cation permeability of the red cell membrane in health and disease€¦ · · 2014-10-27cation...

Joanna Flatt Bristol Institute for Transfusion

Sciences NHS Blood and Transplant

Cation Permeability of the Red Cell Membrane in

Health and Disease

Cation permeability of the red cell membrane in health When cation gradients go wrong... Inherited disorders affecting red cell membrane cation permeability

• The Hereditary Stomatocytoses

Characteristics and causes of increased cation permeability in HSt

Potential implications for blood transfusion services

Talk Outline

Proteins in the red cell membrane

The red cell membrane consists of lipids and proteins

Highly effective barrier between the cell’s cytoplasm and the plasma

Membrane proteins form tightly-packed complexes within the lipid bilayer

Membrane domains of some proteins are involved in the transport of molecules

Proteins form vertical interactions with the cytoskeleton Maintain red cell shape Regulate cellular processes (energy production) Bind haemoglobin

Volume control in normal red cells

When cation gradients go wrong...

Environmental factors • Drugs • Modification of the membrane or its proteins

Hypotonic solution Water moves into the cell

cell swells/lyses

Hypertonic solution Water moves out of the cell

cell dehydrates

What can affect the red cell cation gradients?

Bessis M: Blood Smears Reinterpreted. 1977. Springer-Verlag.

Rozenberg G. Microscopic Haematology: A practical guide for the laboratory. Third Edition 2010. Elsevier.

Genetically inherited conditions • Sickle cell disease • Thalassaemia • Hereditary stomatocytosis

The Hereditary Stomatocytoses - a spectrum of leakiness

Normal red cells; minimal cation leak

Familial Pseudohyperkalaemia; very mild cation leak

Dehydrated Stomatocytosis; mild cation leak

Cryohydrocytosis; cold-induced cation leak

Stomatin-deficient cryohydrocytosis; large cold-induced cation leak

Overhydrated stomatocytosis; very large cation leak

Normal blood film

Stomatocytosis blood film

Rozenberg G. Microscopic Haematology: A practical guide for the laboratory. Third Edition 2010. Elsevier, Chatswood NSW

2067, Australia.

Overhydrated Stomatocytosis

• Very rare - only 7 pedigrees to date

• Cation leak is 40 times greater than normal at 37°C

• Macrocytosis and stomatocytes

• Moderate haemolytic anaemia

• Caused by mutations in RhAG

• Mature OHSt red cells are deficient in the membrane protein stomatin (band 7.2b)

Bruce et al. Blood. 2009;113(6):1350-7

Rh-associated Glycoprotein Novel heterozygous mutations identified in RHAG in Overhydrated stomatocytosis:

- Point mutation (tg) Ile61Arg - Point mutation (tc) Phe65Ser

Bruce et al. Blood. 2009;113(6):1350-7








Normal blood film



2067, Australia.

Stomatin-deficient Cryohydrocytosis

Bawazir et al. J Clin Endocrinol Metab. 2012; 97(6):E987-93

• Extremely rare - only 3 cases to date

• Cation leak 10 times greater than normal at 37°C, increases upon cooling

• Severe pleiotropic phenotype – Seizures – Movement disorder – Developmental delay – Cataracts

• Caused by mutations in Glucose Transporter 1

• Mature red cells are deficient in the membrane protein stomatin

Novel heterozygous mutation identified in SLC2A1 in each case of stomatin-deficient cryohydrocytosis:

- Point mutation (ga) Gly286Asp - 3 nucleotide deletion (atc) Ile435 or 436

Glucose Transporter 1

Flatt et al. Blood. 2011;118(19):5267-77

Mutant GLUT1 does not transport glucose

deox

y-gl

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e up

take

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ur)

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H2O wild type G286D ∆I435 WT/G286D

WT/∆I435

Normal (Wild Type) and mutant GLUT1 proteins were expressed in Xenopus laevis oocytes, then the rate of deoxy-D-glucose influx was measured

Flatt et al. Blood. 2011;118(19):5267-77

Mutant GLUT1 is present in the red cell membrane

Dr. Lesley Bruce

Glut1DS(A)

C1 C1 P1 P1 C2 C2

sdCHC(G286D)

C1 C1 P P C2 C2

Glut1DS(B)

C1 C1 P2 P2 C2 C2

sdCHC Glut1DS (A) Glut1DS (B)

C1 P P C1 C1 P1 P1 C1 C1 P2 P2 C1

Protein 4.2

GLUT1

Stomatin

Flatt et al. Blood. 2011;118(19):5267-77

Mutant GLUT1 is expressed in sdCHC but not Glut1DS red cells

Is unable to transport glucose, resulting in similar neurological condition

Reduction in stomatin expression is not seen in Glut1DS

Stomatin

Small (32 kDa) membrane protein

Self-associates to form scaffold at membrane

Modulates other proteins’ transport activity - binds GLUT1 and changes its transport substrate from glucose to dehydroascorbic acid - also binds Band 3 and aquaporin-1 (water channel)

Stomatin deficiency is shared by OHSt and sdCHC

Both conditions exhibit the most severe red cell cation leaks

In all cases the stomatin gene is normal and intact

Kadurin et al. Biochem J. 2009; 418: 587-594

When is stomatin lost from OHSt and sdCHC cells?

