Identification of 2 novel noncoding variants in patients with Diamond-Blackfan anemia syndrome by whole genome sequencing

Abstract

Diamond-Blackfan anemia syndrome (DBAS) is a rare congenital disorder with variable penetrance and expressivity and is characterized by pure red cell aplasia that typically manifests as early-onset chronic macrocytic or normocytic anemia and is often associated with other congenital anomalies. DBAS is etiologically heterogeneous with >20 known DBAS-associated genes that encode small and large ribosomal protein subunits and an inheritance pattern that is largely autosomal dominant or sporadic. We report 2 DBAS cases with previous negative genetic testing, which included targeted gene panels, karyotype analysis, chromosome breakage analysis, and whole exome sequencing. Although clinical whole genome sequencing (WGS) was initially negative, in-depth reanalysis identified 2 novel noncoding variants in the RPS gene family, namely a maternally inherited splicing variant at the end of the first noncoding exon in RPS7 (NM_001011.4, c.-19G>C) in family 1 and a deep intronic de novo variant in RPS19 (NM_001022.4, c.172+350C>T) in family 2. In family 1, several maternal relatives were identified who shared the same variant through cascade testing; clinically, they exhibited variable degrees of anemia and elevated erythrocyte adenosine deaminase activity, a marker for DBAS. RNA sequencing analysis demonstrated deleterious functional consequences for both noncoding variants. In case 1, hematopoietic stem cell transplant with an unaffected matched sibling donor who did not carry the variant successfully cured the congenital anemia. This study identified novel noncoding variants and underscores the clinical utility of WGS in accelerating diagnosis and improving care for rare genetic disorders, particularly when timely treatment decisions are critically important.

Graphical abstract

Figure 1.

The DBAS8 case with proband carrying the RPS7 variant and the maternal segregation studies. (A) Case 1 (DBAS8) proband at the age of 16 months. (B) The WGS depth and variant allele fractions (AF) in heterozygous subjects revealed by Emedgene software. (C) The maternally inherited RPS7 variant viewed by raw reads in IGV software. (D) The maternal 3-generation pedigree with RPS7 genotype/phenotypes and cascade eADA testing values superimposed. WT, wild-type.

Figure 2.

The genomic map of the RPS7 noncoding variant and in silico prediction on splicing impact. (A) The genomic location map of the RPS7 c.-19G>C variant in reference to the noncoding exon and the start site (ATG/c.1, Alamut software). (B) In silico algorithms unanimously predicted a donor loss and the precise position of the 2 neo-donors at +4 and +98 loci (Alamut). (C) An independent algorithm (SpliceAI) concurrently confirmed a significant donor loss (at c.-19+1) and a robust new donor gain (at c.-19+98) induced by the c.-19G>C variant (framed red). SSF, SpliceSiteFinder.

Figure 3.

The genomic map of the RPS19 noncoding variant and in silico prediction on splicing impact. (A) The pedigree for the DBAS1/RPS19 family with the eADA level superimposed for the solely affected proband. (B) The DBAS1 proband at the age of 1 year. (C) The genomic position depicting the deep intronic nature of the RPS19 c.172+350C>T variant by IGV. (D) The WGS depth and variant AF in heterozygous subjects. (E) In silico splicing prediction algorithms (Alamut and SpliceAI) independently predicted a profound donor gain (red framed) as a consequence of the deep intronic variant. WT, wild-type.

Figure 4.

Functional RNA-seq study demonstrated significant transcriptional stability deficiencies for both noncoding variants. (A) For case 2 (DBAS8/RPS7), robust intron retention extending to RPS7 c.-19+98 (red arrow and frame) was revealed in the proband and mother only when compared with father and controls with the aberrant transcript usage rate shown below. (B) Zoomed-in view of the +98 intron retention demonstrating the new transcript usage rate. (C) RNA functional evidence showing that the c.-19G>C variant is confined to those transcripts with retention. (D) For the DBAS1/RPS19 case, a novel exon 3′ (red arrow) immediately upstream of the RPS19 c.172+350C>T variant was identified uniquely in the proband when compared with controls. (E) New exon 3′ usage rate and sequence analysis depicting unique junctional splicing and several stop-gain codons within the 81-bp novel exon 3′. UDN, undiagnosed diseases network.