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RecQ helicase expression in patients with telomeropathies

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João Paulo L. Silva
João Paulo L. Silva
João Paulo L. Silva
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Flávia S. Donaires
Flávia S. Donaires
Flávia S. Donaires
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Fernanda Gutierrez-Rodrigues at National Institutes of Health
Fernanda Gutierrez-Rodrigues
Davi J. Martins
Davi J. Martins
Davi J. Martins
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Abstract and Figures

Background Telomeropathies are a group of inherited disorders caused by germline pathogenic variants in genes involved in telomere maintenance, resulting in excessive telomere attrition that affects several tissues, including hematopoiesis. RecQ and RTEL1 helicases contribute to telomere maintenance by unwinding telomeric structures such as G-quadruplexes (G4), preventing replication defects. Germline RTEL1 variants also are etiologic in telomeropathies. Methods and results Here we investigated the expression of RecQ (RECQL1, BLM, WRN, RECQL4, and RECQL5) and RTEL1 helicase genes in peripheral blood mononuclear cells (PBMCs) from human telomeropathy patients. The mRNA expression levels of all RecQ helicases, but not RTEL1, were significantly downregulated in patients’ primary cells. Reduced RecQ expression was not attributable to cell proliferative exhaustion, as RecQ helicases were not attenuated in T cells exhausted in vitro. An additional fifteen genes involved in DNA damage repair and RecQ functional partners also were downregulated in the telomeropathy cells. Conclusion These findings indicate that the expression of RecQ helicases and functional partners involved in DNA repair is downregulated in PBMCs of telomeropathy patients.
RecQ helicases are downregulated in telomeropathies. Gene expression of RecQ helicases (RECQL1, BLM, WRN, RECQL4, and RECQL5) and RTEL1 in patients (n = 20) and healthy individuals (controls, n = 20) assessed by RT-qPCR. (A) Patients with gene expression levels similar to the controls in all the five RecQ helicases are represented as red dots; those patients with more pronounced downregulation of all five RecQ helicases are represented as blue dots; black dots represent patients with downregulation of some helicases while levels similar to the controls for other helicases. Data on graphs represent the mean and standard deviation. *P < 0.05 vs. ‘Controls’. (B) Venn diagrams with patients’ identifications; the intersections were defined based on the expression levels; patients with levels similar to the controls for all the five helicases are represented in red (left), and with pronounced downregulation are in blue (right)
RecQ helicases are downregulated in telomeropathies. Gene expression of RecQ helicases (RECQL1, BLM, WRN, RECQL4, and RECQL5) and RTEL1 in patients (n = 20) and healthy individuals (controls, n = 20) assessed by RT-qPCR. (A) Patients with gene expression levels similar to the controls in all the five RecQ helicases are represented as red dots; those patients with more pronounced downregulation of all five RecQ helicases are represented as blue dots; black dots represent patients with downregulation of some helicases while levels similar to the controls for other helicases. Data on graphs represent the mean and standard deviation. *P < 0.05 vs. ‘Controls’. (B) Venn diagrams with patients’ identifications; the intersections were defined based on the expression levels; patients with levels similar to the controls for all the five helicases are represented in red (left), and with pronounced downregulation are in blue (right)
… 
RecQ helicase downregulation in telomeropathy patients is not caused by cell proliferative exhaustion. Peripheral blood mononuclear cells from six healthy donors were collected (day 0, D0) and cultured with T cell activators for four days (D4) to provoke cell exhaustion, with or without thymidine (Thy) for cell cycle synchronization. (A) CD3⁺CCR7⁺CD45RA⁻CD8⁺ T cells were gated by flow cytometry to assess the exhaustion markers LAG3, TIM3, PD1, and CTL4. Bars represent mean and standard error of percentages of cells expressing the markers from three healthy donors. (B) Gene expression of RecQ helicases (RECQL1, BLM, WRN, RECQL4, and RECQL5) in heathy individuals assessed by RT-qPCR before (D0) and after (D4) cell exhaustion with or without Thy synchronization. Data on graphs represent the mean and standard deviation. Differences were not significant
RecQ helicase downregulation in telomeropathy patients is not caused by cell proliferative exhaustion. Peripheral blood mononuclear cells from six healthy donors were collected (day 0, D0) and cultured with T cell activators for four days (D4) to provoke cell exhaustion, with or without thymidine (Thy) for cell cycle synchronization. (A) CD3⁺CCR7⁺CD45RA⁻CD8⁺ T cells were gated by flow cytometry to assess the exhaustion markers LAG3, TIM3, PD1, and CTL4. Bars represent mean and standard error of percentages of cells expressing the markers from three healthy donors. (B) Gene expression of RecQ helicases (RECQL1, BLM, WRN, RECQL4, and RECQL5) in heathy individuals assessed by RT-qPCR before (D0) and after (D4) cell exhaustion with or without Thy synchronization. Data on graphs represent the mean and standard deviation. Differences were not significant
… 
