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Spontaneous remission in a Diamond‐Blackfan anaemia patient due to a revertant uniparental disomy ablating a de novo RPS19 mutation

2018; Wiley; Volume: 185; Issue: 5 Linguagem: Inglês

10.1111/bjh.15688

1365-2141

Emanuela Garelli, Paola Quarello, Elisa Giorgio, Adriana Carando, Elisa Menegatti, Cecilia Mancini, Eleonora Di Gregorio, Nicoletta Crescenzio, Orazio Palumbo, Massimo Carella, Paola Dimartino, Tommaso Pippucci, Irma Dianzani, Ugo Ramenghi, Alfredo Brusco,

Epigenetics and DNA Methylation

Diamond-Blackfan Anaemia (DBA; Mendelian Inheritance in Man reference 105650) is an inherited bone marrow (BM) failure syndrome that, in most cases, shows onset in the first year of life and is characterized by hyporegenerative anaemia, congenital malformations in about one-third of patients and elevated erythrocyte adenosine deaminase activity (eADA) (Da Costa et al, 2018). Anaemia is corrected with steroids in about 50% of patients, with a minority of patients achieving (often inexplicably) clinical spontaneous remission (Vlachos & Muir, 2010). DBA belongs to the large family of ribosomopathies and is usually caused by alterations in one of 20 ribosomal protein (RP) genes that lead to haploinsufficiency (Da Costa et al, 2018). Here, we used high density single nucleotide polymorphism (SNP)-arrays and whole exome sequencing (WES) to solve a clinically and molecularly intricate case of remitted sporadic DBA. The proband was a 35-year-old male, the first of two siblings born full term after an uncomplicated pregnancy from healthy unrelated Caucasian parents (Fig 1A). His parents and brother had normal haematological findings. At birth, he was identified as having a severe macrocytic hyporegenerative anaemia needing red blood cell transfusion (Haemoglobin [Hb] 76 g/l, mean cell volume [MCV] 114.5 fl, absolute reticulocyte count 8 × 109/l). No malformations were present except hypoplasia of the right thenar eminence; eADA was elevated (3 U/g Hb, normal values 1 ± 0.2 U/g Hb) and BM aspirate showed a selective decrease in erythroid precursors. He was clinically diagnosed with DBA. The patient did not respond to steroids and was regularly transfused. At 8 years of age, a few months after a steroid treatment, he achieved stable haematological remission, which was maintained at the last follow-up (age 35 years; Hb 130 g/l; MCV 98.5 fl, absolute reticulocyte count 60 × 109/l, eADA 0.56 U/g Hb). Molecular analysis of the patient and his family was performed after informed consent. High density SNP-arrays (CytoScan HD array, Thermo Fisher Scientific, ‎Waltham, MA, USA) were performed on the proband (II-1) at 7 and 29 years of age, identifying a mosaic segmental 29.7 Mb loss of heterozygosity on chromosome 19q12-q13.43 (chr19:29,348,081-59,097,752; hg19). This pattern was indicative of the presence of multiple clones with a uniparental disomy (UPD) limited to the long arm of chromosome 19 (Fig 1B). When the proband was 29 years old, the UPD involved almost the entire 19q and appeared practically complete (Fig 1C). Microsatellite analysis confirmed an allelic imbalance compatible with paternal chromosome 19q UPD (Fig 1A and Data S1). On blood-extracted genomic DNA of the proband when aged 7 years, WES identified the c.140C>T (p.Pro47Leu) heterozygous change in RPS19 (Data S1) predicted to be damaging according to several bioinformatics tools (https://varsome.com/), and reported to be pathogenic in a Brazilian DBA patient (Ramenghi et al, 2000; Angelini et al, 2007). Sanger sequencing showed the mutation was de novo (Fig 1D). B-allele frequency (BAF) analysis from WES data confirmed the UPD mosaicism detected by SNP-array with a variant allele frequency of 31% (Fig 1E; Data S1). Somatic mosaicism of the c.140C>T variant was further measured by primer extension assay showing a reduction of mutant cells from ≅50% at 7 years in blood to ≅13% at 29 years, when the patient was in full remission (Fig 2A and Fig. S1). Patients with DBA can enter a state of remission, defined by an acceptable Hb level without any treatment, lasting >6 months, independent of prior therapy without no obvious phenotypic or genotypic difference between remission and non-remission patients (Narla et al, 2011). Somatic mosaicism has been