Received: 28 November 2018 | Revised: 4 November 2019 | Accepted: 6 January 2020 DOI: 10.1002/mgg3.1133 CLINICAL REPORT Gene expression profile suggesting immunological dysregulation in two Brazilian Bloom's syndrome cases Marilia M. Montenegro1,2 | Caio R. Quaio2 | Patricia Palmeira3 | Yanca Gasparini1 | Andreia Rangel-Santos3 | Julian Damasceno1 | Estela M. Novak4 | Thamires M. Gimenez5 | Guilherme L. Yamamoto2 | Rachel S. Ronjo2 | Gil M. Novo-Filho1,2 | Samar N. Chehimi1 | Evelin A. Zanardo1 | Alexandre T. Dias1 | Amom M. Nascimento1 | Thais V. M. M. Costa1 | Alberto J. da S. Duarte1 | Luiz L. Coutinho6 | Chong A. Kim2 | Leslie D. Kulikowski1,2 1Laboratorio de Citogenomica, Departamento de Patologia, Faculdade de Abstract Medicina FMUSP, Universidade de Sao Background: Bloom syndrome (BS) is a rare autosomal recessive chromosome insta- Paulo, Sao Paulo, SP, Brazil bility disorder. The main clinical manifestations are growth deficiency, telangiectasic 2 Unidade de Genetica, Departamento de facial erythema, immunodeficiency, and increased risk to develop neoplasias at early Pediatria, Instituto da Crianca, Hospital das Clinicas HCFMUSP, Faculdade de age. Cytogenetic test for sister chromatid exchanges (SCEs) is used as a diagnostic Medicina, Universidade de Sao Paulo, Sao marker for BS. In addition, most patients also present mutations in the BLM gene, Paulo, SP, Brazil related to defects in the DNA repair mechanism. However, the molecular mechanism 3Laboratório de Pediatria Clínica, Departamento de Pediatria, Instituto da behind the pathogenicity of BS is still not completely understood. Crianca, Hospital das Clinicas HCFMUSP, Methods: We describe two patients confirmed with BS by SCE and molecular analy- Faculdade de Medicina, Universidade de sis. Also, we performed the gene expression profile by the RNA-seq methodology Sao Paulo, Sao Paulo, SP, Brazil in mRNA transcripts for differential gene expression analysis using as a biological 4Fundação Pró-Sangue, Hemocentro de São Paulo, Sao Paulo, SP, Brazil condition for comparison BS versus health controls. 5Laboratório de Pesquisa Translacional em Results: We detected 216 differentially expressed genes related to immunologi- Oncohematologia, Instituto de Tratamento cal pathways such as positive regulation and activation of B cells, immune effec- de Cancer Infantil (ITACI), Sao Paulo, SP, Brazil tor process and absence of difference of DNA repair genes expression. In addition; 6Centro de Genomica Funcional, we also observed differentially expressed genes associated with apoptosis control, Departamento de Zootecnia, Universidade such as BCL2L1, CASP7, CDKN1A, E2F2, ITPR, CD274, TNFAIP6, TNFRSF25, de São Paulo, Escola Superior de TNFRSF13C, and TNFRSF17. Agricultura Luiz de Queiroz, ESALQ-USP, Piracicaba, Brazil Conclusion: Our results suggest that the combination of altered expression of genes involved in signaling pathways of immune response and apoptosis control may Correspondence contribute directly to the main characteristics observed in BS, such as recurrent Marília Moreira Montenegro, Laboratorio de Citogenomica, Departamento de infections, growth failure, and high risk of cancer. Transcriptome studies of other Patologia, Faculdade de Medicina FMUSP, instability syndromes could allow a more accurate analysis of the relevant gene inter- Universidade de Sao Paulo, Sao Paulo, SP, BR. actions associated with the destabilization of the genome. This is a first description of Email: [email protected] This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. Mol Genet Genomic Med. 2020;8:e1133. wileyonlinelibrary.com/journal/mgg3 | 1 of 10 https://doi.org/10.1002/mgg3.1133 2 of 10 | MONTENEGRO ET AL. Funding information This work was supported by CAPES the profile of differential gene expression related to immunological aspects detected (Coordenação de Aperfeiçoamento de in patients with BS by RNA-seq. Pessoal de Nível Superior) FINEP/ CT-INFRA-PROINFRA 01/2011 and KEYWORDS Fundação de Amparo a Pesquisa do BLM gene, Transcriptome, Bloom's syndrome, Immunology, RNA-Seq Estado de São Paulo (FAPESP process 2017/08981-1) 1 | INTRODUCTION on RNA-Seq, which allows transcriptome studies, analysis of previously unidentified genes and splice variants, genomic Bloom's syndrome (BS; Online Mendelian Inheritance in variations, and a comprehensive expression profile. Indeed, Man database #21900) is a rare autosomal recessive chromo- the characterization of gene expression in cells via measure- somal instability disorder characterized by severe growth de- ment of mRNA levels is a useful tool in determining how the ficiency (pre- and postnatal), sun-sensitive