The hereditary stomatocytoses and allied disorders: congenital disorders of erythrocyte membrane permeability to Na and K

BaillieÁre's Clinical Haematology Vol. 12, No. 4, pp. 707±727, 1999

6 The hereditary stomatocytoses and allied disorders: congenital disorders of erythrocyte membrane permeability to Na and K Gordon W. Stewart

MD, FRCP

Senior Lecturer in Medicine, Honorary Consultant Physician

E. Jane H. Turner

BSc

Graduate Student Department of Medicine, University College of London, Rayne Institute, University Street, London WC1E 6JJ, UK

The hereditary stomatocytoses and allied disorders are a set of dominantly inherited haemolytic anaemias in which the plasma membrane of the red cell `leaks' sodium and potassium. There are about 10 di€erent forms of these conditions, ranging from a moderately severe haemolytic anaemia to minor conditions in which the haematology is essentially normal, but where the patients present with pseudohyperkalaemia, due to leakage of K from the red cells on cooling to room temperature. Frequently misdiagnosed as atypical hereditary spherocytosis, these conditions can show marked thrombotic complications after splenectomy, which should be avoided. Laboratory studies of these conditions have drawn attention to a 32 kDa membrane protein, stomatin, which seems to act as a regulator of Na and K transport in human and animal tissues generally, but mutations in this gene do not cause these diseases. Genetic mapping in some kindreds, but not all, points to a mutation locus on chromosome 16. Key words: stomatocytosis; xerocytosis; cryohydrocytosis; haemolysis; thrombosis; splenectomy; stomatin; erythrocyte; erythrocyte membrane protein 7.2b; sodium; potassium; pseudohyperkalaemia; membrane transport.

INTRODUCTION In this review we will consider a series of dominantly inherited human red cell disorders in which increased membrane permeability (a leak) to sodium and potassium plays a key role in the pathophysiology. These conditions are conventionally collected under Sir John Dacie's title `hereditary stomatocytoses and allied disorders' (HSt).1 Another recent review on the hereditary stomatocytoses2 is available. An excellent general account of the structure and disorders of the red cell membrane has been given.3 All correspondence to: G. W. Stewart. Tel: 0171 209 6193; Fax: 0171 209 6211; e-mail: [email protected] 1521±6926/99/040707+21 $12.00/0

c 1999 Harcourt Publishers Ltd *

708 G. W. Stewart and E. J. H. Turner

The name stomatocyte was coined to describe the erythrocyte morphology in the ®rst case of this group, in which a dominantly inherited congenital haemolytic anaemia was present, similar to hereditary spherocytosis in all respects testable at the time except in the morphology. The ®lm showed a mouth- or slit-shaped cell form.4 American workers later showed that in a similar case, membrane permeability to Na and K was very dramatically abnormal5 and the link between this anaemia and abnormal ion permeability in the plasma membrane was established. Since these descriptions, stomatocytes have of course been observed in many situations3, such as liver disease, in which ionic permeability is not abnormal, and in some congenital conditions where haemolysis and stomatocytosis are ¯agrant, but where cation leaks are not increased.6 On the other hand, we now know of kindreds, allelic to frankly stomatocytic kindreds, in which there is a de®nite (albeit very mild) abnormality in cation transport across the membrane, in which stomatocytosis is clearly not present. Nevertheless, it remains the case that stomatocytosis and ion leaks are closely associated in many families in which haemolytic anaemia is inherited as an autosomal dominant, and the term hereditary stomatocytosis is a very useful diagnostic term in these cases. We will not consider here the stomatocytic conditions in which cation transport is completely normal. It remains to be seen whether this exclusion is useful, but we have never come across an overtly haemolytic leaky-red-cell condition that was not stomatocytic. But since stomatocytosis is the main diagnostic clue anyway, this argument is perhaps circular. We have come across two families that were originally described as spherocytic, but in which manifest abnormalities of potassium movements prompted measurements of intracellular Na and K levels, which made a diagnosis of leaky red cells. In both of these pedigrees, subsequent blood ®lms showed stomatocytes. It is the purpose of this chapter to review the current state of clinical and scienti®c knowledge of these leaky red cell pathologies. It is ®rst useful to consider the transport of Na and K across the normal human red cell membrane. Red cell Na and K transport In a previous article7 we reviewed the modalities of Na and K transport across the red cell membrane, and have done so again more recently.8 The red cell is like the vast majority of animal cells in that it maintains a low intracellular [Na] and high [K] by virtue of the action of the energy-consuming NaK pump, which expels Na and imports K against their respective concentration gradients. The action of the pump is set against a leak process that is imperfectly understood at the molecular level, and it is this which is increased in the conditions under review here. Other transport modalities can be identi®ed, but in the circulation these either operate simple exchange ¯uxes (loop diuretic-sensitive NaK2Cl co-transport, Na±Na exchange) or are silent (Ca-activated K channel, `Gardos').8 In the stomatocytoses, there is always an increase in the Na/K pump rate and this is interpreted as a compensatory e€ect. Technically, K in¯ux is the most simple and informative measure of transport rates. The convenient isotope 86Rb, which we have validated against actual potassium isotopes (EJ Blackstock and GW Stewart, unpublished results), is used as a tracer. At 5 mM external [K], normal cells show a total in¯ux of about 2±3 mmol/(l cell.h). The addition of ouabain, which inhibits the NaK pump, reduces this in¯ux by about half, re¯ecting the `ouabain-sensitive' fraction mediated by the NaK pump. The further addition of a loop diuretic, preferably bumetanide, which has the highest inhibitory

