Garelli, Emanuela

Illja, J Diets

Marjolijn C, J Jongmans

Quarello, Paola

Roland, P Kuiper

Rolph, Pfundt

English

Institutional Research Information System University of Turin

Somatic reversion events point towards RPL4 as anovel disease gene in a condition resemblingDiamond-Blackfan anemiaSomatic revertant mosaicism is a rare phenomenoncaused by spontaneous correction of a pathogenic alleleand subsequent clonal expansion of the revertant cell. Itexplains patches of healthy skin in conditions like epider-molysis bullosa and improvement or absence of haemato-logical conditions such as bone marrow failure in Fanconianemia, dyskeratosis congenita and the recently identifiedsyndromes caused by mutations in SAMD9 andSAMD9L.1-5 Revertant mosaicism is restricted to condi-tions that fulfil two prerequisites: the diseased cells havethe ability to replicate, and loss of the mutation gives thesecells a selection advantage compared to surroundingmutated cells. Awareness of revertant mosaicism is rele-vant to understanding variable expression of syndromesand to understanding the absence of an expected mutationin DNA diagnostics. A common cause of revertant mosaicism in dominantdisorders is mitotic recombination, resulting in a daughtercell with two paternal copies of a chromosome segmentand one with two maternal copies. Clonal expansion ofthe cells lacking the mutation relative to the mutated cellsresults in mosaic segmental uniparental disomy (UPD),which can be easily visualized in the B-allele frequencyplot of SNP arrays, even when present in a subset of thecells.1 Acquired UPD observations can assist in diseasegene identification, as was illustrated for the conditionIchthyosis with confetti.6Herein, we report about a girl who was born small forgestational age (2440 gr. at term; <-2,5SD), after anuneventful pregnancy. She had a unilateral partial cleft lip,low set ears and toe anomalies consisting of bilateralshortening of the middle phalanges of the 2nd - 4th toe andduplication of the distal phalanges of these toes (Onlinehaematologica 2018; 103:e607CASE REPORTSFigure 1. Acquired 15qUPD assists in theidentification of an RPL4 mutation. (A)CytoscanHD B-allele frequency plots of chr. 15of DNA isolated from blood samples drawn atincreasing ages and from a muscle biopsy. Ablood sample drawn at age 4 shows threestretches of acquired mosaic UPD, with break-points at ~64.5 Mb in 15q22.31, ~40 Mb in15q14, and ~29.5 Mb in 15q13.1, respective-ly (*1, *2 and *3). The samples taken at anearlier (age 1) and later (age 9) time pointshow a normal B-allele frequency pattern on15q in the first year of life, and complete 15qhomozygosity at age 9. In muscle-derivedDNA, a normal B-allele frequency pattern isobserved.  (B) Sanger sequencing analysis ofthe identified mutation in RPL4 (indicated inthe red box) in the patient and the parents (Fwsequences). In the muscle biopsy, the muta-tion is present in a heterozygous state. In theblood sample drawn at age 4, the mutation ispresent in a mosaic state (~20%). In the bloodsample drawn at age 9, the mutation can nolonger be identified. The sequencing analysisshowed that the mutation is absent in thefather and the mother.  (C) Schematic mRNAsequence of the exon 2-3 boundary of wildtype (WT) and mutant (MUT) RPL4, with theinserted nucleotides in pink. AB CSupplementary Figure S1). In her first year of life, hypotoniaand developmental delay were observed. At 11 months ofage, she was admitted to the hospital due to failure tothrive. Hepatosplenomegaly was noted and a routine fullblood count showed mild anemia (hemoglobin 5.7 mmol/l(normal 7.5-10.0 mmol/L), mean corpuscular volume 79fL). A metabolic disease was suspected, for which exten-sive metabolic testing was performed and revealed noabnormalities. Since she showed no clinical signs of severeanemia, hemoglobin count was never followed up and thehepatosplenomegaly improved over time. Later in child-hood, clinical signs of anemia were not observed, andcomplete blood counts and Hb testing were not performedanymore. The girl had a borderline intellectual disabilitywith attention deficit disorder and attends a special school.At ten years of age, she had short stature (123 cm, <-2,5SD) and unilateral hearing loss due to frequent otitis.Informed consent for publication of clinical informationand photographs was obtained from parents. In search of a genomic abnormality underlying this phe-notype, a routine diagnostic genome-wide SNP arrayanalysis was performed on blood and muscle derivedDNA (CytoscanHD; Life Technologies, Carlsbad, CA92008 USA). With a genome-wide SNP array on bloodsampled at age 4, no copy number variations explainingthe phenotype of the girl were identified, but along chro-mosome 15q towards the telomer, at least three flankingregions with increasing imbalances in