CD34+ stem cells were isolated from normal control and patient blood samples

Cultured in a 3 stage medium for 21 days

Proliferation

Overall expansion of cell numbers is ≥ 104 fold

Day 6

Proerythroblast

Terminal differentiation

Enucleation rates can be up to 95%

Day 8

Basophilic normoblast

Day 10

Polychromatic normoblast

Day 13

Orthochromatic normoblast

Enucleating reticulocyte

Reticulocyte


Erythroblasts merge + phase contrast stomatin GLUT1 merge

sdC

HC

co

ntro

l O

HSt

(A)

OH

St (B

)

Stomatin is largely confined in intracellular vesicles in OHSt cells


Reticulocytes merge + phase contrast stomatin GLUT1 merge

sdC

HC

co

ntro

l O

HSt

(B)

OH

St (A

) By reticulocyte stage stomatin can be seen at the plasma membrane in OHSt and sdCHC

Also present in intracellular vesicles

A small number of circulating red cells are stomatin-positive in both conditions

Red cells merge + phase contrast stomatin GLUT1 merge

sdC

HC

co

ntro

l O

HSt

(A)

OH

St (B

)









Normal blood film



2067, Australia.

Cryohydrocytosis

• Rare

• Cation leak 2-4 times greater than normal, increases at lower temperature

• Stomatocytes on blood film

• Haemolytic anaemia

• Cation leak is extremely similar to that observed in South East Asian Ovalocytosis

• Associated with heterozygous mutations in Band 3 Bruce et al. Nat Genet. 2005; 37(11):1258-63

Guizouarn et al. Br J Haematol. 2011;152(5):655-64

Band 3

South-East Asian Ovalocytosis

Reproduced from: Traveldoctor.co.uk

Rozenberg G. Microscopic Haematology: A practical guide for the laboratory. Third Edition 2010. Elsevier, Chatswood NSW 2067, Australia.

Red cell morphology is characteristic ovalocyte

Protects against malaria parasite invasion

High prevalence in Malaysia, The Philippines, Papua New Guinea

Allele frequency of 5 - 20% in certain populations

Heterozygous, dominant

Cation leak is indistinguishable from cryohydrocytosis

Cells leak more at refrigerated temperatures

Impact on blood services?








Normal blood film



2067, Australia.

Dehydrated Hereditary Stomatocytosis (Hereditary Xerocytosis)

• Numerous pedigrees reported

• Cation leak 1-4 times normal

• Clinical presentation is variable

• Red cell morphology may include target cells, stomatocytes, echinocytes, schistocytes

• Cases may also present with perinatal oedema, increased risk of thrombosis and iron overload

• Associated with mutations in PIEZO1, the gene coding for piezo1, mechanosensitive cation channel Zarychanski et al. Blood. 2012;4:1884

• Dehydrated Hereditary Stomatocytosis mutations lead to slower deactivation of PIEZO1 channels

Albuisson et al. Nat Commun. 2013;120(9):1908-15

Syfuss et al. Clin Lab Haematol. 2006; 28(4):270-4

PIEZO1

Adapted from: Zarychanski et al. Blood 2012; 120(9): 1908-15

R2488Q

E2496ELE

R1358P

T2127M

A2020T A2003D

S1117L

G718S

G782S R808Q








Normal blood film



2067, Australia.

Familial Pseudohyperkalaemia

• Most common form of Hereditary Stomatocytosis

• Very mild cation leak, with variable temperature-dependence

• Normal red cell morphology, rare stomatocytes

• Usually asymptomatic

• Shows phenotypic overlap with dehydrated hereditary stomatocytosis

• Red cells lose potassium more rapidly than normal when blood is drawn and stored at temperatures less than 37°C

• Associated with heterozygous mutations in ABCB6, recently identified as the carrier of the Langereis blood group antigen

Andolfo et al. Am J Hematol. 2013; 88(1):66-72

Gore et al. Br J Haematol. 2004;125(4):521-7

ABCB6 - Point mutation: Arg723Gln - Point mutation: Arg375Gln - Point mutation: Arg375Trp

Increased red cell cation permeability Associated with mutations in large multi-transmembrane span proteins The misfolded proteins are expressed in the red cell membrane Haematological phenotype may be accompanied by symptoms in other tissues

South-East Asian Ovalocytosis represents a significant global population of cation-leaky red cells Familial Pseudohyperkalaemia is the most common form of hereditary stomatocytosis in European populations

The Hereditary Stomatocytoses

Why is the type of mutation important in familial pseudohyperkalaemia?