Hereditary stomatocytoses and allied disorders 709

potency, reduces the ¯ux still further, a fraction that is mediated by the NaK2Cl co-transport system. The residual, ouabain-plus-bumetanide resistant (obr) K in¯ux (and e‚ux) is largely mediated by a process that resembles passive di€usion in its linear dependence on incident cation, and it is within this obr fraction that the primary transport defect in the stomatocytoses lies. This obr K in¯ux has a value in normal cells of about 0.015  [K]o , such that at 5 mM it has a value of about 0.075 mmol/ (l cells.h). Since we suppose that this is passive di€usion, the K leak e‚ux (which is the K leak that matters physiologically) can be calculated according to the Ussing ¯ux ratio equation, which takes into account the di€erence in K concentration across the membrane and the membrane potential (9 mV, negative inside), to predict the K e‚ux, which will be roughly equivalent to the rate of the NaK pump at 1.5 mmol/ (l cells.h) in normal cells.9 In the analysis of these leaky cells, it is the case that the physiologically important K leak e‚ux is proportional to the obr K in¯ux. The obr fraction can contain a component of loop diuretic-resistant KCl cotransport10 but this is usually small (although it is enhanced in `young' red cells11) and is dicult to inhibit pharmacologically, although it can be dissected out by the laborious process of Cl-replacement, which is not convenient for routine use. Bernhardt and colleagues have suggested that the passive leak in red cells is mediated by a Na/K±H exchanger12, but this aspect has not been investigated in these abnormal red cells. The ¯uxes of Na are not routinely measured, for a number of reasons: the isotopes are expensive; the simply measured in¯ux contains no NaK pump component; and the obr ¯ux contains a large Na±Na exchange component of no interest. We usually assess Na permeability by incubating the cells in a non-radioactive 150 mM NaCl medium with bu€er, glucose, ouabain and bumetanide, and follow the accumulation of intracellular [Na] over a 6 hour period at 378C.13 The leaky cell usually presents a classic triad of results: a high intracellular [Na] and low [K], the [Na] result being the more signi®cant; an increased obr ¯ux, usually due to the leak; and an increased NaK pump rate, usually interpreted to be compensatory, although there is no proof and the mechanism is quite unknown. If the KCl co-transport ¯ux is increased for some reason, then, although the obr K in¯ux can be high, there is no increase in internal [Na] and the NaK pump rate is only slightly increased. CLINICAL VARIANTS A summary of the main literature on these conditions, which is based on a canon of case reports, is shown in Table 1. Di€erent kindreds can be distinguished on the basis of a series of diagnostic features, summarized in Table 2. In addition to the severity of the cation leak, which roughly correlates with the severity of the anaemia, conditions can be separated on the basis of the degree of hydration of the cells, the temperature dependence of this leak, the presence or absence of the Band 7 membrane protein stomatin, the phospholipid content of the membrane and the degree of hydration of the cell, which can be assessed by either cell water estimations, by measurement of the mean cell Hb concentration, or by osmotic gradient ektacytometry.14 By these means we can at present distinguish in the U.K. eight variants (overhydrated HSt (OHSt), dehydrated HSt (DHSt), cryohydrocytosis (CHC), cryohydrocytosis with stomatin de®ciency, familial pseudohyperkalaemia Edinburgh, familial pseudohyperkalaemia Falkirk/Chiswick (unpublished), HSt Woking (unpublished) and HSt Blackburn) and there are others in the literature, notably DHSt in association with perinatal oedema (see below).

DHSt

OHSt

Diagnosis

Philadelphia San Francisco Gottingen

Zarkowsky et al (1968)5 Mentzer et al (1978)82 Schroter et al (1981)83

Stewart et al (1996)43 Entazami et al (1996)47 Grootenboer et al (1998)48

Boston, MA Rochester, NY Chicago Boston, MA Philadelphia Kawasaki Denver Kawasaki Six families, based on Barcelona Omagh Berlin Paris

Brighton

Meadow (1967)15

Ja€e & Gottried (1968)21 Miller et al (1971)20 Honig et al (1971)86 Glader et al (1974)87 Wiley et al (1975)42 Otsuka et al (1990)88 Lane et al (1990)25 Kanzaki & Yawata (1992)89 Vives Corrons et al (1995)57

London

Location

Lock et al (1961)4

Reference

23

43

49

22, 85 43, 58

17, 49 16, 31 17, 43, 84

19, 43

19, 43, 81

Also reported in reference number:

Decreased 2,3 DPG. Major ¯ux study with 3H-ouabain binding. High PC. No bene®cial e€ect of splenectomy in these cases. Excess of membrane proteins identi®ed. Many di€erent families reported. Resistance to thermal fragmentation: possible diagnostic technique. Included in report on thrombosis; maps to 16q23-qter. First report relating perinatal oedema to DHSt. Perinatal oedema. Maps to chromosome 16.

Original report of HPCHA. No ionic assessment at this time.

First report of stomatocytosis. Word ®rst coined to describe morphology. Ion transport defect not recognized at this stage. Proposita died of post-splenectomy pulmonary hypertension, 1992. Ion transport defect ®rst recognized. First band 7 (stomatin) de®ciency report. Some improvement after splenectomy reported, although thrombotic problems developed later.

Notes

Table 1. Principal case reports and some other contributions in the stomatocytoses.

710 G. W. Stewart and E. J. H. Turner

Toronto

Canine variants Pinkerton et al (1974)39

For further references see Delaunay et al2 and Stewart.7

Brown et al (1994)40 Slappendel et al (1991)97

Philadelphia Blackburn, Lancs

Other forms Oski et al (1969)37 Coles et al (1999)35

Montpellier Cardi€ Falkirk Lille

Edinburgh

Pseudohyperkalaemia presentations Stewart et al (1979)33

Luciani et al (1980)90 Leadbetter & O'Dowd (1982)91 Meenaghan et al (1985)36 Dagher et al (1988)93

Rochester, NY San Francisco Hemel, Watford

Cryohydrocytosis Miller et al (1965)30 Mentzer & Lande (1980)31 Coles et al (1999)13

41, 98

95, 96

94

92

9, 34

16

Recessive. Associated with chondrodysplastic dwar®sm in Alaskan malamutes. Leaky cells, quite similar to OHSt. Recessive. Similar haematology to OHSt. Recessive. Not cation-leaky. Associated with hypertrophic gastritis etc.

