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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 3120-3123
Familial Pseudohyperkalemia Maps to the Same Locus as Dehydrated
Hereditary Stomatocytosis (Hereditary Xerocytosis)
By
A. Iolascon,
G.W. Stewart,
J.F. Ajetunmobi,
S. Perrotta,
J. Delaunay,
M. Carella,
L. Zelante, and
P. Gasparini
From the D Biomed Età Evolutiva, University di Bari, Bari,
Italy; the Department of Medicine, University College London, Rayne
Institute, London, UK; the Department di Pediatria, II University
Napoli, Napoli, Italy; Service d'Hématologie, and INSERM U473,
Hôpital de Bicêtre, Le Kremlin-Bicêtre, France; and
Service di Genetica Medica, IRCCS-CSS, San Giovanni Rotondo (Fg),
Italy.
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ABSTRACT |
Familial pseudohyperkalemia is a "leaky red blood cell"
condition in which the cells show a temperature-dependent loss of potassium (K) from red blood cells when stored at room temperature, manifesting as apparent hyperkalemia. The red blood cells show a
reduced lifespan in vivo but there is no frank hemolysis. Studies of
cation content and transport show a marginal increase in permeability at 37°C and a degree of cellular dehydration, qualitatively similar to the changes seen in dehydrated hereditary stomatocytosis (hereditary xerocytosis). Physiological studies have shown that the passive leak to
K has an abnormal temperature dependence, such that the leak is less
sensitive to temperature than that in normal cells. We performed
genetic mapping on the original family and found that the condition in
this kindred maps to the same locus (16q23-ter) that we have previously
identified for an Irish family with dehydrated hereditary
stomatocytosis, which does not show the same temperature effects.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE TERM FAMILIAL pseudohyperkalemia (FP)
was coined to describe an asymptomatic, dominantly inherited, red blood
cell (RBC) trait in which affected individuals presented with high plasma K+ concentrations.1-3 The
electrocardiogram was normal and no hyperkalemic conditions such as
renal failure or Addison's disease were identified. The hematological
abnormalities were minimal. It was determined that the rise in plasma K
developed when the collected blood was allowed to stand at room
temperature for some hours: plasma K estimations on fresh blood were
normal. Following the first description, other kindreds with
pseudohyperkalemia due to temperature-dependent K loss from RBCs in
association with virtually normal hematology have been
described.4-9
The pseudohyperkalemia in this family was attributable to a
thermotropic abnormality in the so-called "passive leak" to K across the RBC membrane,10 the same flux that is augmented
in the frankly hemolytic hereditary stomatocytoses. This passive leak
can conveniently be assessed experimentally as the residual K flux that
persists in the presence of both ouabain and bumetanide (inhibiting the
NaK pump and NaK2Cl cotransport systems, respectively). In hemolytic
variants of the hereditary stomatocytoses, such as dehydrated
hereditary stomatocytosis (DHS), this ouabain and bumetanide resistant
(OBR) K flux is increased at 37°C. In this kindred with FP, this flux
was only slightly abnormal at 37°C, consistent with the minimal
hemolysis, but the temperature curve showed a shallow slope abnormality
in the interval 37°C to 20°C. Because of the different temperature
sensitivities of the passive leak and the NaK pump, the cells
lost K on cooling to room temperature. Minor abnormalities in cellular
hydration and intracellular [Na] and [K], consistent with the
minimal increase in ion fluxes at 37°C, led us to designate this
kindred as "mild hereditary xerocytosis."10
DHS, also known as hereditary xerocytosis, is the most common variant
of hereditary stomatocytoses and has recently been shown to be
heterogeneous. It was first identified by Glader et al.11 DHS has a dominant inheritance pattern. Typically, it is characterized by a mild anemia, an increased mean corpuscular volume (MCV) and mean
corpuscular hemoglobin concentration (MCHC), and an ektacytometric curve shifted toward the right.12 The most troublesome
complication of DHS is a tendency to thrombosis manifest after
splenectomy, which is contra-indicated.13 The gene
responsible for DHS has been mapped to 16q23-qter in a large Irish
family.14
Recently, frankly hemolytic DHS kindreds have been found to show
similar temperature-related K effects to those first found in
FP.15-17 A survey shows that in about a third of DHS
kindreds, pseudohyperkalemia was present (Grootenboer S, Delaunay J,
unpublished data, January 1999). We have also identified
that, irrespective of the presence or the absence of accompanying
pseudohyperkalemia, perinatal edema (clearly distinguished from hydrops
fetalis) occurred in about half of the French kindreds (Grootenboer et
al, unpublished data). Perinatal edema was preeminent in
the perinatal period and could be fatal; it spontaneously resolved a
few weeks or months after birth, and never relapsed. We hypothesized
that DHS, FP, and perinatal edema, in whatever combination, were caused
by different mutations within the same gene.17
This hypothesis prompted us to map the gene responsible for FP. We did
so in the original Edinburgh family in which FP was first
identified.3 We found that the gene for FP mapped to the
same arm of chromosome 16 as DHS, confirming that FP and DHS are most
likely facets of the same genetically leaky RBC disease, almost
certainly representing distinct mutations within the same gene.
