Hemolytic anemia due to red blood cell enzyme and membrane ... · blood cell enzyme and membrane...

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)


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)


II. Enzyme deficiencies of the hexose monophosphate shuntand glutathione metabolism

Reduced glutathione(GSH)( )

(ϒ-glutamyl-cysteinyl-glycine)


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


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!


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)


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)


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


Normal IE max• Decreased O hyper

-0.2



Neonatal jaundiceNo clinical symptoms, no icterus.Mother heterozygous SLC4A1 c.616-620_delG mutation, icterusChild also affected?


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

[email protected]

[email protected]

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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Hemolytic anemia due to red blood cell enzyme and membrane ... · blood cell enzyme and membrane...

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