Polymorphic Rare Rare

010203040

Oua

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K in

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cel

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)

0.00

0.02

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0.14FP-Chiswick Control

Temperature °C

010203040

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mol

/L c

ells

/h)

0.00

0.02

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0.14FP-Edinburgh Control

Temperature °C 010203040

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cel

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0.00

0.02

0.04

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0.14FP-Cardiff Control

Temperature °C

Arg723Gln Arg375Gln/Trp PIEZO1 mutation

Database European African American

(allele frequency) (allele frequency)

NHLBI Exome Sequencing project 9:8591 2:4404

1000 genomes project 1:757

UK 10K project 10:7500

CSAgilent ClinSeq project 2:1027

Total 22:17,875 (0.1%)

How common is FP-Cardiff (Arg723Gln)?

Interrogation of available databates suggested an allele frequency of 1:1000 (1 in 500 people) in the European population

SNP Genotype Number Genotype Number

Arg375Gln G:G 8708 G:A 1

Arg375Trp C:C 8736 T:C 1

Arg723Gln C:C 8712 T:C 17

Arg723Trp G:G 8712 G:A 0

A screen of the NIHR Cambridge BioResource (NHSBT blood donors) was consistent with this allele frequency

Case 1. • Male donor from Cumbria, previously donated 60 units

• The unit, day-6 post-donation, not irradiated, was used to prime a bypass machine for a neonate due to undergo cardiac bypass.

• The perfusionist preparing the circuit measured sodium and potassium in a blood gas analyser - supernatant potassium was unacceptably high.

• The unused part of the unit had a supernatant potassium of 33.9 mM.

• Minimal haemolysis.

Case 2. • Male donor from London, previously donated 44 units

• The unit, day-5 post-donation, not irradiated, was used to prime a bypass machine for cardiac surgery of four month old infant.

• A blood gas measurement was taken from the bypass circuit before attaching the patient and the potassium was 13.76 mM.

• The unused part of the unit had a supernatant potassium of 41.4 mM.

• There was no evidence of excess haemolysis.

Are there active donors carrying FP-Cardiff (Arg723Gln)?

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 420

10

20

30

40

50

Supernatant potassium mmol/L

Case 2Day 5

41mmol/L

Case 1Day 6

33mmol/L

Median(14.0)

2.5%(6.1)

97.5%(31.8)

Mean(15.5)

Num

ber

of u

nits

Distribution of potassium concentration in red cell units on day 7 of storage (data collected by the Components Development Laboratory, NHSBT)

Bawazir et al. Transfusion. 2014; doi: 10.1111/trf.12757. [Epub ahead of print]

Time (hours)0 10 20 30 40 50 60 70 80

K in

pla

sma

(mm

ol/L

)

4

6

8

10

12

14

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18

20

22Control-RTDonor-RTControl-4°C Donor-4°C

Assessing the potassium leak in FP-Cardiff (Arg723Gln)

Blood samples from the FP donor showed enhanced loss of potassium when stored at cold temperatures

Bawazir et al. Transfusion. 2014; doi: 10.1111/trf.12757. [Epub ahead of print]

Assessing the potassium leak in FP-Cardiff (Arg723Gln)

Potassium in storage solution in blood packs

The increased leak over time was confirmed in FP blood units stored for 35 days at 4°C

0

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Day of storage

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Control 1Control 2Control 3FP1FP2

“Fresh” (< 5 days old) FP units already show high concentrations of supernatant potassium

Final Summary

Cation distributions are important for the volume regulation of the red cell Inherited disorders affecting red cell membrane cation permeability often result in a clinical phenotype - precluding blood donation South-East Asian Ovalocytosis represents the cation-leaky condition with potentially the largest affected population

• Significant cation leak at 37°C and more so at 4°C • Unknown impact on banked blood quality in South-East Asia

FP-Cardiff (ABCB6 - Arg723Gln) individuals may become blood donors

• Asymptomatic • Minimal red cell cation leak at 37°C, significant at 4°C • Relatively common allele frequency

High potassium in blood units is a concern for certain patient groups Work is ongoing to further characterise the effect of the leak on stored blood

This work was supported by the Department of Health, England

Bristol Institute for Transfusion Sciences NHS Blood & Transplant Lesley J Bruce Waleed M Bawazir

Components Development Laboratory NHS Blood and Transplant Rebecca A Cardigan CDL staff

NHS Blood and Transplant Catherine Chapman Lizanne Page Helen New

Newcastle Upon Tyne Hospitals NHS Foundation Trust Jonathan Wallis

The Cambridge BioResource Staff and donors

University College London Gordon W Stewart

APHP, Service d’hématologie biologique

Hôpital Bicêtre, France Jean Delaunay

San Rafael Medical Center

California, USA Bari Levinson

Hôpital Saint-Eloi Grenoble, France

Phillipe Quittet Patricia Aguilar-Martinez

cation permeability of the red cell membrane in health and disease€¦ · · 2014-10-27cation...

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