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MATERIALS AND METHODS |
Clinical case.
A four-generation Scottish kindred consisting of 32 subjects, of which
11 were affected, was studied. The clinical presentation has previously
been described.3 Patients were typed according to a simple
test in which aliquots of heparinized blood were stored on the
laboratory bench (22°C to 25°C) for 0, 2, 4 and 6 hours, at which
times samples were spun and the plasma separated for K estimation.
Methods used for assessing the hematological status and measuring the
cation fluxes have previously been reported.3,10 Briefly,
intracellular Na and K were measured on washed cells by flame
photometry. For flux studies, cells were thrice washed in MOPS-buffered
saline then incubated in (mmol/L): Na, 145; K(86Rb), 5; Cl, 150, MOPS,
15 (pH 7.4 at room temperature [RT]); glucose, 5 and ouabain and/or
bumetanide, 0.1. After 30 to 180 minutes incubation (depending on the
temperature), cells were washed free of extracellular isotope in
ice-cold 106 mmol/L MgCl2, 10 mmol/L tris Cl (pH 7.4 at
RT), packed, lysed, and the trapped beta activity was counted by the
Cerenkov effect. The studies reported in Table 1 were performed at
37°C.
Molecular studies and linkage calculation.
Linkage analysis was performed using nine microsatellite markers from
the DHS locus on chromosome 16 (cen- D16S402 -10 cmol/L- D16S511 1 cmol/L- D16S3037  4 cmol/L- D16S520 1 cmol/L- D16S498 3 cmol/L-
D16S413 3 cmol/L- D16S3026 2 cmol/L- D16S3121-
tel).18 Polymerase chain reactions (PCR) using
fluorescently labeled primers were run according standard protocols. An
aliquot of PCR was run in an ABI PRISM 373 or 377 DNA
sequencer (Applied Biosystems, Foster City, CA) and results were
processed by GENESCAN software. Allele assignation was
performed using the Genotyper software. All living
individuals of the family were genotyped and thus contributed to the
following linkage calculations. Statistical analysis was performed on
the basis of an autosomal disease with complete penetrance. The
disease-gene frequency was set to 0.012, and all marker alleles were
considered to be equally frequent. Two point linkage analysis was
performed using the MLINK program version 5.1 from the
LINKAGE computer package.19 Loops of
consanguinity were accommodated as suggested by Ott.19
Values for maximum LOD score were calculated with the
ILINK program from the same computer package. The
approximate 95% confidence limits for the maximum recombination
fraction (qmax) at the maximum LOD score
(Zmax) were calculated by the 1-LOD-down method.19 Alleles were downcoded without loss of
informativity to reduce computing time.
| |
RESULTS |
To compare temperature effects in the present family with the
previously mapped DHS kindred, we examined the changes in plasma K on
storage of whole heparinized blood at room temperature in FP cells,
normals, and in the previously mapped DHS family (Fig 1A). In the FP cells ( ), there was a
marked increase in plasma K+ in blood stored at 20°C,
reflecting previous results,3 while in normal subjects and
the DHS family ( ,  ), plasma K+ showed no significant
change with time. The OBR K influx was studied as a function of
temperature in the proposita (), three normal subjects, and an
affected member of the previously mapped DHS kindred (Fig 1B). The FP
cells show a slightly increased flux at 37°C, but in the interval
37° to 20°C, the slope of the plot is significantly more shallow
than that in the previously mapped DHS subject and the normal
(confirming previous studies on this) family.10 Table
1 illustrates the minimal ion flux and
content abnormality in these FP cells compared with normal and the DHS kindred. Hemoglobin levels and MCV were within the normal range. Reticulocyte counts were on average slightly high (mean, 2.5%). We
have previously shown a reduced RBC lifespan, slightly abnormal MCHC
and blood film, with anisopoikilocytosis, polychromaphiliia, and few
target cells and stomatocytes.3 The cells showed minimal dehydration.10
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| Fig 1.
