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. 2018 Aug 2;103(2):221-231.
doi: 10.1016/j.ajhg.2018.07.001. Epub 2018 Jul 26.

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder

Free PMC article

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder

Carol-Anne Martin et al. Am J Hum Genet. .
Free PMC article

Erratum in

  • Mutations in TOP3A Cause a Bloom Syndrome-like Disorder.
    Martin CA, Sarlós K, Logan CV, Thakur RS, Parry DA, Bizard AH, Leitch A, Cleal L, Ali NS, Al-Owain MA, Allen W, Altmüller J, Aza-Carmona M, Barakat BAY, Barraza-García J, Begtrup A, Bogliolo M, Cho MT, Cruz-Rojo J, Dhahrabi HAM, Elcioglu NH; GOSgene, Gorman GS, Jobling R, Kesterton I, Kishita Y, Kohda M, Le Quesne Stabej P, Malallah AJ, Nürnberg P, Ohtake A, Okazaki Y, Pujol R, Ramirez MJ, Revah-Politi A, Shimura M, Stevens P, Taylor RW, Turner L, Williams H, Wilson C, Yigit G, Zahavich L, Alkuraya FS, Surralles J, Iglesias A, Murayama K, Wollnik B, Dattani M, Heath KE, Hickson ID, Jackson AP. Martin CA, et al. Am J Hum Genet. 2018 Sep 6;103(3):456. doi: 10.1016/j.ajhg.2018.08.012. Am J Hum Genet. 2018. PMID: 30193137 Free PMC article. No abstract available.


Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects' cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.

Keywords: BLM; Bloom syndrome; RecQ helicases; double Holliday junction dissolution; genomic instability; topoisomerase III.


Figure 1
Figure 1
Variants in TOP3A Are Associated with Prenatal-Onset Growth Retardation and Microcephaly (A) Morphometric data for TOP3A individuals. A global reduction in growth is evident from before birth. Z scores (standard deviations from population mean for age and sex) for birth weight (Wgt), postnatal weight, height (Hgt), and occipital-frontal circumference (OFC). Dashed lines indicate the 95% confidence interval for the general population. Black circles indicate data points for individuals with TOP3A mutations. For comparison, gray bars indicate the mean value for cohorts of 89 (birth weight), 47 (current weight), and 52 (current height) subjects with Bloom syndrome. (B) Photographs of facial features of individuals with TOP3A mutations.
Figure 2
Figure 2
Mutations in TOP3A Markedly Reduce Amounts of TopIIIα but Do Not Affect Its Intrinsic Decatenation Activity (A) Schematic of TopIIIα with locations of variants annotated. Colors are as follows: red, TOPRIM domain; purple, TOPA domain; and blue, GRF zinc-finger domain. (B) TOP3A mutations markedly reduce protein amounts. An immunoblot of a polyacrylamide gel is shown and was generated from total cell lysates from dermal fibroblast lines derived from affected individuals probed with an anti-TopIIIα antibody. siRNA depletion of TOP3A in a fibroblast line from control 2 was used to confirm the specificity of the antibody. C1 and C2 are unrelated control fibroblast lines. TopIIIα antibody was raised against amino acids 652–1,001. (C) Recombinant TopIIIαThr812LeufsTer101 (denoted TopIIIαP1) is less stable than TopIIIαWT. An immunoblot of a polyacrylamide gel is shown and was generated from purified TopIIIαWT and TopIIIαP1 proteins probed with an anti-TopIIIα antibody. Protein size markers are 250, 170, 140, 100, and 70 kDa. (D) Schematic depicting the BTRR complex and its role in dHJ dissolution. Homologous sister chromatids are shown in red and blue. (E and F) TopIIIαP1 is proficient for promoting dHJ dissolution with BLM in combination with RMI1 and RMI2. (E) Representative polyacrylamide gel demonstrating the enzyme-concentration dependence of the dHJ dissolution activity of TopIIIαWT and mutant (TopIIIαP1) TRR complexes with 20 nM BLM at 37°C in 30 min. TRR was included in reactions at increasing concentrations ranging from 20 pM to 10 nM. A labeled circular oligonucleotide (lane 1) and dHJ (lane 2) were included as markers. (F) Quantification of the dHJ dissolution reaction shown in (E). The reaction was repeated twice more with very similar results.
Figure 3
Figure 3
Individuals with TOP3A Mutations Have More SCEs, Reflecting Chromatid Hyper-recombination (A) Schematic of dHJ processing. dHJs are either dissolved by the BTRR complex or alternatively cleaved by structure-specific nucleases that lead to dHJ resolution. (B and C) BrdU strand-specific labeling of sister chromatids shows that TOP3A cells have more SCEs than control and parent cells. (B) Representative images of P1 and parent fibroblast cell lines. (C) Quantification of SCEs in fibroblast cell lines (P1, P5, P7, and P8) or PHA-stimulated lymphocytes derived from peripheral-blood samples (P2 and P3). The median value was plotted with more than ten metaphase spreads counted per subject. Pairwise non-parametric Mann Whitney tests were performed against parental control cells. F1, F2, and F5 SCEs were scored in independent laboratories.
Figure 4
Figure 4
DNA Catenanes Persist into Mitosis in Cells with TOP3A Mutations, Leading to Chromosome Segregation Defects and Genome Instability (A and B) Chromosome segregation is impaired in TOP3A-deficient primary fibroblasts. (A) Representative images of chromatin bridges, lagging DNA (DAPI), and UFBs (detected by the presence of PICH and absence of DAPI stain). (B) Quantification of chromatin bridges, lagging DNA, and PICH-positive UFBs scored in control, parental, and P1 mitotic fibroblasts staged at anaphase B (experiments ≥ 3, n > 50 cells, error bars = SEM). To enrich for mitotic cells, we treated fibroblasts with R03306 for 6 hr and released and fixed them after 45 min. (C) TOP3A P1 fibroblasts display significantly elevated amounts of micronuclei. Top: representative picture of control and P1 fibroblasts. Bottom: quantification of micronucleus containing interphase cells (experiments ≥ 3, n > 500, error bars = SEM). To enrich for G1 cells, we released RO3306-treated cells into fresh media for 30 min and collected, re-seeded, and fixed prometaphase cells after 4–6 hr. (D) P1 fibroblasts with TOP3A mutations display significantly elevated numbers of 53BP1 bodies in G1 nuclei. Top: representative images of 53BP1 foci (red) and DNA (DAPI). Bottom: quantification of cells with at least four 53BP1 foci in G1 nuclei (negative for cyclin A) (experiments ≥ 3, n > 500, error bars = SEM). Scale bar: 2 microns. Two-tailed t test was performed against parent cells.

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