Haemoglobinopathy Case Studies. Dr Jill Finlayson Department of Haematology Pathwest Laboratory Medicine

Transcription

1 Haemoglobinopathy Case Studies Dr Jill Finlayson Department of Haematology Pathwest Laboratory Medicine

2 Case 1 KB, 36y M Refugee Afghanistan Screening bloods Hb 101 g/l RCC 3.75 x10 12 /L MCV 90 fl MCH 27 pg

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5 Iron Stain

6 Heinz body prep Isopropanol stability test positive

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8 PCR testing for the common alpha gene deletions was normal. Alpha-1 Globin Sequencing NG_ (HBA1):[c.119_121delCCA, p.thr40del] + [=] DNA sequencing of the alpha-1 globin gene has detected a heterozygous three base deletion (-CCA) of amino acid 39 (39.2 through 40.1 inclusive) of the mature protein. This deletion mutation results in a protein shortened centrally by one amino acid, known as Hb Taybe. Alpha-2 Globin Sequencing NG_ (HBA2):[c.95+2_95+6 deltgagg] + [=] DNA sequencing of the alpha-2 globin gene detected a heterozygous 5 base pair deletion (-TGAGG) at the 5' donor site of IVS1. This causes abnormal splicing, preventing globin chain synthesis from the affected gene. This results in an alpha thalassaemia phenotype. Beta Globin Sequencing DNA sequencing of the beta globin gene was normal.

9 HBA2:c.95+2_95+6 deltgagg Splice site mutation

10 HBA1:c.119_121delCCA, p.thr40del Hb Taybe Hb Taybe is characterized by the deletion of a Threonine residue in the α1β1 contact area. In addition, Thr39 normally interacts with the Glu35 and Asp36 of alpha haemoglobin stabilising protein (AHSP) This is a chaperone protein which protects the α Hb subunits once formed and participates in their correct folding.

11 Cell, 2004, 119,

12 It is likely that the two mutations are inherited in trans, but can t confirm this with current methods. No family members.

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14 Case 2 NH, 80y M History of myelodysplasia (RCMD, Normal cytogenetics 46,XY) Routine review Hb 69 g/l RCC 3.13 x10 12 /L MCV 74 fl MCH 22 pg

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16 HbH prep

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19 Acquired Alpha Thalassaemia First described in the 1960 s. Registry of cases formed ~1980. No geographic association. Majority of patients are male (57/66 cases). Elderly (median age 68y). Usually associated with MDS. BLOOD, 15 JANUARY 2005 VOLUME 105, NUMBER

20 Typically hypochromic microcytic red cell indices. Dimorphic blood film. In the past, many patients were identified as having ATMDS when a reticulocyte count was performed in the investigation of the anaemia the brilliant cresyl blue staining demonstrated the HbH bodies!

21 Markedly reduced alpha:beta globin synthesis ratio Ratio in ATMDS is 0.2 (N 1.0) Suggests a global reduction in alpha globin synthesis Mutations identified in the ATRX gene, which is located on the X cheomosome. ATRX is involved in chromatin modulation One of the mechanisms may be that loss of ATRX results in increased mh2a1 levels at telomeres, as well as at the a-globin locus in erythroleukemic cells, concomitant with reduced transcription of the HBA genes.

22 Screen for Common Alpha Deletions Normal ATRX Sequencing

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26 Case 3 VG, 50y F Referred by GP for investigation of anaemia -?MDS Hb 82 g/l RCC 2.85 x10 12 /L MCV 108 fl MCH 28.7 pg

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30 Subsequent history: Family history of haemoglobin disorder Previous splenectomy HB F 2.0% Hb A2 0.8%

31 PCR testing for the common alpha gene deletions Normal. Alpha-1 Globin Sequencing Normal Alpha-2 Globin Sequencing NG_ (HBA2):[c.179G>A p.60gly>asp] + [c.427t>c, p.stop>gln] Hb Adana Hb Constant Spring

32 HBA2: c.427t>c, p.stop>gln Hb Constant Spring Mechanisms of reduced levels: Destabilisation of the mrna Failure to bind to AHSP Precipitation of the elongated globin chain on the red cell membrane facilitated by hydrophobic residues in the 31 aa extension

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34 HBA2:c.179G>A p.60gly>asp Hb Adana The Hb Adana mutation is located at amino acid 59 of the mature protein. This is adjacent to the distal histidine in the haem pocket. Its β chain counterpart is Hb J-Calabria β64gly Asp, which is known to be mildly unstable. It may affect the stabilisation of alpha globin chains by AHSP. It may also affect the stability of the haem pocket itself. Alpha: His87, His58 Beta: His92, His63

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36 Case 4 27 y female?middle Eastern FBC: Hb 110 g/l MCV 68 fl MCH 21 pg RCC 5.2 x10 12 /l HbA % HbF 1.2 % 32 y male?middle Eastern FBC: Hb 143 g/l MCV 68 fl MCH 22 pg RCC 6.6 x10 12 /l HbA % HbF 0.1 %