DNA repair genes are modulated in telomeropathies. The expression levels of 84 genes that participate in DNA repair pathways were assessed in peripheral blood mononuclear cells from healthy individuals (n = 5) and patients (n = 7) by qPCR array. (A) Eleven genes were found significantly downregulated in the patients, with fold regulation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge$$\end{document}2 between patients’ and controls’ groups; the clustergram represents the 2-ΔCt of each of those eleven genes across the samples. (B) The protein-protein interactions were predicted for the eleven significantly downregulated genes and are represented in Network 1. Each gene-encoded protein is represented as a circle, and line types represent the level of evidence for prediction of each protein-protein interaction. Network 2 represents the interactions of the same downregulated proteins with the addition of the RecQ helicases (thicker red circle edges) to the network construction. MRE11A substantially interacts with all the five RecQ helicases
DNA repair genes are modulated in telomeropathies. The expression levels of 84 genes that participate in DNA repair pathways were assessed in peripheral blood mononuclear cells from healthy individuals (n = 5) and patients (n = 7) by qPCR array. (A) Eleven genes were found significantly downregulated in the patients, with fold regulation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge$$\end{document}2 between patients’ and controls’ groups; the clustergram represents the 2-ΔCt of each of those eleven genes across the samples. (B) The protein-protein interactions were predicted for the eleven significantly downregulated genes and are represented in Network 1. Each gene-encoded protein is represented as a circle, and line types represent the level of evidence for prediction of each protein-protein interaction. Network 2 represents the interactions of the same downregulated proteins with the addition of the RecQ helicases (thicker red circle edges) to the network construction. MRE11A substantially interacts with all the five RecQ helicases
… 
The accumulation of G-quadruplex structures is altered in telomeropathies. Sequential labelling of telomeres (by interphase nuclei quantitative FISH, IQ-FISH), then G-quadruplex (G4) structures (by immunofluorescence) was performed in dermal fibroblasts of two patients (T21 and T22) and one healthy individual (C21). (A) Representative images of nuclei stained with pan-telomeric (red) and centromere 2 (green) probes (left), and with BG4 antibody for G4 structures (green, right). Nuclear DNA was counterstained with DAPI (blue). (B) Telomere length measured by IQ-FISH; data on graphs represent the mean and standard deviation. (C) G4 intensity of each nucleus was normalized by the respective telomere length, then presented as mean and standard error; the number of nuclei analyzed was 66, 56, and 71, for C21, T21, and T22, respectively. (D) Nuclei were divided into quartiles based on their telomere lengths; then, G4 intensity of each nucleus was normalized by the respective telomere length; the mean and standard deviation of these normalizations are presented for 25% of the cells with the shortest telomeres (< Q1), and for 25% of the cells with the longest telomeres (> Q3). For B, C, and D, *P < 0.05 vs. ‘C21’
The accumulation of G-quadruplex structures is altered in telomeropathies. Sequential labelling of telomeres (by interphase nuclei quantitative FISH, IQ-FISH), then G-quadruplex (G4) structures (by immunofluorescence) was performed in dermal fibroblasts of two patients (T21 and T22) and one healthy individual (C21). (A) Representative images of nuclei stained with pan-telomeric (red) and centromere 2 (green) probes (left), and with BG4 antibody for G4 structures (green, right). Nuclear DNA was counterstained with DAPI (blue). (B) Telomere length measured by IQ-FISH; data on graphs represent the mean and standard deviation. (C) G4 intensity of each nucleus was normalized by the respective telomere length, then presented as mean and standard error; the number of nuclei analyzed was 66, 56, and 71, for C21, T21, and T22, respectively. (D) Nuclei were divided into quartiles based on their telomere lengths; then, G4 intensity of each nucleus was normalized by the respective telomere length; the mean and standard deviation of these normalizations are presented for 25% of the cells with the shortest telomeres (< Q1), and for 25% of the cells with the longest telomeres (> Q3). For B, C, and D, *P < 0.05 vs. ‘C21’
… 
Replication fork processing is preserved in telomeropathies. DNA replication fork progression analysis was performed in dermal fibroblasts of two patients (T21 and T22) and two healthy individual (C18 and C21). (A) Schematic representation of DNA fiber labelling experiments (top); cells were pulse-labelled with CldU for 30 min, irradiated with 20 J/m² ultraviolet C (UVC) light to induce replication fork stalling, then pulse-labelled with IdU for 60 min; the thymidine analogs CldU and IdU were detected after immunostaining. Below, representative images of fibers; white arrows indicate single forks containing tracks from the CldU (red) and IdU (green) pulses. (B) Frequency distribution of CldU/IdU tract length ratios from UVC-treated or untreated fibroblasts
Replication fork processing is preserved in telomeropathies. DNA replication fork progression analysis was performed in dermal fibroblasts of two patients (T21 and T22) and two healthy individual (C18 and C21). (A) Schematic representation of DNA fiber labelling experiments (top); cells were pulse-labelled with CldU for 30 min, irradiated with 20 J/m² ultraviolet C (UVC) light to induce replication fork stalling, then pulse-labelled with IdU for 60 min; the thymidine analogs CldU and IdU were detected after immunostaining. Below, representative images of fibers; white arrows indicate single forks containing tracks from the CldU (red) and IdU (green) pulses. (B) Frequency distribution of CldU/IdU tract length ratios from UVC-treated or untreated fibroblasts
… 
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ORIGINAL ARTICLE
Molecular Biology Reports (2024) 51:754
https://doi.org/10.1007/s11033-024-09678-0
that folds back to the double-strand telomere, creating a loop
(T-loop) that includes a three-strand DNA portion (D-loop).