facial erythema, transcriptional machinery of the cell is affected by external immunological deficiency, and a remarkably increased risk signals, genetic diseases or how cells differ between a healthy of developing neoplasias of various types at a younger age state and a disease state (Huang, Sherman, & Lempicki, than expected in the general population. The neoplasias are 2009). The definition of an accurate map of all genes, with the main cause of death among affected individuals (Bloom, its alternative isoforms, could be critical for biological under- 1954; Chakraverty & Hickson, 1999; Brosh et al., 2001; standing of the disease. In this sense, we used RNA-Seq to Beamish et al., 2002; Bhisitkul & Rizen, 2004; Flanagan and genomic instability in two patients affected by a BS. Cunniff, 2006; Moreira et al., 2013). The cells from patients display genomic instability char- acterized by increased frequency of chromosomal and chro- 2 | PATIENTS AND METHODS matid gaps, breaks, and several chromosomal aberrations, especially quadriradial configurations and increased sister 2.1 | Patients chromatid exchanges (SCEs) which constitute the hall- mark of BS (Cunniff, Bassetti, & Ellis, 2017; Ellis, 1995). We compared gene expression patterns using an assay Chromosomal alterations observed in BS cells may generate based on RNA-Seq of mRNA transcriptome between repositioning of tumor suppressor genes that are important two groups: BS patients versus healthy controls. BS pa- for regulation of development and function of T and B cells, tients group included two patients with definite diagnosis thus predisposing to immunodeficiency and cancer devel- of BS previously confirmed by cytogenetic and molecu- opment (Derks, Hoeijmakers, & Pothof, 2014; Ellis et al., lar study of BLM gene, while healthy controls comprised 1998). Indeed, a recent immunological study of BS patients three individuals (two males and one female) from general demonstrated clinically relevant increased number of infec- population. A summary of main findings of all samples is tions, relatively low serum immunoglobulin, normal range described in Table 1. of T, B, and NK cells and high percentage of CD4+ and The study was approved by ethics committee of the CD8+ effector memory T cells (Schoenaker et al., 2018). University of São Paulo HCFMUSP-CAPPesq 63954/12 and BS is associated with biallelic pathogenic mutations in all patients or their legal guardians signed a consent form. the BLM gene, located in 15q26.1 region. In normal conditions, Patient K1 is a 28-year-old Brazilian woman, fifth child of BLM encodes a 1,417 amino acids protein. It is member of the consanguineous parents of non-Jewish family that was born at RecQ family with DNA helicase activity called BLM helicase 35 weeks of gestation with low birth weight (1800 g) and small or RECQL3 (Elmore, 2007; German, 1969a, 1993). BLM he- licase is a nucleolar protein essential for the integrity and sta- bility of DNA (German, 1997; German & Passarge, 1989). It TABLE 1 Description of samples that compounded this work is considerate anti-recombinant key, because it is a DNA repair Samples DH Gender Age (years) protein able to resolve DNA structures, like a Holliday junction, K1 BS F 28 D-loops, and G-quadruplex generated during HRR caused by a M1 BS F 13 DSB (Ellis et al., 1998; Hickson et al., 2001). Cytogenetic study, previously considered the gold stan- C1 H M 29 dard diagnostic tool for BS (Ellis et al., 1998), has been C2 H F 29 substituted for more accurate molecular analysis of BML C3 H M 31 gene. Recent technological advances with next-generation Abbreviations BS, Bloom´s syndrome; C, Controls; DH, Diagnostic hypothesis; sequencing made possible the rise of methodologies based F: Female; H, Healthy; M: Male MONTENEGRO ET AL. | 3 of 10 length (40 cm) after intrauterine growth retardation. She had alterations. Cytogenetic analysis of peripheral blood revealed normal cognitive development, remarkable failure to thrive and increased frequency of SCE (61,3 SCE per metaphase) and a recurrent infections (otitis, diarrhea, respiratory tract). Physical very rare quadriarradial chromosome configuration (Figure 3). examination revealed microcephaly, facial dimorphisms (prom- Molecular analysis showed two missense heterozygous vari- inent nose, prominent ears, and malar hypoplasia), disseminated ants already described in dbSNP: c.3164G>C:p.Cys1055Ser hyper and hypochromic macules, and decreased anthropomet- and c.3625T>A:p.Ser1209Thr (Figure 4a,b), respectively). ric measurements. Her immunological evaluation (Data S1: The first variant was described as pathogenic, according to the Supplementary