Aged cells were the least dense on centrifugation. Shallow slope temperature abnormality, frank anaemia.

First report of red-cell-based pseudohyperkalaemia. `Shallow slope' temperature abnormality. Maps to 16q23-qter.

First report of cryohydrocytosis. Band 7 protein missing in a case of cryohydrocytosis. U-shaped temperature dependence of ion leak. Band 7 protein present in these cases.

Hereditary stomatocytoses and allied disorders 711

712 G. W. Stewart and E. J. H. Turner Table 2. Diagnostic investigation modalities in the hereditary stomatocytoses. Diagnostic feature

Note

References

Haematologic severity

Very variable. In general OHSt 4 DHSt 4 CHC 4 FP

Blood ®lm

At least some stomatocytes in all haemolytic cases

Cellular hydration

Obsolete measurement. Inversely proportional to MCHC

Osmotic fragility

Parallels cell hydration. Increase in overhydrated forms; decreased in dehydrated forms

Osmotic gradient ektacytometry

Parallels hydration. Particularly simple if apparatus available

Clark et al (1983)14

Phospholipid content

Increased PC usually associated with DHSt

Clark et al (1993)22

Presence or absence of stomatin

Absence usually associated with OHSt. Also seen in some cases of CHC

Lande et al (1982)16; Eber et al (1989)17; Stewart et al (1992)19

Temperature e€ects Pseudohyperkalaemia

Many variants Occurs in many forms

Wiley et al (1975)42; Wiley (1984)99

Never seen without pseudohyperkalaemia as well

Stewart & Ellory (1985)9; Coles et al (1999)35 Coles et al (1999)13; Miller et al (1965)30

Perinatal oedema

Variable association with DHSt

Grootenboer et al (1998)48

Density centrifugation of red cells

`Inverted' density separation seen in one family

Oski et al (1969)37

Cold lysis

OHSt, overhydrated hereditary stomatocytosis; DHSt, dehydrated HSt; CHC, cryohydrocytosis; FP, familial pseudohyperkalaemia; MCHC, mean cell haemoglobin concentration; PC, phosphatidylcholine.

Overhydrated hereditary stomatocytosis The original condition, now referred to as overhydrated hereditary stomatocytosis (OHSt) remains the most severe and dramatic but is rare (three pedigrees in the U.K.4,15, about ®ve others worldwide: see Table 1). In this condition the leak is positively torrential, with ¯ux rates 20±40 times normal, grossly abnormal intracellular Na and K levels with reversal of the normal ratio between intracellular [Na] and [K] (see Table 3). The patients are chronically jaundiced. These families show de®ciency of the integral membrane protein, stomatin, from the red cells.16,17 This protein, and its gene, looked certain to be the key to the whole molecular mystery of these conditions, but when it was puri®ed and the gene sequenced18,19 it showed an anonymous, novel protein, with no obvious functional motifs and the gene in the patients turned out to be normal. De®ciency of stomatin is also seen in a few cases of cryohydrocytosis, which will be discussed below. Genetic mapping has not so far been applicable to available pedigrees, which are all small. The stomatin protein will be discussed further below.

5

4

1

1

1

DHSt (Omagh)

CHC (Watford)

FP Edinburgh

Woking

Blackburn

15

14

12.3

N

13.8

10±12

9.2

51

5±8

5.8

N

5±6

6±12

22

84±96

101

105

N

107

94±108

128

MCV ( ¯)

31.6

32.6

36.6

37.4

34.6

28.6

MCHC (g/dl)

5±11

41

53

9.7±11.9

31.2

11±14

83±95

[Na]i

88±105

44

49

78±83

44.6

83±86

31±21

[K]i

86Rb

0.8±2.0

6.3

6.78

2.6

5.14

4.2±5.2

13±22

0.06±0.10

0.32

0.47

0.082

0.39

0.18±0.21

2.3±4.1

1*

5±6

5±6

1.0

5±6

2

20±40

NaK pump Obr K in¯ux mmol (leak) mmol Multiple (l cells.h) ÿ1 (l cells.h) ÿ1 of normal

K in¯ux at 5 mM [K]o

not 16

?

16

not 16

16

?

Mapping

Coles et al (1999)35

unpublished

Stewart & Ellory (1985)9; Stewart et al (1979)33

Coles et al (1999)13

Stewart et al (1996)43

Lock et al (1961)4; Meadow (1967)15

Reference

Intracellular electrolytes were measured using ¯ame photometry and K in¯ux was measured using as a tracer, as described by Coles et al (1999).35 FP, familial pseudohyperkalaemia; CHC, cryohydrocytosis; DHSt, dehydrated hereditary stomatocytosis; OHSt, overhydrated hereditary stomatocytosis; retics, reticulocyte count; obr, ouabain ‡ bumetanide-resistant; N, normal; MCV, mean cell volume; mapping, chromosome to which family maps in genetic studies. *by de®nition.

Normal

3

OHSt (Brighton)

Variant

Number Haematological of British data families known Hb (g/dl) Retics (%)

Intracellular electrolytes mmol (l cells)ÿ1

Table 3. Comparison of haematological, cation ¯ux and structural data in di€erent hereditary stomatocytosis variants.

