The use of <scp>next‐generation</scp> sequencing in the diagnosis of rare inherited anaemias: A Joint BSH/EHA Good Practice Paper*

Abstract

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-8nm3kGS2D_-Hms9YWMWrrk5K8Eg&u=http%3a%2f%2fb-s-h%2eorg.uk. This guideline group included UK-based medical experts representing the BSH and members of the Red Cell and Iron Scientific Working Group (SWG) of EHA 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 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. 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. Craniofacial Skeletal Cardiac Urogenital tract Distal limb Iron overload Jaundice Hepatosplenomegaly Gallstones Iron overload Progressive myopathy and neurocognitive impairmenta Lymphoedemab Corticosteroids Transfusions and chelation BMT Interferon Transfusions and chelation Often none needed Transfusions and chelation Often none needed Often none needed Splenectomy Transfusions and chelation New agents 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-linked Vesicle trafficking, heterochromatin assembly, nuclear proteins, transcription factors X-linked; autosomal recessive Haem synthesis Autosomal dominant or recessive; X- linked RBC membrane cytoskeleton, RBC transporters and RBC enzymes 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, while 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. Exons of 20–200 genes with some intron/exon boundaries for splice site mutations; 500 000 bp The whole genome (coding and non-coding space) 3 × 109 bp 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 illness. Cost. 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. Excellent comprehensive guidelines exist for the preparation of samples and the quality control that should be followed.8 Likewise, the ACMG and Association of Clinical Genomic Science (ACGS) guidelines detail the interpretation of variants, and all laboratories should follow these criteria to determine pathogenicity of all variants identified.7, 9 The ACGS guidelines are less stringent in their assessment of evidence for pathogenicity. The ACMG system therefore scores more variants as variants of uncertain/unknown significance (VUS)/class 3 than the ACGS guidelines, increasing specificity at the expense of sensitivity. The purpose of this paper is to give guidance on the uses of NGS that are specific to the diagnosis of rare inherited anaemias. This may be useful to laboratories wanting to set up NGS or for ones that have set this up for research and are planning to use it for clinical diagnosis. The type of NGS used, the conditions for which it can be used and the timing of it in the diagnostic pathway will partly depend on each country's healthcare system and funding arrangements. However, we aim to issue general guidance. Most of the guidance below is best suited to t-NGS as this is currently most commonly used, but the principles are equally applicable to the other technologies. Most current NGS approaches include the genes involved in the pathology of DBA, CDA, CSA and disorders of red cell membranes and enzymes.1, 6 The globin genes are frequently but not always included. Firstly, much of globin gene testing required for pre- and neonatal diagnosis requires a rapid turnaround time and analysis of a small number of genes, making it unwieldy and unnecessary to be testing all of the genes on a panel. In most cases, the clinical and laboratory presentation is clear and only a minority have a differential diagnosis of other haemolytic anaemias. Secondly, these are regions of very high sequence homology, potentially resulting in poor specificity and high levels of artefacts and false-positive results on NGS testing, depending on the specific technology selected. In addition, many of the pathogenic genetic abnormalities leading to haemoglobinopathies are CNVs (insertions or deletions), which can be more difficult to detect by t-NGS. In particular, some common alpha globin variants such as the 3.7 kb deletion and triplicated alpha globin gene, are especially challenging as the breakpoint sequences are not unique. However, robust validation of the panel can ensure the reliable detection of most globin variants and some panels have been designed specifically to detect CNVs in globin genes, enabling the option of using NGS for haemoglobinopathy diagnosis. There are circumstances when globin gene sequencing is of particular importance, including in the assessment of microcytic or haemolytic anaemias. Haemoglobin subtype analysis, including the quantitation of haemoglobin A2, can identify or exclude most globin gene variants, but does not reliably identify many cases of unstable haemoglobin, dominant thalassaemia10, 11 or individuals with beta thalassaemia intermedia resulting from heterozygous of beta thalassaemia in the presence of triplicated alpha gene.12 In the case of unstable haemoglobins, the patients may have a mild to severe haemolytic anaemia, including transfusion dependence.13, 14 The unstable haemoglobin is often not detectable using haemoglobin analysis, and