Abstract
Data from a large cohort of individuals referred for NGS testing evaluate the utility of next‐generation sequencing in clinical practice for diagnosing hereditary haemolytic anaemias.
To the Editor,
Current diagnostic techniques for hereditary haemolytic anaemias (HHAs) are diverse, including red blood cell (RBC) indices, reticulocyte counts, osmotic fragility and eosin‐maleimide binding tests or measurement of RBC enzyme activity. 1 , 2 However, some methods are technically complex/unavailable to non‐specialist centres, making definitive diagnoses difficult. 3 , 4 Next‐generation sequencing (NGS) may provide a platform to diagnose a range of HHAs and overlapping clinical entities through extensive, simultaneous evaluation of multiple disease‐causing genes. 5 , 6 , 7 Inaccurate diagnosis of HHAs can have serious implications, highlighting the potential clinical utility of NGS in supplementing routine diagnostic techniques. 1
A real‐world diagnostic programme for patients with a suspected HHA disease or carrier status, excluding haemoglobinopathies, has been available since 2018 (ARUP Laboratories® 2018–2020, AnemiaID®/Revvity, Inc.® 2020–present) to provide a diagnosis using an NGS panel of HHA‐associated genes. 8 Here, we describe genetic testing results from this real‐world programme to evaluate the clinical utility of NGS in supporting the diagnosis of suspected HHAs. In total, this programme evaluated 2003 individuals between 3 December 2020 and 23 February 2023.
Clinical indication for testing varied among patients, ranging from unexplained/unspecified anaemia or hyperbilirubinaemia to specific diagnoses indicated by prior clinical testing. It also included patients with a family history of haemolytic anaemia. Individuals were referred for testing based on clinicians' judgement, and the submission of prior clinical workup results was non‐mandatory. Overall, 47.6% of the patient cohort were adults (≥18 years) and 52.0% were female.
The programme employed an exome sequencing‐based virtual gene panel, analysing 51 genes (Table S1). Details of sample preparation and sequencing can be found in Appendix S1. Most specimens tested were whole blood (54.4%) or saliva (45.2%). Variants were classified according to American College of Medical Genetics and Association for Molecular Pathology guidelines (see Appendix S2 for further details). 9
A ‘diagnostic’ or ‘positive’ panel report (termed a ‘genetic diagnosis’) was presumed when identified genetic findings were aligned with known gene–disease associations related to anaemia as shown in Table S1. This included homozygous/hemizygous pathogenic/likely pathogenic (P/LP) variants (including recessive X‐linked variants in males); two P/LP variants (without confirmatory testing proving the variants were inherited from different parents) for autosomal or X‐linked recessive conditions; or heterozygous P/LP variants for autosomal dominant conditions. An ‘inconclusive’ panel report was presumed when the reportable panel findings were insufficient to imply a genetic diagnosis. This category included individuals with one P/LP variant for a recessive condition (suggestive of carrier status) and/or a variant of uncertain significance in an autosomal dominant or X‐linked panel gene, or individuals heterozygous for Gilbert's syndrome (GS)‐associated variants and with no other reportable panel gene variants. ‘Negative’ reports refer to those with no reportable genetic findings. Additionally, reports indicating genetic findings consistent with GS, but not qualifying as diagnostic for HHA‐associated disorders, were classified as ‘clinically beneficial’ reports. No formal statistics were employed; data were summarized descriptively.
Genetic diagnoses were identified in 554/2003 (27.7%) patients tested and were more common in males than females (32.5% vs. 23.2%) and paediatric patients than adults (36.2% vs. 19.9%). A genetic diagnosis involving only one anaemia gene was identified in 97.1% of these patients, and 2.9% were found to have two different concomitant genetic diagnoses (Table 1). Inconclusive reports were issued to 47.1% of all individuals tested (see Appendix S3 for further details). Genetic diagnoses were found in 20/51 panel genes (Figure 1A). The most common genes with diagnostic findings were ANK1 and SPTB (both 19.5%), followed by G6PD (17.1% [55% G6PD‐A and 45% G6PD‐B]), SPTA1 (12.6%), SLC4A1 (9.2%) and PKLR (9.0%). Together, these genes accounted for 86.9% of all genetic diagnoses.