Hereditary stomatocytoses and allied disorders 713

714 G. W. Stewart and E. J. H. Turner

Dehydrated hereditary stomatocytosis By far the commonest of the conditions is dehydrated hereditary stomatocytosis (DHSt), also known as hereditary xerocytosis. First described by Miller et al20, many pedigrees have been identi®ed (see Table 1) and we know of ®ve in the U.K. This condition is haematologically milder than OHSt and the cation ¯uxes are much less abnormal at only about 2±3 times normal. The membranes show an excess of phosphatidylcholine (PC) and it is now recognized that this condition is identical to what was ®rst described21 as high phosphatidylcholine haemolytic anaemia (HPCHA).22 The Band 7 stomatin protein is normal in these cases. Some European cases of DHSt are associated with an unusual condition of transient, self-limiting, perinatal oedema, discussed below. This complication has not been seen in other forms of HSt. All DHSt families so far tested by us map to a locus on chromosome 16q23-qter.23 Studies of cell lipids show increased proportions of phosphatidylcholine in DHSt, and Shohet has demonstrated that there is a block in the transfer of labelled acyl chains from PC to phosphatidylethanolamine and phosphatidylserine.24 It is possible that there is a complex system of lipid movements in the membrane involving a circulation process between the two lea¯ets of the bilayer and metabolic change, and that this is blocked or somehow altered in the stomatocytoses. Two di€erent groups have reported that membrane surface area is increased in this condition.25,26 In one report, intravascular haemolysis occurred after exercise27, a clinical result that was associated with increased shear sensitivity in the dehydrated erythrocytes. It has also been reported that these cells show an increased sensitivity to oxidant stress, in the form of hydrogen peroxide.28,29 Cryohydrocytosis The next most prevalent condition in the U.K. is cryohydrocytosis (CHC)13,30, but case reports of this condition in the literature are very few31 (Table 1). This is a mild anaemia with a reticulocytosis of about 5% and normal or nearly normal haemoglobin levels. The cells, if examined fresh, show mild dehydration with a high mean cell haemoglobin concentration (MCHC) (see Table 3), but if left to stand at room temperature, they begin to lose K, gain Na and become overhydrated with a low MCHC. If left in the cold, these changes become very marked indeed and overnight storage in EDTA or heparin leads to marked lysis. The e€ect of storage at low temperatures on the mean cell volume is shown in Figure 1. The mean (red cell) volume (MCV) rises and lysis occurs. Autohaemolysis at low temperatures is very strikingly high. The loss of K gives pseudohyperkalaemia, factitious hyperkalaemia due to this temperature e€ect (loss of K by membrane leakage precedes lysis).13 These patients recount tales of recall to hospital by anxious physicians after routine plasma electrolyte estimations, in which the ®rst sample has shown a potassium level of, say, 6±10 mM. A repeat with urgent analysis is normal. All of these e€ects can be attributed to the bizarre temperature dependence of the Na/K leak, which will be discussed below (see Temperature E€ects). Some cases of cryohydrocytosis are associated with stomatin de®ciency16, but no British cases show this abnormality.13,30 Cryohydrocytosis does not map to chromosome 16 (A Iolascon & P Gasparini, pers. comm.). Cryohydrocytosis cells show an unusual susceptibility to membrane phospholipid degradation by phospholipases at low temperatures32, which may re¯ect abnormal packing in the lipids at these temperatures.

Hereditary stomatocytoses and allied disorders 715

Figure 1. E€ect of cold storage on cell size in cryohydrocytosis (CHC) and control (Ctrl) cells, as determined using Sysmex technology. Cells were anti-coagulated in EDTA and kept on ice. At the times shown, the samples were passed through the counter for determination of cell volume. A gradual increase in mean cell volume, with a concomitant increase in the relative distribution width, occurred only in the cryohydrocytosis cells. We thank Professor S. Machin at the Department of Haematology, UCH, for access to this apparatus.

In spite of the relative mildness of the anaemia, it is clear that cryohydrocytosis red cells are more leaky than those in DHSt (Table 2). In general, among these conditions, the haemolysis is proportional to the magnitude of the leak, but the comparison of these cases opposes this trend.

Familial pseudohyperkalaemias (a heterogeneous group) Familial pseudohyperkalaemia is a mild condition in which both the haematology and the leak at 378C are essentially normal. These patients present via the chemical pathology service with pseudohyperkalaemia, due to the same kind of net loss of K on storage described in the section on cryohydrocytosis, above. The original Edinburgh family that we found9,33 had a mildly dehydrated condition and maps to chromosome 16 along with common DHSt.34 Isotopic ¯ux studies show the passive leak has a shallow slope variant form of temperature dependence, quite di€erent to the U-shaped form seen in cryohydrocytosis. This shallow slope abnormality was also seen in a frankly haemolytic Blackburn pedigree35, which also showed very marked pseudohyperkalaemia. Further families with this combination of virtually normal haematology and pseudohyperkalaemia have been described, also under the label familial pseudohyperkalaemia, and curiously it is clear that they are not the same as this pedigree. Meenaghan et al36 investigated a kindred in Falkirk and found a U-shaped rather than a shallow slope temperature dependence for the obr K ¯ux. In mapping investigations, we noted that after overnight storage (our unpublished results), the Falkirk red cells swell, like a mild version of cryohydrocytosis, while the red cells from the Edinburgh pedigree did not. We have a further pedigree, of Austrian extraction but now living in Chiswick, with an identical condition (PG Haines, C Crawley & GW Stewart, unpublished results). Thus even this apparently simple syndrome is heterogeneous and variants can be distinguished by temperature studies of ¯ux and cell swelling. Further cases are listed in Table 1.