the presence of transfused blood also makes phenotypic diagnosis more difficult, particularly if started neonatally. Globin gene variants are the commonest cause of inherited anaemia, and all patients should be formally assessed for their presence, using a combination of haemoglobin analysis and specific genetic tests for suspected variants, and by inclusion on NGS panels, depending on local practice. Particular consideration needs to be given to excluding CNVs of the alpha globin genes, which may require specific assays using a gap polymerase chain reaction (Gap-PCR) or multiplex ligation-dependent probe amplification (MLPA). The number of genes to include in a panel must balance inclusivity, to reduce false-negative rates, with increasing workload from needing to review and critically assess a large number of variants. For any laboratories wishing to set up t-NGS for rare inherited anaemias, Appendix 1 contains our suggested list of genes. Any published list is rapidly out of date as new evidence accumulates. However, the majority of known genes will be valid for some time. In England, genetic testing has been harmonised nationally and all the genes on each panel offered are available on PanelApp: https://panelapp.genomicsengland.co.uk/. This list of genes has been determined and curated by specialists in the field and is updated yearly to ensure that newly published genes are included. It is worth considering if there are conditions in which NGS is of no added value and whether the reluctance to use NGS in some cases is purely due to its cost. There are rare anaemias that are often straightforward to diagnose without recourse to DNA analysis, e.g., hereditary spherocytosis (HS). Nevertheless, for such cases the advantage of carrying out molecular analysis is that it facilitates genetic counselling. This can be especially helpful in some HS cases without a clear family history, to distinguish between recessive inheritance and a de novo variant. Conversely, laboratory tests for HS reach a sensitivity/specificity of >98%/90%, which is higher than for t-NGS. Although these are often mild conditions, they can result in significant morbidity including fetal anaemia, kernicterus and transfusion dependence, and genetic counselling is useful, particularly in families who wish to avoid further affected pregnancies. It is particularly important to be certain of the precise diagnosis before performing splenectomy for presumed HS, to avoid ill-advised splenectomy in dehydrated hereditary stomatocytosis as this procedure is accompanied by a greatly increased risk of thromboembolic disease.15 Phenotypically these conditions can be very similar unless some assessment of red cell hydration is performed, such as osmotic gradient ektacytometry or osmotic fragility measurement. In general, genetic diagnosis should be confirmed before recommending splenectomy in HS, and this will typically involve analysis using an NGS panel. Additionally, documenting genetic variants will eventually lead to some genotype–phenotype correlations.16, 17 This is the case with pyruvate kinase (PK) deficiency, where response to the new drug AG-348 depends on whether the mutations are missense or not.18 For some conditions, NGS is far superior to Sanger sequencing of specific genes, due to the phenotypic variability and the unreliability of phenotypic tests such as enzyme assays for rare enzymopathies, making it difficult to target genes precisely, particularly when the patient is transfusion dependent. Because of frequent misdiagnosis of 'dyserythropoiesis' in some haemolytic anaemias,1, 6 genetic analysis should always be used to confirm a 'CDA'. One condition where genetic analysis is particularly useful is dehydrated hereditary stomatocytosis (xerocytosis) due to autosomal dominant mutations in the gene Piezo-type mechanosensitive ion channel component 1 (PIEZO1), a mechanosensitive calcium channel. Patients with this condition are probably at high risk of developing post-splenectomy thrombosis and splenectomy in these cases is generally contra-indicated.15, 19 This condition is difficult to diagnose, and can be associated with only occasional stomatocytes on the blood film; genetic diagnosis should usually be performed before splenectomy when there is a possibility that the diagnosis could be dehydrated hereditary stomatocytosis; this will include most cases of presumed HS. Finally, NGS-based genetic testing is useful for the identification of complex modes of inheritance that are recognised to account for at least 4% of diagnosed Mendelian conditions.20 The use of NGS will partly depend on each country or hospital system's technical and reimbursement characteristics. The traditional investigative pathway is to take a history and examination, full blood count (FBC), reticulocyte count, and haemolytic markers, before selecting specialised tests (enzyme assays, osmotic gradient ektacytometry, eosin-5-maleimide [EMA] test, erythrocyte adenosine deaminase [eADA] etc.). In some cases, this may lead to a bone marrow biopsy or aspiration, with