This telomere structure prevents the chromosome natural
ends from being recognized as damaged DNA, pathogenic
double-stranded breaks, or infectious nucleotide material
by the DNA repair machinery [2]. However, the telomeric
sequences are eroded during mitosis due to the DNA poly-
merase’s inability to fully replicate the template strand [3].
Thus, the newly synthesized strand is shorter than the tem-
plate, shortening the telomeres with each cell division – the
“end-replication problem” [4]. When telomeres reach criti-
cally short lengths, they engage cell proliferation arrest or
apoptosis, which explains the nite proliferation capacity
of human cells in culture – the “Hayick limit” [5]. Cells
with high proliferative demand circumvent telomere erosion
by activating telomerase, a reverse transcriptase (TERT)
that uses an RNA template (TERC) to synthesize telomere
repeats [6].
Other proteins contribute to telomere maintenance and
stability. The RTEL1 helicase disrupts the D-loop forma-
tion during homologous recombination [7] and unwinds the
Introduction
Mammalian linear chromosome termini, called telo-
meres, are composed of hundreds to thousands of tandem
5’-TTAGGG-3’ nucleotide repeats coated by specialized
proteins, collectively termed shelterin [1, 2]. The very end
of telomeres culminates in a 3’ single-stranded overhang
Rodrigo T. Calado
rtcalado@usp.br
1 Department of Medical Imaging, Hematology, and Oncology,
Ribeirão Preto Medical School, University of São Paulo, Av.
Bandeirantes, 3900 – 7 o andar, sala 743 – HCRP, Ribeirão
Preto, SP 14049-900, Brazil
2 Hematology Branch, National Heart, Lung, and Blood
Institute, National Institutes of Health, Bethesda, MD, USA
3 Department of Microbiology, Institute of Biomedical
Sciences, University of São Paulo, São Paulo, Brazil
4 Department of Hematology, Aarhus University Hospital,
Aarhus, Denmark
Abstract
Background Telomeropathies are a group of inherited disorders caused by germline pathogenic variants in genes involved
in telomere maintenance, resulting in excessive telomere attrition that aects several tissues, including hematopoiesis. RecQ
and RTEL1 helicases contribute to telomere maintenance by unwinding telomeric structures such as G-quadruplexes (G4),
preventing replication defects. Germline RTEL1 variants also are etiologic in telomeropathies.
Methods and results Here we investigated the expression of RecQ (RECQL1, BLM, WRN, RECQL4, and RECQL5) and
RTEL1 helicase genes in peripheral blood mononuclear cells (PBMCs) from human telomeropathy patients. The mRNA
expression levels of all RecQ helicases, but not RTEL1, were signicantly downregulated in patients’ primary cells. Reduced
RecQ expression was not attributable to cell proliferative exhaustion, as RecQ helicases were not attenuated in T cells
exhausted in vitro. An additional fteen genes involved in DNA damage repair and RecQ functional partners also were
downregulated in the telomeropathy cells.
Conclusion These ndings indicate that the expression of RecQ helicases and functional partners involved in DNA repair is
downregulated in PBMCs of telomeropathy patients.
Keywords Telomere · Helicase · Telomerase · Telomeropathy
Received: 28 February 2024 / Accepted: 24 May 2024 / Published online: 14 June 2024
© The Author(s), under exclusive licence to Springer Nature B.V. 2024
RecQ helicase expression in patients with telomeropathies
João Paulo L.Silva1· Flávia S.Donaires1· FernandaGutierrez-Rodrigues2· Davi J.Martins3· Vinicius S.Carvalho1·
Barbara A.Santana1· Renato L. G.Cunha1· SachikoKajigaya2· Carlos F. M.Menck3· Neal S.Young2· EigilKjeldsen4·
Rodrigo T.Calado1
1 3
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