716 G. W. Stewart and E. J. H. Turner

Other conditions Miscellaneous other conditions are also seen. Oski et al37 investigated a pedigree by centrifugation of the erythrocytes to delineate fractions of di€erent density. Unusually, the young, reticulocyte-rich fraction, which normally have the least density were found in this family to be the most dense and to show the least abnormality in intracellular Na and K. The more mature cells were the least dense and showed a very major abnormality in ion content, all suggesting that the cells became progressively more and more leaky as they aged in the circulation, which would be an important ®nding. We have never found a family like this in the U.K. The Woking case cited in Table 3 is unique, in that the intracellular Na and K levels and leak ¯uxes are both more abnormal than those in simple DHSt, while the temperature dependence of the ¯ux is di€erent from both that in the Blackburn kindred and that in cryohydrocytosis. A Japanese family has been reported in which dominantly inherited stomatocytosis ran through the maternal line.38 Two daughters of the a€ected mother had stomatocytosis and the Djerine±Sottas syndrome, a neuropathy attributable in this family to a point mutation in the myelin protein zero gene. In the two daughters who inherited the myelin protein zero mutation, which was thought to be due to a germline mutation mosaicism in the mother, the severity of the stomatocytosis was reportedly worse, although no ®gures were given. Myelin protein zero is not known in the red cell and this is a very curious observation indeed. No studies of either ion ¯ux or stomatin protein were reported: but this observation is reminiscent of the involvement of the homologues of the stomatin protein in neuronal function in Caenorhabditis elegans and neurological associations in dogs (see below). Stomatocytosis in dogs Stomatocytosis is reported in dogs. A simple form of stomatocytosis exists with abnormal intracellular cations39 and is associated with chondrodysplastic dwar®sm. Marked stomatocytosis with macrocytosis and increased relative distribution width, but without frank haemolysis, was found in a colony of miniature Schnauzers40, but intracellular Na and K were not reported. This was inherited as an autosomal recessive. A further congenital form of stomatocytosis is associated with hypertrophic gastritis, which resembles Menetrier's disease in man. The red cells in this canine stomatocytosis display normal cation content, decreased membrane PC content, unlike DHSt, and increased sphingomyelin. In addition, the dogs show liver disease, neurological disturbance and in some cases, polycystic renal disease. The genetic cause of this interesting syndrome, which seems to be a disorder of phospholipid metabolism, is unknown41 and is reminiscent of the non-leaky, presumably recessive case that we described in association with pseudo-homozygous hypercholesterolaemia.6 CLINICAL DIAGNOSIS The blood ®lm remains the vital clue, but it is clear that appearances can be dicult to recognize in many cases. Wiley has stressed the importance of a wet ®lm.42 All families go through stages where they are diagnosed as `atypical hereditary spherocytosis', but this is dangerous because of the potential thrombotic complications after splenectomy in DHSt and OHSt43 (see below). Variable hyperkalaemia, which can be very marked in some cases13,35 can be a useful pointer to the existence of leaky cells, but is not

Hereditary stomatocytoses and allied disorders 717

always present. Splenectomy failure in an a€ected family member can be a useful distinguishing feature from hereditary spherocytosis. Once the diagnosis is suspected, the optimum approach is to measure isotopic ¯ux rates, but a simple measure of intracellular [Na] and [K] on cells washed in cold isotonic tris-bu€ered magnesium chloride will usually be abnormal in the frankly haemolytic cases (see Table 2).

CLINICAL PROBLEMS Thrombosis after splenectomy In 1992, a British OHSt patient15 died of pulmonary hypertension secondary to multiple pulmonary emboli. The signi®cance of this event was not immediately clear but later it emerged that, worldwide, thrombotic problems were not uncommon in OHSt and DHSt. The common link in these thrombotic cases was prior splenectomy, and within pedigrees it could be seen that those who were splenectomized developed one or more of a number of thrombotic problems, including deep and super®cial venous thromboses, portal vein thrombosis, episodes of abdominal pain and arterial thromboses.43 OHSt and DHST can be considered to be examples of an e€ect ®rst described by Hirsh and Dacie, that if the spleen is removed in a congenital haemolytic anaemia and the anaemia does not respond to the procedure, then the patient can develop thrombocytosis and a tendency to venous thrombosis.44 The cause of this e€ect is not clear, although evidence has been presented that suggests that the red cells in the splenectomized individuals are more adherent to endothelial cells cultured in monolayers than normal.45 In this study, one patient was successfully treated with a chronic transfusion programme and another with pentoxifyllin. It is our recent experience that splenectomy in cryohydrocytosis is not complicated by thrombotic problems.13 This may re¯ect the fact that this is a milder anaemia, but there may be other di€erences. No patient with any of the familial pseudohyperkalaemia conditions has been splenectomized. This problem highlights the importance of accurate diagnosis. A case of alleged spherocytosis associated with pulmonary hypertension due to intra-pulmonary thrombosis46 causes concern. Studies of intracellular electrolytes in such families might point to a diagnosis of stomatocytosis and avert subsequent events in other a€ected family members. Perinatal oedema Entazami et al47 described a child born to a DHSt family in which polyhydramnios and marked fetal ascites were found antenatally. Due to the strong family history of DHSt, a prenatal diagnosis of DHSt was made by cordocentesis at 25 weeks gestation: 400 ml of ascitic ¯uid was removed. A cousin of the fetus who had a diagnosis of DHSt had also had intrauterine ascites, which had resolved a few days postpartum. An attempt was made to treat the fetal ascites by intrauterine blood transfusion, but this was without e€ect. At 33 weeks, Caesarian section was performed because of preeclampsia. Before this, 900 ml of ascites was removed and in the ®rst 6 weeks postpartum six aspirations of about 300 ml each time were made and a partial exchange transfusion was performed, again without e€ect on the ascites. The birth weight was greater than the 95th percentile of weight per gestational age. The ascites