TABLE 1.
Genetic diagnostic outcomes in patients who underwent NGS testing.
| Test result breakdown | Patients, n/N (%) |
|---|---|
| Total tested | 2003 (100.0) |
| Patients with molecular findings consistent with anaemia a | 554/2003 (27.7) |
| Genetic diagnosis in a single anaemia‐related gene | 538/554 (97.1) |
| Diagnosis in one anaemia‐related gene | 473/538 (87.9) |
| Diagnosis in one anaemia‐related gene and GS | 65/538 (12.1) |
| Genetic diagnosis in two distinct anaemia‐related genes (dual genetic diagnosis) leading to HHA | 16/554 (2.9) |
| Dual genetic diagnosis of HHA | 12/16 (75.0) |
| Dual genetic diagnosis of HHA and GS | 4/16 (25.0) |
| Patients with a negative report b | 286/2003 (14.3) |
| Patients with diagnosis of GS only | 220/2003 (11.0) |
| Inconclusive reports c | 943/2003 (47.1) |
| Reports with only heterozygous GS‐associated variants d | 226/943 (24.0) |
| Reports with one P/LP variant for a recessive condition e | 156/943 (16.5) |
Abbreviations: GS, Gilbert's syndrome; HHA, hereditary haemolytic anaemia; NGS, next‐generation sequencing; P/LP, pathogenic/likely pathogenic.
A genetic diagnosis resulted upon identification of (1) homozygous/hemizygous P/LP variants; or (2) two P/LP variants (without successive confirmation of the variants being on opposite chromosome [in‐trans]) for autosomal/X‐linked recessive conditions; or (3) heterozygous P/LP variants for autosomal dominant conditions.
Negative reports were defined as no variants identified in any of the panel genes tested.
Any other report that is not diagnostic, negative or diagnostic for GS only was considered an ‘inconclusive report’.
Individuals heterozygous for reduced function UGT1A1 promoter variants A(TA)7TAA or A(TA)8TAA.
A diagnosis of only one P/LP variant in a recessive condition is suggestive of carrier status.
FIGURE 1.
Distribution of genetic diagnoses,a excluding GS, among diagnosed patients (A) and distribution of presumed clinical diagnoses,b excluding GS, among diagnosed patients (B). aA genetic diagnosis was classified by the identification of (1) homozygous/hemizygous P/LP variants; or (2) two P/LP variants (without successive confirmation of the variants being on opposite chromosome [in‐trans]) for autosomal/X‐linked recessive conditions; or (3) heterozygous P/LP variants for autosomal dominant conditions. bIndividuals were given a presumed clinical diagnosis of the condition if they had a genetic diagnosis in the respective genes appropriate for diagnosis, as per the genotype/phenotype correlation. AK1, adenylate kinase 1 deficiency; CDA, congenital dyserythropoietic anaemia; DBA, Diamond–Blackfan anaemia; G6PD, glucose‐6‐phosphate dehydrogenase deficiency; GS, Gilbert's syndrome; HE, hereditary elliptocytosis; HPP, hereditary pyropoikilocytosis; HS, hereditary spherocytosis; PK, pyruvate kinase; P/LP, pathogenic/likely pathogenic; SA, sideroblastic anaemia.
While a comprehensive assessment of the clinical utility of NGS testing was limited due to insufficient clinical data, which hindered the ability to correlate genetic findings with clinical presentations, presumed clinical diagnoses were evaluated based on known gene–disease associations (Table S1). Special consideration for inferring genotype–phenotype correlation was applied for patients with molecular findings in the SPTA1 and SPTB genes (Appendix S4).
The most common presumed clinical diagnoses were hereditary spherocytosis (51.3%), G6PD deficiency (17.1%) and pyruvate kinase deficiency (9.0%). Together, these conditions accounted for 77.4% of all presumed clinical diagnoses (Figure 1B). A presumed clinical diagnosis of GS was identified in 14.4% of those tested (see Appendix S3 for further details).