B-allele frequencywere encountered, suggestive of multiple subclonal mitot-ic reversion events resulting in acquired mosaicUPD15qter (Figure 1A).We hypothesized that these clonal UPD events resultedfrom a selective advantage of cells in which an as of yetunidentified heterozygous mutation, potentially explain-ing the symptoms in our patient, had reverted to wildtype. Indeed, SNP array analysis on blood DNA samplestaken at an earlier (age 1) and later (age 9) time pointrevealed progression from a normal B-allele frequency pat-tern on 15q in the first year of life towards complete 15qhomozygosity at nine years of age (Figure 1A). SNP arrayanalysis on DNA derived from a muscle biopsy taken as adiagnostic procedure when the patient was three years olddid not show UPD15qter (Figure 1A), illustrating the tis-sue-specificity of this phenomenon. The emerging 15q homozygosity in blood cells over-lapped with the disappearing features of anemia, suggest-ing that the region distal from the most telomeric recom-bination breakpoint at 15q22.31 carries a genetic mutationhaematologica 2018; 103:e608CASE REPORTSFigure 2. Impaired rRNA pro-cessing in the RPL4-mutatedpatient. Agilent Bioanalyser2100 electropherogramsobtained from total RNA offibroblasts taken at age 3 of thepatient carrying a RPL4 muta-tion (A) and from a healthy con-trol (B). The electropherogramdata demonstrated two promi-nent peaks corresponding to18S and 28S rRNA. Based onthe quantified peak surfaces,we detected a reduced28S/18S ratio (C) andincreased 32S/28S ratio (D) ofthe patient compared to thehealthy control. Depicted arethe values determined in threeindependent measurements.   A BC Dwith growth disadvantage in the hematopoietic system.Whole exome sequencing was performed on musclederived DNA (WES; SureSelect All Exon DNA enrichment;Solid 5500 XL sequencing). The region distal of 15q22.31contains five OMIM genes that could possibly explain(part of) the phenotype of the patient. However, none ofthese genes carried mutations (Online Supplementary TableS1). A search for rare coding variants with allele frequen-cies in dbSNP and gnomAD <1% revealed four potentialcandidate genes (Online Supplementary Table S2). One ofthese variants was a de novo mutation (c.176-7A>G) inRPL4 (MIM: 180479; NM_000968.3; Figure 1B), a riboso-mal protein gene highly intolerant for mutations (ExACpLI score=1.00). Sanger sequencing confirmed that thismutation was present in a full heterozygous state in mus-cle derived DNA, subclonal (~20%) in the blood sample atage 4, and completely absent in blood at age 9 (Figure 1B).The variant identified had not been reported before in ourin-house database, nor in the databases ExAC, gnomAD,GoNL or the Exome Variant Server (ESP). Sequencing of cDNA from fibroblast-derived RPL4 RNAof the patient showed that the c.176-7A>G variant affectssplicing, creating a six-nucleotide insertion(r.175_176insUUUUAG; p.(Ala58_Gly59insValLeu);Figure 1C) in the transcript, which was not affected bynonsense-mediated decay (NMD; Online SupplementaryFigure S2). The RPL4 gene encodes ribosomal protein L4. So far,mutations in RPL4 have not been linked to human dis-eases, but mutations in other ribosomal protein (RP) genescause Diamond-Blackfan anemia (DBA).7 A substantialportion of patients with a clinical diagnosis of DBA remaingenetically unexplained.8 DBA is mainly characterized bychronic hyporegenerative anemia, and approximately 50%of patients with DBA have additional anomalies, includingdevelopmental delay, short stature, cleft lip and low setears. These features overlap with the symptoms describedin our patient. The most frequent skeletal anomaly in DBAare thumb defects, but anomalies of the toes as identifiedin our patient have also been reported.9Next, we studied whether this splice site mutation dis-rupts ribosome biogenesis similar to other DBA causing RPgene mutations. Ribosome biogenesis involves the assem-bly of RPs and ribosomal RNAs (rRNAs), which togethercomprise the structural components of the 40S and 60Sribosomal subunits. In patients with mutated RPs of thelarge ribosomal subunit (RPLs), characteristic pre-rRNAprocessing signatures are found, characterized by lower28S/18S ratios and higher residual levels of the 32S precur-sor.10 We analysed rRNA processing using capillary elec-trophoresis analysis of rRNA maturation of the 40S and60S ribosomal subunits as described before.10 RibosomalRNA processing analysis on our patient’s fibroblasts inwhich the mutant allele is expressed, showed a reduced28S/18S ratio and an increased 32S precursor peak com-pared to a healthy control (Figure 2). These results suggestan impairment of rRNA processing resulting from theRPL4 splice site mutation in fibroblasts of our patient, sim-ilar to what is observed for other DBA-causing RP genes.Recently, another