hemolytic anemia due to red blood cell enzyme and membrane ... · blood cell enzyme and membrane...
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Hemolytic anemia due to red blood cell enzyme and membrane disordersmembrane disorders
BelangenverklaringBelangenverklaringIn overeenstemming met de regels van de Inspectie van de Gezondheidszorg (IGZ)
Naam: REG Schutgens
Organisatie: UMC UtrechtOrganisatie: UMC Utrecht
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BelangenverklaringBelangenverklaringIn overeenstemming met de regels van de Inspectie van de Gezondheidszorg (IGZ)
Naam: HA van Wijk
Organisatie: UMC UtrechtOrganisatie: UMC Utrecht
Ik heb geen 'potentiële' belangenverstrengeling
Type van verstrengeling / financieel belang Naam van commercieel bedrijf
Ontvangst van subsidie(s)/research ondersteuning:
Ontvangst van honoraria of adviseursfee:
Lid van een commercieel gesponsord ‘speakersbureau’:
Financiële belangen in een bedrijf (aandelen of opties):Financiële belangen in een bedrijf (aandelen of opties):
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Wetenschappelijke adviesraad:
Classification hemolytic anemia
Hereditary Acquired
Hemoglobinopathy Immune mediated
Membrane disorders
Enzyme deficiency
Micro-angiopathic
PNH
Extravascular Intravascular
Macrophages DICp g
Liver/spleen AHTR
Intrinsic Extrinsic
Hemoglobin structure
Membrane
Antibody
Infection/toxin
Metabolism
Diagnostic strategy
1. Diagnose or exclude immune mediated hemolysis
– Auto-immune hemolytic anemia (AIHA)All i h l ti i– Allo-immune hemolytic anemia
– Drug-induced
2 Hereditary or acquired?2. Hereditary or acquired?
– Family historyTime of onset– Time of onset
– Concommitant symptoms
Hereditary hemolytic anemias
• Intracorpuscular defect of the red bl d llblood cell
• Premature removal from the• Premature removal from the circulation by the spleen andmonocyte-macrophage system
• Extravascular hemolysis
Hereditary hemolytic anemias
General clinical features
• Highly variable degree of anemia (palor, fatique)• Presentation at variable age
Ch i h l i i di h l ti• Chronic hemolysis vs periodic hemolytic episodes
• Exacerbation of hemolysis during infection/stressSplenomegal• Splenomegaly
• Neonatal jaundice / intermittent jaundice• Gall stones
A l ti i• Aplastic crises• Iron overload• Leg ulcers• Failure to thrive• Generally good response to splenectomy
Case 1
• Male, 1966• Admitted 9-1984 with headache, tiredness and
abdominal pain.S l l d d i f l• Spleen enlarged and painful
• Laboratory:Hb 6 2 mmol/L– Hb 6.2 mmol/L
– Reticulocytes 280– Bilirubine 81 (direkt 16)( )– LDH 790– Haptoglobine 0.2– Directe antiglobine test (Coombs): negatief
• Family history: several members with splenomegaly
Case 1
• Hb-electroforesis: normal(PCR α thal normal)(PCR α-thal normal)
• Enzymes:– G6PD: 10,9 (nl 7.1-11.5)– HK: 1.98 (nl 1.02-1.58) – PK: 3.2 (nl 6.9-14.5)( )
• Conclusion: hereditary hemolysis due to PK-deficiencyConclusion: hereditary hemolysis due to PK deficiency
Enzymes of the red blood cell
AcetylcholinesteraseAd i d i
Lactate dehydrogenaseMonophosphoglycerate mutaseAdenosine deaminase
Adenylate kinaseAldolaseBisphosphoglycerate mutase
Monophosphoglycerate mutaseMultiple inositol polyphosphate phosphataseNADPH diaphorasePhosphofructokinaseNucleoside phosphorylaseCatalase
NADH-cytochrome b5 reductaseEnolaseGalactokinase
Nucleoside phosphorylasePhosphoglucomutase6-Phosphogluconate dehydrogenase6-Phosphogluconolactonase
Galactose-4-epimeraseGlutamate cysteine ligaseGlucose phosphate isomeraseGlucose-6-phosphate dehydrogenase
Phosphoglycerate kinasePhosphoglycolate phosphatasePhosphomannose isomerasePyrimidine-5’-nucleotidasep p y g
Gluthathione peroxidaseGluthathione reductaseGlutathione synthetaseGlutathione-S-transferase
yPyruvate kinaseRibosephosphate isomeraseSuperoxide dismutaseTransaldolaseGlutathione S transferase
Glyceraldehyde 3-phosphate dehydrogenaseHexokinase
