The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper

Abstract

METHODOLOGY The British Society for Haematology (BSH) produces Good Practice Papers to recommend good practice in areas where there is a limited evidence base but for which a degree of consensus or uniformity is likely to be beneficial to patient care. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) nomenclature was used to evaluate levels of evidence and to assess the strength of recommendations. The GRADE criteria can be found at http://www.gradeworkinggroup.org. This Good Practice Paper was produced as a collaboration with the European Hematology Association (EHA) compiled according to the BSH process at http://scanmail.trustwave.com/?c=8248&d=68DV3b1jbPPsVn. This guideline group included UK-based medical experts representing the BSH and members of the EHA Red Cell and Iron Scientific Working Group (SWG). Literature review details MEDLINE, EMBASE and PubMED were searched systematically for publications in English from 2000 to 2019 using the following key words. ‘NGS’ and ‘next-generation sequencing’ or ‘high throughput sequencing’ AND ‘haemolytic anaemia’ or ‘DBA’ or ‘Diamond Blackfan anaemia’ or ‘CDA’ or ‘congenital dyserythropoietic anaemia’ or ‘sideroblastic anaemia’ or ‘HS’ or ‘hereditary spherocytosis’ or ‘red cell membrane disorders’ or ‘red cell enzyme disorders’ or ‘PK deficiency’ or ‘PKD’. References from relevant publications were also searched. Conference abstracts were included if deemed to be of particular relevance. Review of the manuscript Review of the manuscript was performed by the BSH Guidelines Committee General Haematology Task Force, the BSH Guidelines Committee and the General Haematology sounding board of the BSH. It was also on the members section of the BSH website for comment. It has also been reviewed by members of the EHA Red Cell and Iron SWG and the EHA Guidelines Executive Committee. INTRODUCTION The use of next-generation sequencing (NGS) in the diagnosis of rare inherited anaemias is increasingly common, as evidenced by a growing number of publications describing its clinical utility.1–6 Excluding disorders of globin synthesis, rare anaemias include Diamond-Blackfan anaemia (DBA), congenital dyserythropoietic anaemias (CDA), congenital sideroblastic anaemias (CSA), and disorders of red cell membrane and enzymes. Other forms of genetic anaemias can also be considered while establishing NGS panels, in particular genetic syndromes, where anaemia comprises one of the constellation of symptoms. Table 1 briefly summarises the key aspects of these conditions. Table 1. - Key Aspects of the Rare Anaemias Not Due to Disorders of Haemoglobin Synthesis DBA CDA Sideroblastic Anaemia Red Cell Membrane/Cation Leaking and Enzyme Disorders Age at presentation Usually 2–3 mo of age or <first year of life Usually child/young adult Usually child/young adult Foetal/neonate/child/young adult Associated features CraniofacialSkeletalCardiacUrogenital tract Distal limbIron overload Ring sideroblasts on bone marrow aspiration JaundiceHepatosplenomegalyGallstonesIron overloadProgressive myopathy and neurocognitive impairmentaLymphoedemab Severity Moderate to severe Usually mild to moderate Mild to severe Mild to severe Treatment CorticosteroidsTransfusions and chelationBMT InterferonTransfusions and chelationOften none needed Transfusions and chelationOften none needed Often none neededSplenectomyTransfusions and chelationNew agents Genetics Autosomal dominant (45%) or de novo (other inheritance for DBA-like disease)Ribosomal proteins or other genes affecting ribosome biogenesis (other genes for DBA-like disease) Autosomal recessive or dominant, X-linkedVesicle trafficking, heterochromatin assembly, nuclear proteins, transcription factors X-linked; autosomal recessiveHaem synthesis Autosomal dominant or recessive; X- linkedRBC membrane cytoskeleton, RBC transporters and RBC enzymes aAssociated with some rare enzymopathies or rare form of glucose transporter type 1 (GLUT1) variants.bAssociated with some severe form of hereditary stomatocytosis.CDA = congenital dyserythropoietic anaemia; DBA = Diamond-Blackfan Anaemia; RBC = red blood cell. The advantages of using NGS over single-gene testing, in addition to the cost effectiveness, is that clinical and laboratory features are often not specific for a particular condition, and a large number of large candidate genes might need to be analysed before making a diagnosis. A proportion of the patients also present with overlapping phenotypes, and it has been shown that in 10%–40% of cases, there is a degree of misdiagnosis or no diagnosis when this is based purely on phenotype and traditional non-NGS testing.1,6 This can result in incorrect or inadequate treatment, causing anxiety and adversely affecting quality of life and potentially cost. The term ‘NGS’ refers to all types of high-throughput sequencing, and for the purposes of this good practice guideline will include targeted resequencing (t-NGS), whole exome sequencing (WES) and whole genome sequencing (WGS). Table 2 shows the advantages and disadvantages of each type of NGS. A detailed description of NGS techniques is beyond the scope of this paper; however, this is summarised in Figure 1. In t-NGS, only the genes selected are sequenced, whereas