Temperature effects in FP and DHS. (A) Test for
pseudohyperkalemia. From the original FP proposita and a representative
affected individual of the previously mapped DHS family, whole
heparinized blood was stored at room temperature for up to 6 hours
before centrifugation, separation of the plasma, and estimation of K. Key, to both panels: , FP; , DHS; , normals (error bars, A,
denote mean ± 2 SEM, n = 22). Only in FP does the K rise with
time at this temperature. (B) OBR K influx versus temperature in the
same three cases. In FP () the OBR flux is marginally greater than
normal at 37°C, but shows the shallow slope in the interval 37° to
20°C characteristic of this family, while in DHS, the flux at 37°C
is significantly greater than normal (consistent with the frank
hemolysis) and shows a temperature profile which is parallel to
normal.
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The four-generation kindred was analyzed using nine different
microsatellite markers of DHS locus. A summary of the results is
reported in Table 2. Significant LOD scores
have been obtained with most of the markers used (D16S511, D16S3037,
D16S520, D16S498, and D16S3026). The highest LOD score (4.14) was
obtained with marker D16S3037 at theta ( ) of recombination
frequency. Additional positive LOD scores were also detected with the
remaining four markers. These findings clearly showed that FP disease
locus in this family maps at the same position as DHS on the long arm
of chromosome 16 (16q23-qter).
| |
DISCUSSION |
Positive LOD scores have been obtained in this family with FP with a
series of microsatellite markers from the DHS locus. In particular,
significant LOD scores higher than three have been obtained for the
following markers: D16S402, D16S511, and D16S3037. These results
allowed us to colocalize FP and DHS loci at the same chromosomal
position. While in DHS the marker showing the highest LOD score was
D16S520, here we obtained the highest score with marker D16S3037. This
finding can be most likely explained by the different informativity of
the marker itself in the present family as compared with the DHS
family.14 We conclude that FP and DHS are almost certainly
caused by alterations of the same gene. The possibility of two adjacent
alleles cannot formally be dismissed, but seems unlikely. In five
families showing this combination, we have never observed any
recombination (Grootenboer S, Delaunay J, unpublished data, January 1999).
The incidence of FP is unknown. Its occurrence must certainly be
underestimated, because it is asymptomatic and its discovery is always
accidental. On functional grounds, the slight hematological and ion
flux abnormalities at 37°C allow it to be classified as a mild
variant of the hemolytic conditions gathered under the generic label
hereditary stomatocytosis syndromes. It is the temperature effects that
define this syndrome, and in particular, the shallow slope profile of
the OBR fluxes. The present mapping work confirms this relationship,
and the comparison of temperature effects shows that different
thermotropic variants can be due to mutations of what is almost
certainly the same gene.
It should be noted that the temperature profiles of the OBR K fluxes in
other kindreds with FP may be different from the shallow slope variant
seen here. In particular, the ouabain-plus-furosemide K efflux in the
family described by Meenaghan et al6 showed a U-shaped
curve, similar to that seen in normal RBCs suspended in media in which
either Cl is replaced by salicylate or
thiocyanate,20 or in which Na+ is replaced by
an organic cation.21 The French family described by Dagher
et al8 was different again. Given these heterogeneities, it
may not be appropriate to extrapolate the present mapping results to
other families with the FP combination of almost-normal hematology and
temperature-dependent pseudohyperkalemia.
Biochemical data and laboratory findings suggest that the function of a
candidate gene should be related to monovalent cation movements across
the membrane. Unfortunately, there are no known genes resembling a
Na+K+ transporter or channel located within the
FP/DHS locus. However, several gene fragments (expressed sequence tags)
whose function remains unknown have already been mapped within this
locus. Work is in progress to define the possible function of each of
them and to see if one could be involved in determining FP/DHS.
Recombinants with more centromeric markers (D16S503, D16S515, D16S516,
and D16S3091) spanning the region of LCAT and KCC genes exclude these two genes as candidates and reduce the region for pseudohyperkalemia to
20 cM. This region has a minimum of overlapping of approximately 1.5 cM
with that for xerocytosis, and most likely support the hypothesis of allelism (one gene with different mutations).
In conclusion, this work shows that a variant of that group of
genetically leaky RBC conditions maps to a locus on chromosome 16 to
which we have already mapped DHS. As previously suggested by
hematological studies,17 FP and DHS may be variants of the same genetically leaky RBC disease. Much work will be necessary to
indentify the assumed "FP/DHS gene," and to account for the simple or compound phenotypes arising from the various mutations within
this gene.
| |
ACKNOWLEDGMENT |
We are grateful to the patients for their cooperation.
| |
FOOTNOTES |
Submitted November 3, 1998; accepted January 4, 1999.
Supported in part by Telethon to MC (E783). We also thank the Wellcome
Trust and Action Research for support.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to A. Iolascon, MD, PhD, Dipartimento di
Biomedicina dell'Età Evolutiva, Piazza Guilio Cesare 11-70124, Bari, Italy; e-mail: a.iolascon{at}bioetaev.uniba.it.
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ABSTRACT
INTRODUCTION