718 G. W. Stewart and E. J. H. Turner

were minimal at 8 weeks postpartum and had spontaneously resolved after 10 weeks. By 6 months she was normal except for mild anaemia with reticulocytosis. Subsequently, Grootenboer et al48 reported a genetic syndrome associated with DHSt, pseudohyperkalaemia and fetal ascites in a French family, which was transmitted in a dominant fashion. They too found that the transfusions did not cure the oedema and that it resolved spontaneously after a few months. Spontaneous delivery occurred at 31 weeks gestation. At birth, in addition to the ascites and haemolytic anaemia, there was an enlargement of the liver and the spleen. As with the case reported by Entazami, the clinical picture was dominated by perinatal oedema. Two transfusions of packed red cells were necessary within the ®rst 10 days postpartum. Ascites (which acquired a chylous character) was drained three times. After 8 months the ascites resolved spontaneously though the haemolytic anaemia persisted and reticulocyte count rose. Similar to the case reported by Entazami there was a family history of DHSt, pseudohyperkalaemia and perinatal oedema in the mother and very possibly one of her twins which both aborted spontaneously. In the cases described, the perinatal oedema was not caused by the haemolytic anaemia since transfusions did not cause resolution of the oedema. Grootenboer speculated that the cation transport abnormality was also expressed perinatally in some cells of the lymphatic vessels. They also suggested that given the inheritance patterns, DHSt, pseudohyperkalaemia and certain forms of perinatal oedema stem from a variety of alterations within the same locus. They suggest that perinatal oedema disappears due to tissue speci®c mechanisms (such as gene silencing). Iron overload In the Denver DHSt pedigree25, problems with iron overloading were very prominent43, and although iron saturations tend to be high in many of these cases, we have not, in the U.K., come across severe iron-related damage. Pseudohyperkalaemia and pseudomacrocytosis As described in the section on cryohydrocytosis, above, some of these patients can present with spuriously high plasma [K] readings due to loss of K from red cells on storage. This applies particularly to the familial pseudohyperkalaemia and cryohydrocytosis variants. In cryohydrocytosis, the cells can swell before the blood count is performed (Figure 1) and the MCV can be reported to be high, prompting questions about the patient's ethanol intake and unnecessary measurements of serum vitamin B12 and folate. Patients (and their physicians) should be reassured that there is no abnormality in potassium handling in the body and that this is simply a bothersome test-tube e€ect. Tiredness Never investigated and purely anecdotal, these patients tend to complain of tiredness which is out of proportion to the degree of anaemia. It is possible that these red cells are inecient in their role in the respiratory cycle. Their transit through capillaries may be delayed; gas exchange may be sub-optimal; Wiley et al have reported that 2,3 diphospho-glycerate (2,3 DPG) concentrations are abnormally low49. This is presumed to be the result of increased ATP consumption by the hyperactive NaK pump, and this could modify the oxygen anity. Betticher et al50 have reported that

Hereditary stomatocytoses and allied disorders 719

when normal red cells are made stomatocytic by treatment with chlorpromazine, the red cells become sti€er and the oxygen di€using capacity in the lungs decreases by 18%, a change that they attributed to the increased viscosity of the red cells and their general reluctance to pass through narrow capillaries. Fatigue is a notoriously dicult symptom to investigate (RF Robertson, pers. comm.), but these preliminary results may form the basis for more detailed physiological studies. TEMPERATURE EFFECTS The ®rst temperature observation in the stomatocytoses was that of Miller and others, who described the cryohydrocytosis phenotype.30 To investigate a number of families with di€erent temperature-related e€ects, we have made a study of the dependence of the obr K ¯uxes in these conditions. The results are shown in Figure 2A. It is ®rst pertinent to point out that the dependence of this ¯ux on temperature in normal cells is not trivial, showing a minimum at about 108C.51 This minimum is not seen in OHSt cells, which show a simple monotonic curve. DHSt shows a curve that is essentially parallel to normal but shifted up the y-axis. Cryohydrocytosis, by complete contrast, shows a minimum at 208C, such that the ¯ux at 08C exceeds that at 378C. The familial pseudohyperkalaemia Edinburgh and HSt Blackburn cases show a shallow slope variation in which the ¯ux shows a considerably lower Q10 than normal. The Woking case shows an identical ¯ux to cryohydrocytosis and Blackburn at 378C, but has a steeper temperature dependence. Thus those cases in which pseudohyperkalaemia is most prominent (cryohydrocytosis, familial pseudohyperkalaemia, Blackburn) have the most strikingly abnormal temperature pro®le for this leak ¯ux. In all cases, the NaK pump shows a simple (and steep) monotonic temperature dependence. When the cells are cooled, the steady state is disturbed and the leak is not balanced by the pump: K loss (and Na gain) occurs, with pseudohyperkalaemia and, in cryohydrocytosis with very severe leaks, full scale lysis. Very interestingly, the U-shaped pro®le seen in cryohydrocytosis almost exactly mimics the pro®le seen when normal cells are incubated in media from which either Na has been replaced by an organic cation such as choline or N-methyl-D-glucamine (NMDG)52; or where Cl has been replaced by an organic cation, such as thiocyanate or salicylate.53 Thus it appears that these cells have some kind of genetic defect that mimics this ionic e€ect on normal cells. These temperature pro®les (and the related cellular phenomena that accompany them such as cell swelling and pseudohyperkalaemia) can be very useful diagnostic indicators. For instance, at one time we thought that the families Woking, Blackburn and Watford (cryohydrocytosis) had identical anaemias, on the basis of intracellular Na and K levels and isotopic ¯ux studies at 378C. But when the temperature e€ects were investigated, it became clear that the three kindreds are quite di€erent, a piece of information that was useful in genetic mapping. In e€orts to diagnose di€erent family members with cryohydrocytosis, we found that storage of blood overnight on ice elicited a characteristic macrocytosis (Figure 1) that was diagnostic. The ¯ux curves shown in Figure 2A show the temperature dependence of the ¯ux rate itself. The temperature dependence of this ¯ux rate is in¯uenced by the e€ect of low temperatures on the di€usion rate of the ions and is not a true re¯ection of the physical state of the membrane as a function of temperature. We get the impression that the membrane gets leaky at low temperatures, but exactly how does it change? To try to get a better representation of the dependence of permeability on temperature,

720 G. W. Stewart and E. J. H. Turner A

B 1000

Relative corrected permeability

Ouabain+bumetanide-resistant K influx (mmol/(l cells.h))

10

1

0.1

0.01 40

30

20

10

0

100

10

1

0.1

40

30

20

10

0

Temperature (oC)