TransketolaseTriosephosphate isomerase
...causing hematological disease
AcetylcholinesteraseAd i d i
Lactate dehydrogenaseMonophosphoglycerate mutaseAdenosine deaminase
Adenylate kinaseAldolaseBisphosphoglycerate mutase
Monophosphoglycerate mutaseMultiple inositol polyphosphate phosphataseNADPH diaphorasePhosphofructokinaseNucleoside phosphorylaseCatalase
NADH-cytochrome b5 reductaseEnolaseGalactokinase
Nucleoside phosphorylasePhosphoglucomutase6-Phosphogluconate dehydrogenase6-Phosphogluconolactonase
Galactose-4-epimeraseGlutamate cysteine ligaseGlucose phosphate isomeraseGlucose-6-phosphate dehydrogenase
Phosphoglycerate kinasePhosphoglycolate phosphatasePhosphomannose isomerasePyrimidine-5’-nucleotidasep p y g
Gluthathione peroxidaseGluthathione reductaseGlutathione synthetaseGlutathione-S-transferase
yPyruvate kinaseRibosephosphate isomeraseSuperoxide dismutaseTransaldolaseGlutathione S transferase
Glyceraldehyde 3-phosphate dehydrogenaseHexokinase
TransketolaseTriosephosphate isomerase
Associated with decreased red blood cell survival – hemolysis
Red cell enzyme disorders disturb cellular metabolism
Cellular metabolism:
keeps the iron of hemoglobin in the functional, ferrous (Fe2+) formmaintains intracellulair potassium (high)maintains intracellulair potassium (high), sodium and calcium (low) levelskeeps sulhydryl groups of red cell p y y g penzymes, hemoglobin, and membranes in the active, reduced formsmaintains the red cell’s biconcave shapemaintains the red cell s biconcave shape
Red blood cell metabolism
Lack of energy shortens the red blood cell’s life-span
Energy deprivationEnergy deprivation
Metabolic depletion/accumulation
Metabolic depletion/accumulation
Altered shape / Altered shape / deformabilitydeformability
Premature removal from the circulation(RES)
Premature removal from the circulation(RES)
Hereditary Nonspherocytic Hemolytic Anaemia
Non-hematological manifestations
Hereditary red cell enzymopathies
I E d fi i i f l l i d l id b liI. Enzyme deficiencies of glycolysis and nucleotide metabolism
ATPATP(adenosine triphosphate)
Hereditary red cell enzymopathies
I E d fi i i f l l i d l id b liI. Enzyme deficiencies of glycolysis and nucleotide metabolism
Continuously impaired ATP maintenance: chronic HNSHA
Associated deficiencies: Hexokinase (HK)
Continuously impaired ATP maintenance: chronic HNSHAExacerbated by infection, physiological stress
Associated deficiencies:- Hexokinase (HK)- Glucosephosphate isomerase (GPI)- Phosphofructokinase (PFK)- Aldolase (ALD)( )- Phosphoglycerate kinase (PGK)- Triosephosphate isomerase (TPI)- Pyruvate kinase (PK)
Pyrimidine 5’ nucleotidase (P5N)- Pyrimidine 5 -nucleotidase (P5N)- Adenylate kinase (AK)- Adenosine deaminase (ADA)
Autosomal recessive disorders, except forPGK deficiency (X-linked) and ADA hyperactivity (AD)
Hereditary red cell enzymopathies
II. Enzyme deficiencies of the hexose monophosphate shuntand glutathione metabolism
Reduced glutathione(GSH)( )
(ϒ-glutamyl-cysteinyl-glycine)
Hereditary red cell enzymopathies
II E d fi i i f h h h h h dII. Enzyme deficiencies of the hexose monophosphate shunt and glutathione metabolism
Inadequate levels of reduced glutathione: acute HNSHA
Associated deficiencies:
Inadequate levels of reduced glutathione: acute HNSHAInduced by oxidant drugs, food (favism), infection, physiological stress
Associated deficiencies:
- Glucose-6-phosphate dehydrogenase (G6PD)- Glutathione reductase (GR)- Glutamate-cysteine ligase (GCL)- Glutathione synthetase (GSH-S)
Autosomal recessive disorders, except for G6PD deficiency (X-linked)
Laboratory diagnosis of red cell enzymopathies
Demonstration of the specific enzyme defect p yby measuring red blood cell enzyme activities
In general, little correlation between residual enzyme activity and clinical expression
Pitfalls: i.e. reticulocytosis, neonatal RBCs
DNA analysis
Case 1: case solved?