in WES ~30 000 genes are sequenced and in WGS all genes and intergenic regions are sequenced. However, in WES and WGS, the coding sequences of only a subset of genes are analysed, what is frequently referred to as a ‘virtual panel’. In addition, coverage of genes is best in WGS where no DNA amplification step is required. Large duplications and deletions, involving one or more whole genes, known as copy number variants (CNVs), are more difficult to identify, but can be detected using appropriate analysis, particularly using WGS, but also WES and targeted resequencing. Table 2. - Comparison of Different Types of Next-Generation Sequencing t-NGS WES WGS Target of sequencing; size (base pairs [bp]) Exons of 20–200 genes with some intron/exon boundaries for splice site mutations; 500 000 bp The ‘exome’ ~30 000 exons of known coding genes (~1.5% of genome but 80%–90% of known disease-causing mutations) with some intron/exon boundaries for splice mutations; 2 × 107 bp The whole genome (coding and noncoding space)3 × 109 bp Method Capture of chosen exons, amplification steps and sequencing or amplification of chosen exons and sequencing Capture all the exons, amplification step and sequencing DNA is fragmented randomly, ligation of adaptors and direct sequencing (no capture or amplification) Advantages Cost, relative ease of interpretation, few unsolicited findings, more challenging to identify CNVs Cost lower than WGS Entire genome interrogated including non-coding region; more potential to identify CNVs. Can add genes to virtual panel. Relatively even coverage Disadvantages Will only identify mutations in targeted regions, coverage is often uneven, so mutations may be missed. Harder to detect some CNVs Interpretation can be challenging, high chance of unsolicited findings, will only find mutations in coding regions, coverage is often uneven, may not detect CNVs. Ethical issues of incidental findings in genes that predispose to serious illness Interpretation challenging unless there is a trio, non-coding region cannot easily be interpreted. Ethical issues of incidental findings in genes that predispose to serious illnessCost CNV = copy number variant; t-NGS = targeted resequencing panels; WES = whole exome sequencing; WGS = whole genome sequencing. Figure 1.: (A) Cartoon of the process of creating an NGS report from arrival of sample in the laboratory. (Usually includes clinical scientists and clinicians.) (B) American College of Medical Genetics variant classification, with examples of further studies that can be carried out to determine the pathogenicity of class 3 variants of uncertain significance. This includes family studies to investigate segregation, as well as functional assays such as red cell enzyme activities, EMA dye binding for hereditary spherocytosis, and osmotic gradient ektacytometry (Osmoscan), which investigates red cell deformability for membrane disorders. This list is not exhaustive and includes other functional assays (eg, electron microscopy for CDA, ribosomal profiling, or northern blots for DBA); EMA dye binding. CDA = congenital dyserythropoietic anaemias; DBA = Diamond-Blackfan anaemia; Ekta = ektacytometry; EMA = eosin-5’-maleimide; MDT = multidisciplinary team; NGS = next-generation sequencing.It is important to note that, depending on the size of the panel, a number of variants will always be identified after all of the filters are applied, even in normal individuals. This number will depend on the number of genes, the inherent polymorphic potential of the gene and the ethnic origin of the individual tested. All variants identified post-filtering need to be assessed against strict criteria to determine their pathogenicity, based on the guidelines of American College of Medical Genetics (ACMG).7 It is good practice to assess all variants even after a pathogenic variant has been found, to help with interpretation if this variant is identified again in the future. 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variants identified NGS that variants are with to the phenotype of the patient sample form can be found in Figure further are to the diagnosis (eg, family by the variant these can be on the report are the important in the appropriate NGS and which quality criteria be are carried out as t-NGS, some out of regions, the variants genes that been virtual some all genetic in the form of WGS, virtual will be increasingly The of using t-NGS over virtual is to cost and may be lower for WGS, this a beyond the scope of However, a of using t-NGS is that if genes are found to be with a known a gene to the and This and process t-NGS to a WGS is also for of a genetic of a number of inherited with globin gene a particular for all to CNV from targeted are their capture and may be with to determine the from the selected will depend on it is that a laboratory is of the of the and that steps are to some of these (eg, by or that the report produced is on the of the This may (eg, to CNVs that may not be 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In some cases, the variants are by functional evidence to this is not the it is that publications are reviewed to assess All relevant regions of known genes be included and at of the regions be by the sequencing. 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