reduced ssDNA decatenation activity of the BTRR complex. Persistence of unresolved hemicatenanes into mitosis leads to the formation of chromatin and ultrafine mitotic bridges that have previously been reported in Bloom syndrome19 and in individuals with microcephalic dwarfism and condensin mutations.39 The resulting genome instability arising from persistent chromatin bridges40, 41 and micronuclei42 could therefore impair cell viability during development, causing microcephaly and global growth restriction. In summary, we demonstrate that mutations in TOP3A cause microcephalic dwarfism with increased SCE, implicating the BTRR complex as a cause of growth disorders.