Figure 2. A. Temperature e€ects on obr (leak) K in¯ux in normal red cells and di€erent stomatocytosis variants. K in¯ux was measured at 5 mM external [K] using 86Rb as a tracer. The medium contained: K, 5 mM; Na, 145 mM; Cl, 150 mM; MOPS, 15 mM (pH 7.4 at room temperature); glucose, 5 mM; and ouabain and bumetanide, both 0.1 mM. The dotted lines are designed to highlight the di€erence in slope between the two `shallow slope' variants (Blackburn, *; and familial pseudohyperkalaemia (FP) Edinburgh, *) and the normals (&). The DHSt pro®le (&) is essentially parallel to the normal pro®le, while cryohydrocytosis (CHC, ~), shows a minimum of 208C. OHSt Brighton (^) shows a monotonic pro®le with no obvious minimum. Woking (q +) shows a slightly shallow curve but is clearly di€erent from all other curves, especially CHC and Blackburn, with which it is coincident at 378C. B. Representation of relative membrane permeability in di€erent stomatocytosis pedigrees corrected for e€ects of temperature. The raw ¯ux data in A was treated as follows: Relative corrected permeability ˆ Flux…temp T†=0:08*exp…ÿEa =R…273 ‡ 37††=exp…ÿEa =RT† where Ea is the Arrhenius activation energy chosen for each pro®le, R is the gas constant and T the absolute temperature in degrees Kelvin, and 0.08 is a typical normal ¯ux for this external [K] at 378C. This gives some idea of the way in which actual membrane permeability changes with temperature rather than simply the ¯ux. The values of Ea for each pro®le are: normal (&) 65 kJ/mol; OHSt Brighton (^) 90 kJ/mol; DHSt Omagh (&) 70 kJ/mol; CHC Watford (~) 18 kJ/mol; FP Edinburgh (*) 40 kJ/mol; Blackburn (*) 24 kJ/mol; Woking (q +) 45 kJ/mol. It can be seen that the kindreds shown fall into two groups: those in which there is a marked in¯ection at 208C (normal, cryohydrocytosis and DHSt), and those in which the in¯ection is less marked (familial pseudohyperkalaemia Edinburgh, Blackburn and Woking). In spite of the monotonic pro®le in the OHSt curve, the permeability curve for this pedigree shows an upward in¯ection at about 20±238C.

we have recalculated the data from some of these kindreds in another form in Figure 2B. The data have been multiplied by a temperature-normalizing factor based on the Arrhenius equation for reaction rates: Rate ˆ Ao exp…ÿEa =RT† where Ao is a constant, R is the gas constant (8.31 J8Kÿ1molÿ1), T is the absolute temperature and Ea is the Arrhenius activation energy, a measure of the supposed

Hereditary stomatocytoses and allied disorders 721

energy barrier which the ion must surmount to traverse the membrane. From the data in Figure 2A, we can de®ne a value for Ea for each kindred over the range 37±238C (in which they are all linear) and apply this to the data (see legend to Figure 2B) to factor out the slowing e€ect on transport of cooling. If the process obeys the Arrhenius law, then the resulting plot will be ¯at, as it is in all pedigrees in the range down to 238C. Below this temperature, the pro®les tend to slope upward, re¯ecting the increase in permeability as the temperature falls. This is admittedly arbitrary in its application and without ®rm physical basis, but it does yield some striking results. It can be seen (Figure 2B) that the pedigrees considered break down into two groups: those in which the curve deviates upwards sharply (normal, cryohydrocytosis and DHSt) and those in which it is more or less ¯at (Blackburn, Woking, Edinburgh, OHSt). What is perhaps most striking is how the pro®les of normal and cryohydrocytosis, which appear so totally di€erent in the simple ¯ux data in Figure 2A, look quite similar in this plot. Both show an upward deviation that begins at about 208C, and although the cryohydrocytosis plot rises to a much higher value, it is evident that they are basically similar, in that the in¯ection in the permeability occurs at more or less the same temperature in both. The implication of this graph is that, if one was to look for a physical change in the membrane that might reveal a structural change in the membrane, this graph would predict that it would occur at the same temperature in both normal, DHSt and cryohydrocytosis, in spite of the major di€erence in the appearance of the actual ¯ux pro®les in Figure 2A. It is the greater degree of basic leakiness in the cryohydrocytosis cells which moves the ¯ux minimum up. The explanation for these e€ects is not at all clear. Since some of these can be elicited in normal cells by manipulation of the external ionic environment with a number of di€erent ionic species, a physical e€ect based on charge is suggested. The e€ects of external ions can be sen in the context of the so-called lyotropic series of ions, that ranking of ions in aqueous solution that was ®rst recognized by Hofmeister when studying the ability of di€erent ions to salt out starch molecules from solution.54 Thus small highly charged ions (H, Na, Mg) are more e€ective in such processes than larger ions with more di€use charge, such as NMDG and choline. Similarly, the anions can be ranked in this way and it is those lower in the series (thiocyanate, salicylate) that make red cells leaky at low temperatures. The rankings are related to the entropy of hydration of the relevant ion in solution: it is possible that the salting out phenomena relate to the monopolization of water molecules by the ions higher in the series. These water molecules then become unavailable for solubilization of the macromolecule, which then precipitates. (It is this e€ect which is used in the laboratory to precipitate proteins using ammonium sulphate and DNA using sodium acetate.) Some kind of parallel physical e€ect on the organization of the erythrocyte bilayer is a possible explanation for these temperature e€ects in normal cells: the abnormal cells are somehow resistant to the stabilizing e€ect of NaCl, and exposure to NMDG or thiocyanate has no further e€ect on them.13 These considerations suggest physical studies of the structure of the bilayer that might prove interesting. Perhaps most pertinent are the studies of Moore et al55, in which perdeuterated dimyristoylphosphatidylcholine was introduced into the membrane, and its conformational order was assessed using Fourier transform infrared spectroscopy, which reports the CD2 stretching vibrations in the molecules. Since only PC is deuterated, this technique selects out this species.55 By this means a cooperative phase transition was observed in the region of 208C, suciently close to the in¯ection in the curves in Figure 2B to be signi®cant. It would be most interesting to subject these pathological erythrocytes to this kind of study.

