Introduction:
Inherited Red Blood Cell Disorders (IRBCD) in children and young adults are a heterogenous group of disorders caused by underlying genetic defects affecting various cellular aspects of red blood cell structure and function. These defects commonly involve enzymatic pathways, cell membrane structure and elasticity, and ribosomal proteins. Clinical symptoms include anemia, reticulocytopenia or reticulocytosis, jaundice and splenomegaly, and are often accompanied by other features such as congenital anomalies. Identification of the underlying etiology is critical for management and providing anticipatory guidance in patients with IRBCD. 

Methods:
We retrospectively reviewed the genetic and clinical findings of 90 patients who underwent a next-generation sequencing (NGS) panel interrogating 52 genes associated with IRBCD at Children’s Hospital of Philadelphia from 2017 to 2024. Testing was performed using either targeted capture-based NGS panel (n=68) or slice-based testing on genome sequencing (n=24) to detect both sequence and copy number variants. When available, additional genetic findings from other tests were extracted from medical records.

 

Results:
The age at testing ranged from newborn to 20 years, with an average age of 4 years. Patients presented with various types and severities of anemia; 37% (33/90) had a family history of anemia and 30% (27/90) exhibited additional features, primarily neurodevelopmental disorders (n=14), malformations (n=5) and other miscellaneous issues (n=8). Panel analysis identified a diagnosis or likely diagnosis in 42% (38/90) of cases. Excluding 11 individuals with findings limited to G6PD, diagnoses in 27 patients involved 11 additional genes associated with anemia, including membrane skeleton disorders (SPTA1x7, SPTBx6, ANK1x4, SLC4A1), hydration defects (PIEZO1x2), Diamond-Blackfan anemia (DBA) (RPL5, RPL11, RPS19), pyruvate kinase deficiency (PKLR), glutathione synthetase deficiency (GSS) and congenital nonspherocytic hemolytic anemia (AK1, NT5C3A). Three of these 27 patients also had coincidental G6PD findings. Follow-up testing, including familial segregation, variant phasing, or functional analyses (for some variants) was performed in 22 initially inconclusive cases, which yielded likely positive results in 32% (7/22) and likely negative results in 9% (2/22). Results of eight patients remained inconclusive (9%, 8/90). The remaining 49% (44/90) had likely negative or negative results. The diagnostic yield of panel testing varied by anemia types on test indication: 92% (12/13) in spherocytosis, 80% (4/5) in macrocytic anemia, 60% (3/5) in aplastic anemia and suspected DBA, 39% (14/36) in hemolytic anemia, 30% (3/10) in unspecified/other types, 11% (1/9) in normocytic anemia, and 8% (1/12) in microcytic anemia. Additional testing including exome or genome sequencing was performed in 22 individuals, leading to further diagnoses related to anemia in four individuals (4/22, 18%) either due to genes not covered by the panel (FAS, GFI1, and TALDO1) or a variant involving the promoter region of HK1 erythroid-specific isoform undetected by the panel. Alterations related to non-anemia disorders were observed in five individuals (5/22, 23%). Except for two cases with copy number variants, the variants associated with anemia were small sequence variants.



Conclusion:
Our study demonstrated that a targeted IRBCD panel with follow-up familial and functional studies can effectively facilitate the diagnosis of IRBCDs. Understanding the underlying pathophysiology of these conditions guides patient management and prevents complications in affected individuals. Beyond informing prognosis and management, genetic diagnoses also enable accurate recurrence risk counseling. However, it is important to recognize the limitations of panel-based testing. A targeted approach does not cover all IRBCDs and may miss certain variant types. Expanding to exome or genome sequencing can capture variants not detectable by targeted analysis, which is especially valuable for patients with non-hematologic features. The variability in diagnostic yield across anemia types as well as additional findings from various tests underscore the importance of clinical correlation and careful test selection.