reported to explain remission (Biesecker & Spinner, 2013). Farrar et al (2011) described three DBA individuals with mosaic copy loss on chromosomes 3q and 15q, containing two well-established DBA genes, RPL35A (3q29) and RPS17 (15q25.2). Interestingly, the two patients with low-level mosaicism experienced spontaneous remission of DBA in the second decade of life, whereas the subject with a higher fraction of mosaicism remained transfusion-dependent. These findings suggest that the fraction of blood mosaicism may correlate with prognosis, and may also impact on the clinical outcome of patients with a mosaic point mutation in DBA genes. Our case parallels two recently described patients with a reversion of DBA, involving a deletion spanning the RPS26 gene in one (Venugopal et al, 2017), and RPL4, a novel pathogenic gene, in the other (Jongmans et al, 2018). These authors have suggested revertant mosaicism (RM) as a second rescue mechanism in DBA. RM refers to the co-existence of cells carrying disease-causing mutations with cells in which the inherited mutation is genetically corrected by a spontaneous event. Restoration of gene functions can be obtained by different genetic mechanisms, including gene conversion, intragenic crossover, back mutation and second-site mutation (Biesecker & Spinner, 2013; Venugopal et al, 2017). The case described here confirms RM in patients with DBA remission. Our data suggest that soon after zygote formation, a segmental paternal UPD of chromosome 19q reverted the maternal de novo mutation in a subset of embryonal cells (Fig 2B). SNP-array data show that the UPD had different extensions, suggesting the presence of different clones spanning RPS19, thus supporting the hypothesis of a selective advantage (Fig 1B–C). This biological process slowly led to a stable state of remission. Remarkably, we noted that our patient reached stable normalization of eADA levels at remission, showing complete rescue of the haematological phenotype. Indeed, eADA usually remains elevated in DBA cases, even in patients who have achieved remission or are haematologically stable on steroids (Vlachos & Muir, 2010). On this basis, we strongly suggest the monitoring of eADA activity, especially in patients who experience remission, in order to identify a possible RM. An alternative assay to monitor remission is the analysis of rRNA processing using Bioanalyzer, as described in (Quarello et al, 2016). In conclusion, we have finally solved a 28-year-old diagnostic puzzle, showing that the disease in our patient was due to a de novo maternal missense mutation in RPS19, and the rescue to a paternal UPD. However, unanswered questions remain on this case: for example, how and why did the revertant UPD occur and which factors determined the outgrowth of the reverting cells? Further work is required to understand the hidden mechanisms of this "natural gene therapy" phenomenon. We gratefully acknowledge the family who participated in this study. Elisa Giorgio and Cecilia Mancini were supported by Fondazione Umberto Veronesi fellowship 2017 and 2018. This research received funding specifically appointed to Department of Medical Sciences from the Italian Ministry for Education, University and Research (Ministero dell'Istruzione, dell'Università e della Ricerca - MIUR) under the programme "Dipartimenti di Eccellenza 2018 – 2022", Project code D15D18000410001 Fondazione Europea per l'Anemia Diamond Blackfan – Onlus (ID and UR), Diamond Blackfan Anemia Foundation-USA (ID) and Banca del Piemonte (UR). Brusco A., Garelli E., Giorgio E. conceived the experiments; Quarello P., Ramenghi U. performed the clinical evaluation and the follow-up of the patient; N. Crescenzio performed BFU-E evaluation; Carando A., Carella M., Dimartino P., Garelli E., Di Gregorio E., Giorgio E., Mancini C., Menegatti E., Palumbo O., Pippucci T. performed and interpreted molecular biology experiments; Brusco A., Garelli E., Giorgio E., Quarello P. interpreted final data, wrote the manuscript and prepared the figures; all authors critically revised the manuscript. The authors have no competing interests. Data S1. Materials and methods Table SI. Primers used for microsatellites genotyping. Table SII. WES prioritized genes on chromosome 19. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.