722 G. W. Stewart and E. J. H. Turner

These temperature e€ects in di€erent kindreds do not co-segregate in a simple way with the results of gene mapping (see below). It is of interest that cells showing south-east Asian ovalocytosis show a cold-injury e€ect56, but this seems to be an irreversible phenomenon in which permeability to many solutes is increased after cold storage, whereas these e€ects in the stomatocytoses are speci®c to Na and K and are reversed by return to 378C after cooling. In addition to these ¯ux e€ects, it has been observed that in DHSt, the cells are resistant to the e€ects of heating in the test tube.57 GENE MAPPING In a European collaboration, DHSt, the commonest form of these diseases, has been mapped to a locus on chromosome 16q23-qter.23 This is consistent with smaller studies aimed at exclusion of speci®c candidate genes.58,59 The Edinburgh form of familial pseudohyperkalaemia (shallow slope variant) also maps to this locus.34 The mutant gene itself is not yet known; no obvious candidate is found in this area. It is also clear that not all families map to this locus: neither the family labelled Blackburn35 (which also shows the shallow slope temperature abnormality, in this case with frank haemolysis) nor cryohydrocytosis, map to this locus (P Gasparini, A Iolascon, J Delaunay & GW Stewart, unpublished results), showing that more than one mutant gene can cause this general phenotype. THE STOMATIN PROTEIN This protein is missing from the red cells in all cases of OHSt and some cases of cryohydrocytosis, but no mutation has been found in the gene itself, which is located on chromosome 960 and is clearly not related to the mutation in the chromosome 16 gene on DHSt. Its very striking absence must almost certainly imply that it is somehow involved in the pathophysiology. Its real function remains unclear. A mouse knock-out is without obvious phenotype61, perhaps because of degeneracy in that species. The human genomic structure has been clari®ed.62,63 Our studies show that some protein is present in red cells (about 1/50 of normal), while in a liver sample from an OHSt patient, normal amounts were present (MC Chetty & GW Stewart, unpublished results) con®rming that the protein de®ciency is not general. The single most likely reason why it should be missing in a dominant condition is that it forms part of a multi-molecular protein complex, in which its binding site is abnormal, by virtue of the presence of another mutant protein in the complex. The mutant protein would be expected to be expressed only in erythrocytes. The protein oligomerizes.64 Anity studies indicate binding to a putative G-protein coupled receptor65; it is palmitoylated.66 Stomatin is very widely distributed in the human body67 and in the animal kingdom as a whole. Three homologues have been found in the simple nematode Caenorhabditis elegans, and mutations in all of these cause some form of neuronal dysfunction. Mutations in one of these homologues, mec-2, causes mechanoinsensitivity68, the inability to detect simple touch stimuli. Also, in the small group of 18 genes that can cause this phenotype are found mec-4 and -10, which code for subunits of what is now known to be an ion channel, homologous to the mammalian amiloride-sensitive Na channel, or ENaC.69 It is immediately striking that this stomatin homologue should be associated, in a quite di€erent manner and quite di€erent biological context, with a

Hereditary stomatocytoses and allied disorders 723

sodium channel. The genetics on C. elegans suggest that mec-2 interacts closely with the ion channel and it is natural to suggest that it is an ENaC-type molecule that leaks in the stomatocytoses. However, the leak in OHSt cells is not inhibited by amiloride or analogues70, and co-expression studies in which stomatin and ENaC channel subunits were injected into Xenopus oocytes (CM Canessa, ME Chal®e and GW Stewart, unpublished) were negative. The original form of the ENaC channel71 is expressed in kidney, gut and lung (although homologous proteins are well described in many other mammalian tissues) and the protein is not recognized in the normal human red cell. Nevertheless, it is possible that a few copies are present, for in Liddle's syndrome, the human condition in which the kidney±gut±lung ENaC channel is congenitally leaky72, ouabain plus loop diuretic insensitive Na and K transport in red cells is accelerated, suggesting that a few copies of the functioning protein may be present in red cells.73,74 Tavernerakis et al75 have suggested, on the basis of sequence homology comparison, that stomatin is somehow related to a complex that manages some membrane proteins by regulation of their turnover. In Liddle's syndrome itself, it is clear that the mutation in the channel gene destroys a domain that is associated with protein turnover, by Nedd4mediated ubiquitination.76 It is possible that in the stomatocytoses, the membrane leaks because a channel, which is normally silenced by simple removal from the membrane, spends too long in the membrane and functions too much. The export of unwanted membrane proteins is a major feature of the maturing red cell77 and it has been established that DHSt red cells, at least, show an excess of membrane proteins in general.25,26 In red cell studies, the stomatin protein has been associated, in binding studies, with lipids.78 Given that stomatocytosis implies an imbalance between the two lea¯ets of the bilayer, it might be suggested that stomatin might have a role in inter-lea¯et phospholipid movement. We have examined the movements of ¯uorescently labelled lipid across the red cell membrane in stomatin-de®cient cells and there is no major abnormality, although we did ®nd that the ¯op movements of phosphatidylserine from inner lea¯et to outer were reduced in a semi-quantitative assay.79 The ¯ipase, ¯opase and scramblase activities80 were all present. CONCLUSION The hereditary stomatocytoses represent a set of rare human haemolytic anaemias in which the membrane permeability of the plasma membrane of the red cell is pathologically increased for reasons that are as yet poorly understood. Nevertheless, the molecular pathology implicates a membrane protein, stomatin, of general distribution which appears to be important in the regulation of an ion channel in the nervous system, and it seems possible that the molecular system at fault in the stomatocytoses is of universal importance with implications far beyond haematology. Acknowledgements We thank Jean Delaunay, Achille Iolascon, Britta Fricke, Monica Driscoll, Gary Lewin and Mike Harvey for useful discussions, and Margaret Chetty for invaluable technical assistance. We are grateful to The Wellcome Trust for support. Jane Turner is funded by a fellowship from the International Journal of Experimental Pathology. We thank our patients for their co-operation.

724 G. W. Stewart and E. J. H. Turner

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