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DBA genotype-phenotype associated with RPL35A
Figure 2. Distribution of RPL35A pathogenic variants reported in patients with Diamond-Blackfan anemia (DBA). All pathogenic single nucleotide and small indel variants are represented once per family. cDNA position based on transcript NM_000996.3. Numbers and letters indicate amino acids. Dotted lines indicate exons. Gray circles: literature, HGMD, and ClinVar cases. Black circles: collaborator cases. Cluster, defined as >5 unrelated cases by family in one area, is located between codon 28 to 33 (*). The numbers within the circles indicate the number of unique families with that specific pathogenic variant.
Table 2. Pathogenic germline RPL35A variant type.
erature. We characterized the pathogenic variants in all 66 cases from 62 unrelated families. Two families had three affected family members each sharing a variant c.305 G>C or c.82_84CTT (NM_000996.3), respectively.
Twenty-eight of the 62 pathogenic RPL35A variants from unrelated families were large deletions (45%) (Table 2). Genomic co-ordinates were not available for six cases with large deletions. The size of the large deletions in 22 cases with available data ranged from 5Kb to 13.42 Mb (Online Supplementary Table S5). The number of genes deleted in these cases ranged from two to 153. Large dele- tions extending upstream and downstream of the 3q29 deletion syndrome region were observed in 13 cases, four cases included part but not all of the region, and five did not include the region at all (Figure 3 and Online Supplementary Table S5).
Thirty-four of the 62 pathogenic variants were non-large deletions from 19 unique collaborator families and 15 unique families from literature cases. Fifty-six percent (19 of 34) of the unique family variants were observed more than once (Online Supplementary Table S6). The inframe deletion c.82_84delCTT was the most common pathogen- ic variant (n=10) (Online Supplementary Table S6), followed by missense c.125A>G (n=3); three other variants were reported in two families each. All other variants were spread across the gene without clear mutational hotspots and no additional clusters were identified using the patho- genic variants from the cases in the literature (Figure 2). Most of the variants were located in the middle and 3’ end of the gene with only one pathogenic variant located near the beginning of the gene.
Discussion
We have compiled the largest cohort of patients with DBA due to pathogenic germline RPL35A variants. This international multi-institutional collaboration allowed us to combine well-curated genomic information with extensive clinical data to better characterize the genotype and DBA phenotype associated with variants involving RPL35A. We conclude that cases with RPL35A large deletions are pheno- typically different from cases with other variant types in RPL35A and have a more complex clinical phenotype.
Large deletions in RPL35A were frequent, comprising 45% of the pathogenic variants in this cohort. The fre-
Variant type
Large deletion
Inframe deletion
Missense
Small frameshift
Splice
Nonsense
N. Percent of families
28 45%
10 16%
14 23%
4 6%
4 6%
2 3%
Pathogenic variants were only counted once per family. Percent is based upon a total of 62 unique unrelated families in the study. Pathogenic variants must have been reported by collaborators as pathogenic and have minor allele frequency <1% in gnomAD.Variants of unknown significance including two UTR and two duplication variants were excluded. Large deletion was defined as entire RPL35A deleted, small frameshift included insertion/deletion that was not large or inframe, and no large insertions were identified.
either group. Delayed development and intellectual dis- ability including learning problems requiring an individual- ized education program in school were significant among cases with RPL35A large deletions compared with other pathogenic variants (n=13 vs. n=2; P=0.0004) (Table 1); one case also had a diagnosis of autism spectrum disorder. Chronic gastrointestinal (GI) problems, including chronic diarrhea, gastrostomy feeding tube-dependence, and chronic enterocolitis, were also significantly associated with large deletions compared with all other pathogenic variants (n=9 vs. n=0; P=0.0006) (Table 1).
In an attempt to evaluate the phenotypes of cases with non-large deletion pathogenic variants based on their genomic location, we noted that the variants were spread across the RPL35A gene with one area of clustering from codon 28 to 33 (Figure 2). A cluster was defined as >5 unre- lated cases in one area. There were nine cases from seven different families from collaborators in this cluster. The variants in six of the seven families were the same inframe deletion (c.82-84delCTT NM_000996.3). We did not find any difference in the hematologic or physical phenotype between the cases in the cluster and those spread across the RPL35A gene (data not shown).
RPL35A pathogenic variant characterization
In addition to the 45 cases obtained from collaboration with DBA registries/investigators used in the above geno- type/phenotype analysis, we identified 21 other cases of DBA due to RPL35A pathogenic variants reported in the lit-
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