• PKLR analysis: heterozygous mutation (5’-splice sitePKLR analysis: heterozygous mutation (5 splice site intron 11)
– PK deficiency is an autosomal recessive disease– We would not expect severe hemolysis
Did i hi ?– Did we miss something?
Laboratory diagnosis of red cell membrane disorders
1. Screening tests- Morphology- Reti’s ↑- DAT neg- Direct bilirubin ↑- Osmotic Fragility (OF)
Case 1: abnormal osmotic fragilityCase 1: abnormal osmotic fragility
Laboratory diagnosis of red cell membrane disorders
2 Specialized tests2. Specialized tests- Spectrin (RIA)- EMA (Eosine-5-maleimide); binds Lys430 of Band 3, Rh(AG), CD47(HS: sensitivity 93% specificity 99%)(HS: sensitivity 93%, specificity 99%)
Case 1: Spectrin: 100% (reference range 85-115%) EMA: 40% relative fluorescence (reference >80%)( )
Red blood cell membrane disorder
Membrane disorders
Integral proteins
Anchoring proteins
Cytoskeletal proteins
Salomao et al. PNAS 2008
A d fi i d f ti i f th b t i kA deficiency or dysfunction in any one of these membrane proteins can weaken or destabilize the membrane, ultimately resulting in reduced life span (hemolysis)
Membrane disorders
Red blood cell membrane disorders are characterized by altered d bl d ll h lred blood cell morphology
Integral membrane defectsHereditary Elliptocytosis (HE)Hereditary Pyropoikilocytosis (HPP)Hereditary Pyropoikilocytosis (HPP)Hereditary Spherocytosis (HS)
“Ch l thi ”“Channelopathies”Hereditary Stomatocytosis (DHSt, OSt, pseudohyperkalemia, crohydrocytosis)Southeast Asian Ovalocytosis (SAO)
Disturbed vertical interactions lead to spherocytosis
onl i
nter
actio
Vert
ical
Disturbed vertical interactions, i.e. disturbed anchoring and membrane h i h dit h t icohesion, cause hereditary spherocytosis
Disturbed horizontal interactions lead to elliptocytosis and pyropoikilocytosis
Major protein: spectrin(heterodimer, α and β subunits)( , β )
Association protein 4.1R:junction with actin network
Horizontal interaction
Disturbed horizontal interactions cause hereditary elliptocytosis (HE) y p y ( )and hereditary pyropoikilocytosis (HPP)
Channelopathies: Hereditary stomatocytosis
Channelopathies: abnormal cation permeability and cellular hydration
Hereditar eroc tosis/deh dratedHereditary xerocytosis/dehydrated stomatocytosis (PIEZO1)Overhydrated stomatocytosis (RHAG)Cryohydrocytosis (SLC4A1)Cryohydrocytosis (SLC4A1)
Rare, dominantly inherited disorder
Mild to severe hemolysis
Pseudohyperkalemia, MCHC may be i d
Numerous stomatocytes
increased
Increased risk of thrombotic complications after splenectomy!complications after splenectomy!
Laboratory diagnosis of red cell membrane disorders
2 Specialized tests2. Specialized tests- Spectrin (RIA)- EMA (Eosine-5-maleimide) binds Lys430 of Band 3, Rh(AG), CD47(HS: sensitivity 93% specificity 99%)(HS: sensitivity 93%, specificity 99%)
Case 1: Spectrin: 100% (reference range 85-115%) EMA: 40% relative fluorescence (reference >80%)
Band 3 deficiency?Band 3 deficiency?
