Gene targeted NGS to improve diagnosis of rare anaemia. Frances Smith 16 th Annual Meeting of Saudi Society of Haematology Jeddah 24 th February 2018

Transcription

1 Gene targeted NGS to improve diagnosis of rare anaemia Frances Smith 16 th Annual Meeting of Saudi Society of Haematology Jeddah 24 th February 2018

2 Outline: Introduction to rare inherited red blood cell disorders Difficulties with diagnosis New diagnostic approach using nextgeneration sequencing Overview of cases analysed so far Interesting cases

3

4 Functional components - haemoglobin Schechter, Blood Nov 15; 112(10):

5 Functional components the membrane Narla Mohandas, and Patrick G. Gallagher Blood 2008;112:

6 Functional components the membrane Spherocyte Normal red blood cell Elliptocyte Poikilocyte Narla Mohandas, and Patrick G. Gallagher Blood 2008;112:

7 Functional components enzymes

8 Production - erythropoiesis Kouray and Hasse, 2015

9 Difficulties with diagnosis: Many pathology tests undertaken Transfusion Bone marrow not representative Phenotypic variation

10 Using next-generation sequencing: Bring genetics up the diagnostic pathway DNA extracted from white blood cells Representative of patients true genotype Able to analyse large number of genes in one test Reduces the need for repeat sampling Ability to diagnose unusual or new presentations

11 212 genes involved in red cell and other related disorders Version control - Gene Selection 15 Sub Panels: Membranopathy Enzymopathy Haemoglobinopathy Congenital dyserythropoietic anemia (CDA) Diamond Blackfan Anaemia (DBA) Congenital Erythrocytosis Megaloblastic anaemia Bone Marrow Failure Thrombocytopenia Neutropenia Porphyria Sideroblastic anaemia Iron regulation Hemophagocytic lymphohistiocytosis (HLH) Lymphoedema

12 Next generation sequencing

13 Data analysis: Analysed as sub-panels Average patient has~250 rare or novel sequence variants Filtered on frequency Interpretation aided by inhouse variant database called SNPpy Integration of multiple sources of evidence to classify variants Single report written Rachel Mayhew

14 Interpretation: Literature search ClinVar NCBI variant database Previously identified in the laboratory VEP variant effect predictor Annovar annotations Alamut conservation Copy number variations OMIM known disease mutations Previously identified in another laboratory ExAC population frequency SNPpy Professional judgement

15 Review of cases analysed: Total Diagnosed Not diagnosed Uncertain Diagnostic yield % Haemolytic anaemia Non-haemolytic anaemia Unexplained anaemia Thalassaemia Congenital erythrocytosis Total

16 Haemolytic anaemia positive diagnoses SPTA1 SPTB ANK1 CDAN1 CYB5R2 CYB5R3 SLC4A1 PKLR G6PD PIEZO1 PGK1 KLF1 HK1 GPI G6PD, SPTB G6PD, HBB

17 Red cell disorders presenting with liver failure Case 1: Phosphoglycerate kinase deficiency presenting with: - jaundice - hypoglycaemia - hepatosplenomegaly - acute liver failure

18 Blood film PGK1 Film comment: Many nucleated red cells, polychromasia, acanthocytes

19 Previously reported mutation in PGK1 p.l89p: markedly reduced enzyme stability Enzyme activity ~5% in RBCs and nucleated cells 1 case described - Haemolytic anaemia and neurological complications (no myopathy) died age 9 years from pneumonia associated with neurological problems Liver disease not reported previously Hepatitis reported in older patient but different mutation

20 Red cell disorders presenting with liver failure Case 2: Alpha spectrin mutation presenting with: liver failure profound anaemia conjugated hyperbilirubinaemia antenatal hydrops

21 SPTA1 Heterozygous c.2659c>t; p.arg887* SPTA1 Heterozygous c.2659c>t; p.arg887* SPTA1 Homozygous c.2659c>t; p.arg887*

22 Sideroblastic anaemia case 3 42 year old female 44% ring sideroblasts Compound heterozygous YARS2 variants Known syndrome: MLASA Myopathy Lactic acidosis Sideroblastic anaemia Respiratory chain defects Cytochrome C oxidase

23 Haematology Sideroblastic anaemia Genetics Compound heterozygous YARS2 Uncertain significance Neurology Muscle biopsy Loss of OXPHOS Research Yeast model Rob Taylor Claudia Donnini

24 Moving to the age of genomics Targeted sequencing Red Cell panel (212 genes) or Focused exome: 6, 200 genes Whole exome sequencing 1-2% of genome 22,000 genes Whole genome sequencing

25 Conclusions: Red blood cell disorders are complex Genetic diagnosis can help in most cases Sharing data between diagnostic laboratories, clinicians, research laboratories is essential Feedback is required

26 Acknowledgements: King s College Hospital: Barnaby Clark David Rees Subarna Chakravorty Austin Kulasekararaj David Brawand Pete Rushton Laura Steedman Nick Parkin Chris Wall Rachel Mayhew Eva Moldes Beiro Alison Bybee Oxford: Noiemi Roy Mel Provan Newcastle: Rob Taylor Parma: Claudia Dunnini All the referring clinicians All the patients and families