NGS provides a rapid, practical approach to the genetic diagnosis of HHAs and similar conditions, especially for patients in whom direct RBC testing may be inconclusive. 6 Performing the test in the physician's office using saliva/cheek swabs, without phlebotomy, offers a practical diagnostic option, especially for neonates. 8 In some clinical cases, such as ambiguous aetiology of neonatal hyperbilirubinaemia, NGS testing can allow physicians to make rapid diagnoses (Appendix S5). In addition, kits can be mailed to, and samples generated at, patients' homes for wider access to testing 8 ; the lack of costs incurred by patients/insurers with the programme further improves testing access. 8 There was widespread interest in the programme, with now over 6800 patients tested since 2020.
In conclusion, the programme testing results presented here demonstrate that NGS is a valuable platform for supporting HHA diagnostic workups, which may allow for earlier diagnosis and intervention, and more appropriate genetic counselling of patients with these conditions. However, as detailed phenotypic data were not available for this study, further research is needed to determine the correlation of NGS results with clinical characteristics, both in the United States and globally.
AUTHOR CONTRIBUTIONS
All authors contributed to the study conception or design, acquisition, analysis, or the interpretation of data. All authors critically revised the manuscript and approved the final version.
FUNDING INFORMATION
This testing programme was funded by Agios Pharmaceuticals, Inc.
CONFLICT OF INTEREST STATEMENT
JB is an employee at Revvity, Inc. and has received consulting fees from Agios Pharmaceuticals, Inc. SY and BMG are employees and shareholders of Agios Pharmaceuticals, Inc. SC is a consultant for and receives research funding from Agios Pharmaceuticals, Inc., Alexion, Amgen, Novartis, Pfizer, Roche/Genentech and Takeda Pharmaceuticals. RFG receives research funding from Agios Pharmaceuticals, Inc., Novartis and Sobi, and is a consultant for Agios Pharmaceuticals, Inc., Sanofi and Sobi. GAS is an advisory board member for Agios Pharmaceuticals, Inc., Janssen Scientific Affairs, LLC, Novartis, Sanofi and Sobi/Dova Pharmaceuticals, Inc. YR, AMA and GP have no conflicts to disclose. BG is a consultant for Agios Pharmaceuticals, Inc. PB is a scientific advisor for Agios Pharmaceuticals, Inc.
PATIENT CONSENT STATEMENT
Physicians provided certification of patients' informed consent by signing a test requisition form.
Supporting information
Appendix S1–S5.
ACKNOWLEDGEMENTS
The authors would like to thank the patients, their families and all investigators involved in this study. Medical writing support was provided by Alex Watson, MSc, and Sean Booth, MRes, both of Adelphi Group, Macclesfield, United Kingdom, and was funded by Agios Pharmaceuticals, Inc., in accordance with Good Publication Practice.
Archana M. Agarwal and Geetha Puthenveetil are co‐senior authors.
Saliha Yilmaz: HEOR and Data Science; Bryan McGee: Medical Science.
DATA AVAILABILITY STATEMENT
Qualified researchers may request access to related clinical study documents. Please send your data sharing requests to datasharing@agios.com. The following considerations will be taken into account as part of the review: (1) The ability of external researchers to re‐identify trial participants such as in small, rare disease trials or single‐centre trials. (2) The language used in data and requested documents (e.g., English or other). (3) The informed consent language with respect to the allowance for data sharing. (4) The plan to re‐evaluate safety or efficacy data summarized in the approved product labelling. (5) Potential conflicts of interest or competitive risks.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Appendix S1–S5.
Data Availability Statement
Qualified researchers may request access to related clinical study documents. Please send your data sharing requests to datasharing@agios.com. The following considerations will be taken into account as part of the review: (1) The ability of external researchers to re‐identify trial participants such as in small, rare disease trials or single‐centre trials. (2) The language used in data and requested documents (e.g., English or other). (3) The informed consent language with respect to the allowance for data sharing. (4) The plan to re‐evaluate safety or efficacy data summarized in the approved product labelling. (5) Potential conflicts of interest or competitive risks.