Case 1 – SLC4A1 analysis
exon 8, partial DNA sequence
Heterozygous 1bp-deletionc.616 620delG (p.Gly206fs)_ (p y )
Premature stop at codon 206:Band 3 Elst
Case 1 – family screening
* * *
AH‐V (1956)
LV (1941)
DvD‐V (1950)
DV (1952)
LV (1943)
MV‐B (1944)
HB (1943)
BB (1946)
* * *
(1956) (1941) (1950) (1952) (1943) (1944) (1943)(1946)
***
DV (1966)
SB (1988)
AS (1978)
HV (1969)
MM‐vdV (1976)
DJV (1986)
PK 1618_IVS11+1delGBand 3, delG, 9125‐9129 LV
(1999)CV
(1996)
New developments in the diagnosis of red blood cell membrane disorders
Osmotic gradient ektacytometryOsmotic gradient ektacytometry
Laser-assisted Optical Rotational Cell Analyzer (LoRRca)
Osmotic gradient ektacytometry
EI max (mean surface area)
EI hyper
AUC
EI
EI min
O Hyper (hydration state)O (EI max)
O Min (surface area-to-volume ratio)
Osmotic gradient ektacytometry
0.8• Increased Omin• Decreased IE max
(Hereditary) spherocytosis
0.6
)
• Decreased Ohyper
0.4Langermans day 2
Langermans
on In
dex
(EI)
Patient
Patient
0.2Normal
Control
Elon
gatio
100 200 300 400 500 6000.0
Osmolarit (mOsm/kg)
-0.2
Osmolarity (mOsm/kg)
Osmotic gradient ektacytometry
0.8 Dehydrated stomatocytosis / Hereditary xerocytosis
0.6Normal
Control A0.4
Michelsen H.
Control A
Control B
on In
dex
(EI)
Patient
0.2
Elon
gatio
• Decreased O min• Normal IE max
100 200 300 400 500 6000.0
Osmolarity (mOsm/kg)
Normal IE max• Decreased O hyper
-0.2
Osmolarity (mOsm/kg)
Osmotic gradient ektacytometry
Neonatal jaundiceNo clinical symptoms, no icterus.Mother heterozygous SLC4A1 c.616-620_delG mutation, icterusChild also affected?
Osmotic gradient ektacytometry
0.6
Normal Area
Patient 2Control
0.4
dex
(EI) O min increased
IE max normal
0.2Elon
gatio
n In
d IE max normalO hyper (low) normal
100 200 300 400 500 6000.0
Osmolarity (mOsm/kg)Osmolarity (mOsm/kg)
(mild) spherocytosis
Heterozygous SLC4A1 c.616-620_delG (p.Gly206fs)
Next Generation Sequencing (NGS)
Next Generation Sequencing (NGS) in red
no 95% of mutations are unique yes to confirm recessive HS
blood cell membrane disordersno 95% of mutations are unique yes to confirm recessive HSno technically challenging yes prenatal and perinatal diagnosisno expensive yes transfused patientsno clinical management unaffected (?) yes genotype–phenotype correlationno clinical management unaffected (?) yes genotype phenotype correlation
P t b 2012Per september 2012 NGS-based analysis for: SPTA1SPTBSPTBANK1SLC4A1EPB41EPB41EBP42RHAG
Next Generation Sequencing (NGS) in red
33 patients studiedFamily no. Sex d.o.b.
Inheritance
Mutated gene genotype LELY
HA027 F 05 09 1996 AD? SPTA1 4453C>T ( L 1485Ph ) h
blood cell membrane disorders
Molecular diagnosis in 27/33 (85%) of patients analyzed
HA027 F 05‐09‐1996 AD? SPTA1 c.4453C>T (p.Leu1485Phe) homozygousHA030 M 20‐07‐1959 AD? SPTB entire gene deletion heterozygousHA074 F 17‐08‐1998 AR SPTA1 c.83G>A (p.Arg28His) heterozygousHA083 F 03‐08‐1999 AD ‐ normal normalHA100 M 08‐06‐2000 AR ‐ normal normalHA105‐1 F 09‐07‐2003 AR ANK1 c.5201_5202insTCAG (p.Thr1734fs) normalHA105‐2 F 09‐07‐2003 AR ANK1 c.5201_5202insTCAG (p.Thr1734fs) normalHA108 F 05 06 1988 AD RHAG 808G A ( V l270Il ) l
29 unique mutations
26 l i
HA108 F 05‐06‐1988 AD RHAG c.808G>A (p.Val270Ile) normalSPTB c.3233C>T (p.Ser1078Phe)
HA109‐1 F 09‐06‐1996 AR ‐ normal normalHA111 M 08‐02‐2007 unknown SPTA1 c.101G>C (p.Arg34Pro) heterozygousHA115‐1 M 04‐07‐1967 AD ANK1 c.971T>C (p.Leu324Pro) normalHA129‐1 M 10‐07‐1976 AD SPTA1 c.678G>A (splicing) homozygousHA134 F 18‐04‐1963 AD SPTA1 c.83G>A (p.Arg28His) heterozygous