genetic analysis being kept at the end of the pathway. In other places, genetic analysis may occur much earlier in the pathway.21 The advantages are that this may lead to a more rapid diagnosis, may be cost effective in reducing delay in diagnosis (at the expense of a higher cost upfront) and may (in some conditions) preclude the need for a bone marrow biopsy. Figure 2 shows examples of aspects of the history and examination that should be sought when evaluating the patient, as well as standard blood tests. The requirement for specialised tests, bone marrow aspiration and biopsy, and genetic analysis and the order in which they are requested, will differ between services, but in time, genetic analysis is likely to be carried out earlier in the pathway, with specialised functional analysis used to confirm the genetic diagnosis. Most panels are currently carried out as t-NGS, although some diagnostic laboratories carry out target enrichment across thousands of regions, then analyse the variants among genes that have been grouped together into virtual panels. As some countries move towards conducting all genetic analysis in the form of WGS, virtual panels will be increasingly used. The choice of using t-NGS over virtual panels is mostly due to availability, cost and turnaround time. While cost-per-base may be lower for WGS, this requires a capital investment beyond the scope of most diagnostic laboratories. However, a major disadvantage of using t-NGS is that if any new genes are found to be associated with a known phenotype, adding a gene to the panel requires complete redesign and revalidation. This time-consuming and expensive process limits updating t-NGS panels to about once a year. WGS is also better suited for determination of CNVs, a common genetic cause of a number of inherited anaemias, with alpha globin gene deletions remaining a particular challenge for all technologies. New bioinformatic protocols to improve CNV assessment from targeted panels are improving their detection across modalities. Bait capture and unique molecular indexed amplicon methods may be combined with bioinformatic algorithms to determine the breakpoint mapping from short reads.22 As the selected method will depend on many factors, it is critical that a laboratory is aware of the limitations of the technique, and that additional steps are taken to either overcome some of these limitations (e.g., gap-filling by Sanger Sequencing) or that the report produced is explicitly clear on the limitations of the analysis. This may require suggesting alternative methods (e.g., MLPA) to address CNVs that may not be detected reliably by t-NGS. The availability of complementary diagnostic tools such as erythrocyte morphology, red cell and reticulocyte indices, EMA dye binding or osmotic gradient ektacytometry for red cell membrane disorders, may allow a phenotypic confirmation of the diagnosis in the absence of a definitive genetic diagnosis. Once variants have been identified and graded for pathogenicity, a multidisciplinary team meeting (MDT) is carried out, where variants are discussed in the context of the clinical presentation and a final report is written. In cases of an established pathogenic variant that fits with the phenotype, a report can be issued by the clinical scientists in the absence of an MDT meeting. The ACMG guidelines must be followed for pathogenicity of single-nucleotide variants (five classes) - pathogenic (class 5) and likely pathogenic (class 4) variants related to the clinical suspicion should be included in the report. This also includes circumstances where two very rare VUSs are identified in a gene(s) implicated in the phenotype, and family studies indicate they are in trans and functional this gene as being of significance or variants that a complex of inheritance can form the of research with the that this requires a form of to that for diagnostic The of variants between laboratories a very important in and cost However, this is often much more difficult to than might be with issues such as and the of variants being significant One of the for variant is that laboratories use the system for variant of variants is difficult the to be includes the clinical phenotype, the pathogenicity was assessed including individual of the and of the other variants found in the A potentially pathogenic variant where a definitive genetic cause has also been found in the patient the one is less likely to be However, the more variants are the more the the possibility that individuals may be identified according to specific Other to variant across laboratories include technical and in the and time need to the up to date and with to take for any is also using the nomenclature (e.g., Society and against the In cases of the specific must be used, but this is not always should to ensure that the they are using is in may be useful in this assessment For a laboratory to a number of must be which to the laboratory the panel the pathway and the report. Finally, it may be useful to a with patients at the of the of of and that they