37 Female multiplex Normal MLPA Normal HBA1 Normal HBA2:c.178G>C,p.Gly60Arg Hb Zurich-Albisrieden Male multiplex Normal MLPA Not done HBA1 Normal HBA2:c.178G>C,p.Gly60Arg Hb Zurich-Albisrieden Fetal death at 26 weeks Fetus: HBA2:c.[178G>C, p.gly60arg] + [178G>C, p.gly60arg] T / T

38 Hydrops Fetalis associated with homozygosity for Hb Adana 3 Indonesian families (unrelated) Fetal death at 22, 21 and 25 weeks gestation Homozygous for Hb Adana HBA2:c.179G>A,p.Gly60Asp The authors postulate that the severity of the phenotype might not be due to the decreased α-globin chains synthesis but due to the variant α-globin chains interfering with normal tetramer formation. Hb Zurich-Albisrieden affects the same amino acid as Hb Adana, and the cause for Hydrops may be very similar. Hemoglobin, 34(4): , (2010)

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40 Case 5 LN, 6y F Cyanosis? Methaemoglobinaemia Hb 106 g/l RCC 3.39 x10 12 /L MCV 96 fl MCH 31 pg Abnormal peak 18.9% (Retention time 4.86mins)

41 Alpha Globin Sequencing Normal Beta Globin Sequencing NG_ (HBB):c.[277C>T,p.His93Tyr] ; [=] DNA sequencing of the beta globin gene has detected a heterozygous, single base change (C>T) in amino acid 92 (92.1) of the mature protein. This causes an amino acid change from Histidine to Tyrosine (CAC>TAC). This result is consistent with Haemoglobin M-Hyde Park, and methaemoglobinaemia.

42 Disorders of Oxidised Haemoglobin Alpha: His87, His58 Beta: His92, His63 There is a low level oxidation of Fe 2+ to Fe 3+ resulting in limited methaemoglobin formation. This is caused by the intermittent discharge of oxygen as a superoxide ion (O 2- ) leaving the haem iron in the Fe 3+ state. Methaemoglobin reductases (NADH - cytochrome b5 reductase) present in the red cells ensure that the proportion of methaemoglobin normally does not exceed approximately 1% of the total haemoglobin. Blood Reviews (2005) 19, 61 68

43 Methaemoglobinaemia arises from the production of nonfunctional haemoglobin containing oxidised Fe 3+. This results in reduced oxygen supply to the tissues and manifests as cyanosis in the patient. There are three mechanisms: Mutations in the globin chains (Haemoglobin M) Deficiency of methaemoglobin reductase enzyme/s Toxin-induced oxidation of haemoglobin. Blood Reviews (2005) 19, 61 68

44 Hb M-Saskatoon and Hb M-HydePark (Milwaukee-2) Histidine is substituted by Tyrosine There is a covalent link between the phenolic moiety of Tyr and the haem iron, so that the haem iron is stabilised in the Fe 3+ form. (Presumably the same mechanism with the other Tyr substitutions) Hb M Milwaukee-1 Val 67 is substituted by Glu in the chain, a position four residues or one helical turn away from the distal histidine. In this case interaction of the C-carboxyl group of the glutamic acid with haem stabilises iron in the Fe 3+ form.

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46 Case 6 ZD, 3y M Thalassaemia Hb 62 g/l RCC 3.10 x10 12 /L MCV 68 fl MCH 20 pg

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51 HB F 93.5% Hb A2 3.3% Hb A 6.0%

52 Screen for Common Alpha Deletions 3.7 kb deletion of a single alpha globin gene. Beta Globin Sequencing NG_ (HBB):c.[92+5G>C] DNA sequencing of the beta-globin gene has detected a hemizygous, single base change (G>C) in IVS1 position 5. This mutation greatly reduces the efficiency of the donor splice site, resulting in a severe Beta (+) thalassaemia.

53 MLPA for Beta Globin: NG_ (HBG1;HBBP1;HBD;HBB):[g.(48959-?)_(81707+?)del] ; [70691G>C]. Heterozygous deletion of the gamma1, beta pseudogene1, delta and beta globin genes

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55 Gamma Delta Beta Thalassaemia G ( A ) 0 Heterozygotes: Same phenotype as Delta Beta Thalassaemia G A ( ) 0 Hypochromic microcytic indices Normal Hb A2 Elevated Hb F Hb F in thalassaemia comprises almost entirely G. Hb F levels fairly similar for all cases: 11 14%

56 Homozygotes Hb levels g/l (Thalassaemia Intermedia) Compound Heterozygous G ( A ) 0 and Thalassaemia Variable phenotype, presumably dependent on the severity of the beta thalassaemia mutation Mild to severe beta thalassaemia intermedia.

57 Acknowledgements PathWest Haematology Paula Holmes Dianne Grey Reza Ghassemifar PathWest Diagnostic Molecular Genomics John Beilby Chris Newbound Emma North Nicole Pell Maxine Dennis Laura Greenwood Michelle Jennens