26 novel mutations
(Only) one mutation identified in 3 l t d f ili (SPTA 83G>A)
HA135 M 02‐03‐2001 unknown normal normalHA142 F 31‐08‐2003 unknown SPTA1 c.779T>C (p.Leu260Pro) heterozygousHA143 F 12‐11‐2011 unknown SPTB c.2137C>T (p.Gln713X) heterozygousHA149 M 11‐09‐2004 AD ‐ normal normalHA155 M 17‐01‐2006 AD ANK1 c.3649_3650insT (p.Val1217fs) normalHA156 M 18‐05‐1979 AD SPTA1 c.4240C>T (p.Arg1414Cys) normalHA157 F 01‐07‐1947 unknown SPTA1 c.3569+2T>C (splicing) homozygous
unrelated families (SPTA c.83G>A)
Most mutations are missense mutations in ANK1 and SPTA1
HA162 M 27‐05‐1986 AD SPTA1 c.1750C>A (p.Leu584Met) homozygousANK1 c.341C>T (p.Pro114Leu)
HA165 M 04‐07‐1965 AD SPTA1 c.1850dup (p.Glu618fs) normalc.5081G>A (p.Arg1694His)
ANK1 c.3932G>A (p.Arg1311Gln)HA170 F 07‐09‐1969 AR? SPTA1 c.5124A>T (p.Lys1708Asn) heterozygous
ANK1 c.344T>C (p.Leu115Pro) mutations in ANK1 and SPTA1
No mutations in SLC4A1 (band 3)
HA173 M 05‐10‐2012 unknown SPTA1 c.83G>A (p.Arg28His) heterozygousHA182 M 22‐03‐1954 unknown RHAG c.808G>A (p.Val270Ile) homozygousHA183 M 12‐05‐2011 AD? SPTA1 c.4339‐99C>T (LEPRA) normal
SPTA1 c.6769G>T (p.Glu2257*)HA195 F 18‐08‐1978 unknown ANK1 c.3985G>T (p.Val1329Leu) heterozygousHA201 M 07‐05‐1964 AD? SPTA1 c.1982G>A (p.Arg661His) normal
SPTA1 c.3357G>C (p.Lys1119Asn)HA202 M 16‐05‐1956 AD ANK1 c.245T>C (p.Leu82Pro) normalHA202‐1 V 29‐12‐1997 AD ANK1 c.245T>C (p.Leu82Pro) normalHA215 F 12‐02‐1965 unknown ANK1 c.4105‐1G>A (splicing) normalHA227 F 24‐09‐2008 unknown SPTB SPTB exon 5_8dup normalHA229 M 15‐05‐1989 unknown SPTA1 homozygous c.6843‐2A>G (splicing) normal
Next Generation Sequencing (NGS) in red blood cell membrane disorders
Feasible and reliable diagnostic method to detectFeasible and reliable diagnostic method to detect mutations in patients affected by various disorders of the RBC membrane
Particularly important in young children with congenital anemia, t f i d d t ti t d i f ili ith i bl li i ltransfusion-dependent patients, and in families with variable clinical expression or complex inheritance patterns
Understanding the molecular mechanisms involved in disturbed RBC membrane function will contribute to a better understanding of normal RBC physiologynormal RBC physiology
Genotype-phenotype correlation in red blood cell membrane yp p ypdisorders
Frequentie of red blood cell enzymopathies
Red cell enzyme disorder No. of reported cases
Glucose 6 phosphate dehydrogenase >400 000 000 (class I: >60 families)Glucose-6-phosphate dehydrogenase deficiency
>400,000,000 (class I: >60 families)
Pyruvate kinase deficiency >500 families, >200 mutations
Pyrimidine-5’-nucleotidase deficiency >60 families 26 mutationsPyrimidine 5 nucleotidase deficiency >60 families, 26 mutations
Glucose-6-phosphate isomerase deficiency >50 families, 31 mutations
Glutathione synthetase deficiency >50 families, 32 mutations
T i h h t i d fi i 50 100 18 t tiTriosephosphate isomerase deficiency 50 – 100 cases, 18 mutations
Phosphofructokinase deficiency 50 – 100 cases, 17 mutations
Phosphoglycerate kinase deficiency 40 cases, 19 mutations
Hexokinase deficiency 20 cases, 5 mutations
Adenylate kinase deficiency 12 families, 7 mutations
Glutamate cysteine ligase deficiency 12 families, 6 mutations
Aldolase deficiency 6 cases, 4 mutations
Glutathione reductase deficiency 2 families, 3 mutations
Adenosine deaminase hyperactivity 3 families, no mutationsyp y
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