example of revertant mosaicism in apatient with DBA, based on a deletion encompassingRPS26, was reported.11 In the patient described here, weobserved three separate events of mitotic recombinationresulting in acquired mosaic UPD15q-tel. This indicatesthat these events provoke a selection advantage resultingin clonal outgrowth and replacement of RPL4 mutatedcells. Somatic reversion events in blood may occur morefrequently in DBA as, in 20% of these patients, the anemiagoes in remission.12,13 Such events hamper the identifica-tion of mutations in blood, which may explain why muta-tions in RPL4 have not previously been reported.8,14In conclusion, we here report the occurrence of mitoticrecombination-based somatic reversion in a girl with aDiamond-Blackfan anemia-like phenotype and show howthis genetic event directed us to identify RPL4 as a noveldisease-causing gene. Marjolijn C. J. Jongmans,1,2,3 Illja J. Diets,1 Paola Quarello,4Emanuela Garelli,5 Roland P. Kuiper3 and Rolph Pfundt11Department of Human Genetics, Radboud university medical cen-ter and Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands; 2Department of Medical Genetics, UniversityMedical Center Utrecht, the Netherlands; 3Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands; 4Paediatric Onco-Haematology, Stem Cell Transplantation and Cellular TherapyDivision, Regina Margherita Children's Hospital, Torino, Italy and5Department of Public Health and Paediatric Sciences, University ofTorino, ItalyFunding: Illja J. Diets is funded by the KiKa Foundation (project 127).Correspondence: M.C.J.Jongmans-3@umcutrecht.nldoi:10.3324/haematol.2018.200683Information on authorship, contributions, and financial & other dis-closures was provided by the authors and is available with the onlineversion of this article at www.haematologica.org.References1. Jongmans MC, Verwiel ET, Heijdra Y, et al. Revertant somaticmosaicism by mitotic recombination in dyskeratosis congenita. AmJ Hum Genet. 2012;90(3):426-433.2. Jonkman MF, Scheffer H, Stulp R, et al. Revertant mosaicism in epi-dermolysis bullosa caused by mitotic gene conversion. Cell.1997;88(4):543-551.3. Waisfisz Q, Morgan NV, Savino M, et al. Spontaneous functionalcorrection of homozygous fanconi anaemia alleles reveals novelmechanistic basis for reverse mosaicism. Nat Genet. 1999;22(4):379-383.4. Bluteau O, Sebert M, Leblanc T, et al. A landscape of germ linemutations in a cohort of inherited bone marrow failure patients.Blood. 2018;131(7):717-732.5. Tesi B, Davidsson J, Voss M, et al. Gain-of-function SAMD9L muta-tions cause a syndrome of cytopenia, immunodeficiency, MDS, andneurological symptoms. Blood. 2017;129(16):2266-2279.6. Choate KA, Lu Y, Zhou J, et al. Mitotic recombination in patientswith ichthyosis causes reversion of dominant mutations in   KRT10.Science. 2010;330(6000):94-97.7. Clinton C, Gazda HT. Diamond-Blackfan Anemia. In: Pagon RA,Adam MP, Ardinger HH, et al, eds. GeneReviews(R). Seattle(WA),1993.8. Mirabello L, Khincha PP, Ellis SR, et al. Novel and known ribosomalcauses of Diamond-Blackfan anaemia identified through compre-hensive genomic characterisation. J Med Genet. 2017 Jun;54(6):417-25.9. Errichiello E, Vetro A, Mina T, et al. Whole exome sequencing in thedifferential diagnosis of Diamond-Blackfan anemia: clinical andmolecular study of three patients with novel RPL5 and mosaicRPS19 mutations. Blood Cells Mol Dis. 2017;64:38-44.10. Quarello P, Garelli E, Carando A, et al. Ribosomal RNA analysis inthe diagnosis of Diamond- Blackfan Anaemia. Br J Haematol.2016;172(5):782-785.11. Venugopal P, Moore S, Lawrence DM, et al. Self-reverting mutationspartially correct the blood phenotype in a Diamond Blackfan ane-mia patient. Haematologica. 2017 ;102(12):e506-e509.12. Chen S, Warszawski J, Bader-Meunier B, et al. Diamond-blackfananemia and growth status: the French registry. J Pediatr. 2005;147(5):669-673.13. Vlachos A, Ball S, Dahl N, et al. Diagnosing and treating DiamondBlackfan anaemia: results of an international clinical consensus con-ference. Br J Haematol. 2008;142(6):859-876.14. Doherty L, Sheen MR, Vlachos A, et al. Ribosomal protein genesRPS10 and RPS26 are commonly mutated in Diamond-Blackfananemia. Am J Hum Genet. 2010;86(2):222-228. haematologica 2018; 103:e609CASE REPORTS

Somatic reversion events point towards RPL4 as a novel disease gene in a condition resembling Diamond-Blackfan anemia.

Jongmans, Marjolijn C.J.

Diets, Illja J.

Kuiper, Roland P.

Pfundt, Rolph

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Somatic reversion events point towards RPL4 as a novel disease gene in a condition resembling Diamond-Blackfan anemia

Somatic reversion events point towards RPL4 as a novel disease gene in a condition resembling Diamond-Blackfan anemia.

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