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D.M. Gianferante et al.
Introduction
Diamond Blackfan anemia (DBA) is a rare inherited bone marrow (BM) failure syndrome (IBMFS) with an esti- mated incidence of 5-10 per million live births.1-5 It is char- acterized by failure of red blood cell (RBC) production, congenital malformations, and cancer predisposition. Classic DBA consists of profound anemia diagnosed before one year of age, macrocytosis, reticulocytopenia, and a paucity of erythroid precursors in the BM.6 However, DBA is a heterogenous disorder with many cases having additional symptoms or no symptoms at all. Congenital abnormalities are variable but include mostly midline craniofacial defects, renal, cardiac, and thumb abnormalities.1 Malignancies associated with DBA include myeloid neoplasia, colorectal adenocarcinoma, osteogenic sarcoma, and genitourinary cancers.7,8 Standard treatment for anemia in DBA is long-term corticosteroids. Nearly 40% of people with DBA who respond to initial treatment become steroid-dependent, and those who fail to respond to corticosteroids require chronic RBC transfusions and iron chelation or hematopoietic cell transplant (HCT).1,9
DBA is predominately an autosomal dominant disorder caused by pathogenic germline variants in genes encoding ribosomal proteins.10 Twenty-six ribosomal genes have been linked to DBA etiology as well as two X-linked genes, TSR2 and GATA1, which encode a ribosome chap- erone and a hematopoietic transcription factor targeted by altered ribosome levels, respectively.10,11 Although all known DBA genes are typically included in gene discov- ery analysis or descriptive cohort studies of DBA, geno- type-phenotype studies aimed to find clinical association by gene are currently limited. Only the most frequent ribosomal genes have been examined from a genotype- phenotype perspective (e.g., RPS19, RPL11, RPL5) with lit- tle to no information available for the majority of disease- causing genes, including RPL35A.10,12 RPL35A was first associated with DBA in 2008 and has been reported to cause about 3.5% of DBA cases.13,14 Typically, data on DBA due to RPL35A have been confined to case reports, or as part of a larger DBA study with limited to no phenotyp- ic information.10,13-20
RPL35A codes for a large ribosomal subunit protein located at the telomeric end of chromosome 3q (3q29- qter); pathogenic germline variants have been reported as single-nucleotide variants (SNV), small frameshifts, inframe deletions, and large deletions involving the entire RPL35A gene with or without multiple contiguous genes deleted in the 3q29 region.14,15 Patients with DBA caused by RPL35A have been reported to have severe anemia as well as additional phenotypes not usually shared by other DBA patients, such as immunodeficiency and autism spectrum disorder.12,13,15 It is unclear whether these pheno- types are associated with the deletion of RPL35A itself or other genes deleted in the region. Some of these pheno- types overlap with 3q29 deletion syndrome, a clinical syn- drome in which the contiguous deleted region is near, but does not include, RPL35A.15 The phenotype of 3q29 dele- tion syndrome is thought to be related to the genes within the deleted region and includes dysmorphic facial fea- tures, intellectual disability, musculoskeletal problems, and neuropsychiatric issues.15,21,22 The deletion in 3q29 deletion syndrome is typically about 1.5 megabase (Mb) in size, and the consistent size is thought to be related to low copy repeat regions at each end of the deletion.21,23,24
We conducted a multi-institutional international collab- orative study of patients with DBA due to RPL35A to determine the clinical consequences of germline large deletions versus other pathogenic RPL35A variants. We also assembled variant data from additional published cases of RPL35A-associated DBA to better characterize pathogenic germline variants in this disease. The pheno- types of these patients were compared with those of the 3q29 deletion syndrome to elucidate the similarities and the differences related to variants in this region of the genome.
Methods
Study population
Patients with DBA due to RPL35A were identified within the National Cancer Institute (NCI) IBMFS cohort (clinicaltrials.gov identifier: NCT00027274),25 and through collaboration with investi- gators from the DBA Registry of North America (DBAR: clinicaltri- als.gov identifier: NCT00106015),26 DBA registries from Germany, France, Italy, the Czech Republic, and Greece, and through inves- tigators from Alabama Children’s Hospital, Arkansas Children’s Hospital, and Boston Children’s Hospital (Figure 1 and Online Supplementary Table S1). All individuals were participants in Institutional Review Board approved protocols and had signed informed consents for participating in research studies.
Additional cases of DBA for RPL35A pathogenic variant charac- terization were identified through a search of PubMed and review of ClinVar27 and Human Gene Mutation Database (HGMD).28 All data were extracted as of February 22, 2019. ClinVar variants were restricted to pathogenic or likely pathogenic RPL35A DBA variants that met the minimum requirements for data sharing and quality assurance,29 and HGMD was restricted to RPL35A DBA disease- causing mutation. Any case that was a potential duplicate of a col- laborator case was excluded (Figure 1).
Clinical data extraction
Data extraction focused on clinical criteria associated with DBA
and with 3q29 deletion syndrome.22,30 A positive finding was
counted as present, and a clinical finding marked absent or not
stated was considered absent. Standard criteria for defining
cytopenia and immunodeficiency were used, and definitions of
phenotypes studied are outlined in Online Supplementary Table S2.31
Pathogenic variant calling and variant annotation
Pathogenic variant locations (i.e., genomic or chromosomal co- ordinates) were used as provided by collaborators or from publi- cations. Methods used to identify pathogenic variants included targeted sequencing, panel testing, exome sequencing, multiplex ligation-dependent probe amplification, SNP array, or array com- parative genomic hybridization (Online Supplementary Table S3). Inclusion of a variant in the study required it to be reported as pathogenic, be rare (minor allele frequency [MAF] <1% within any gnomAD ethnic subgroups32), missense variants needed to be predicted pathogenic by meta in silico predictor programs CADD (>25)33 and REVEL (>0.5)34 (Online Supplementary Table S3), and the same case could not be included more than once. Any variants of unclear pathogenicity, including untranslated regions (UTR) and duplication, were excluded. A “large deletion” was defined as a deletion of the entire RPL35A gene. “Small frameshift” included all insertion and deletions that were not large or inframe. SnpEff was used to annotate missing chromosomal, genomic, or protein posi- tions.35 ANNOVAR was used to annotate MAF from publicly
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