may be to further samples to help some of the In addition they can be aware that other family members may be to of a variant of The laboratory must be involved in an (e.g., Clinical Association for and must in quality assessment for NGS. none the laboratory should a to carry out some with other similar laboratories to ensure there is an of the laboratory carrying out the NGS does on DNA samples it from these laboratories should also be Clinical NGS will ensure that variants are against the phenotype of the patient and where this is the laboratory should to it from or issue a limited report if there is no phenotypic pathogenicity in the laboratory according to the ACMG guidelines as is critical as publications pathogenicity in the the recommendations. In some cases, the variants are accompanied by functional evidence to confirm pathogenicity. As this is not the it is our that publications are reviewed to assess All relevant regions of known genes should be included and at least of the regions must be by the The coverage at individual genes may be included in the or and in the laboratory and available sequencing into the is needed to identify splice variants and should bp to ensure mutations are All types of known variants should be detected by the for this include variants in the region (e.g., 2 binding site in or for of coverage should be to detect a heterozygous including single-nucleotide variants, and variants including common pathogenic variants should be reliably have to be large to detect phenotypic 6 In cases of where anaemia may be the these genes can also be included in the panel (e.g., 1 and or that are not known to be pathogenic should not be included unless specific research has been As with the of any new test, robust validation should be carried out before into the diagnostic should be based on the criteria and are summarised in Appendix on the some laboratories only need to the while need to each individual panel. The report should also the following sequencing coverage coverage is generally set to exclude variants at an of in the general although this may depending on the ethnic which should be available for each Any variant with a of pathogenicity should be included of the In HS, common pathogenic are present the (e.g., and should be specifically sought in patients where is a diagnosis, especially in the presence of other 1 variants. It should be that while is usually included in most t-NGS panels, is not usually with standard t-NGS However, is frequently with the variant that is by standard t-NGS Sanger sequencing is not needed to confirm individual variants if the panel has been and the whole pathway is assessed as of However, in some cases Sanger sequencing is before a report can be This includes any variant or (e.g., any CNVs should be confirmed where as should any variants that are present in only or as this may result from and Sanger sequencing can also be used to confirm that the has been and therefore the report is being issued to the of patient may be by DNA analysis of a new analysis of confirmation or additional functional tests may or pathogenicity of individual variants as the ACMG It is best practice to samples from relevant family members that family studies may be carried out, including testing for de novo a pathogenic variant is identified in only one of a recessive gene that is associated with the clinical phenotype, it is common practice to report that the pathogenic variant may be to the The report should be clear on the limitations of the and further can be suggested where this be The genes that have been the coverage of genes and the types of genetic abnormalities must be This is to ensure that if a report that no pathogenic variants were the is clear about what of the condition have not been This includes common pathogenic variants not detected by the current Other of up deletions e.g., and single-nucleotide The false-negative will be for each phenotype that is being assessed by the panel, depending on what proportion of the phenotype is known to be at the molecular This is likely to improve in time as genes are The clinical report should be to and be by The technical of the should be available which was used should be The BSH General Haematology Task members at the time of this good practice paper were and The to the BSH sounding and the BSH guidelines for their in this good practice The also to the EHA Guidelines Committee as well as EHA Executive for their in and this good practice and all in the synthesis and of the The BSH the the of this good practice All have a of to the BSH and Task which may be on There were no of that on the of this of the group will the group if any new evidence available that the strength of the in this or it The will be and from the BSH current guidelines website if it new are an will be published on the BSH guidelines website While the and in this guidance is to be and at the time of to the the the any for the of this guidance. number of required to the criteria site tools

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