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RED CELLS
Pleiotropic syndrome of dehydrated hereditary stomatocytosis,
pseudohyperkalemia, and perinatal edema maps to 16q23-q24
Sabine Grootenboer, Pierre-Olivier Schischmanoff, Ingrid Laurendeau, Thérèsa Cynober, Gil Tchernia, Jean-Paul Dommergues,
Didier Dhermy, Mireille Bost, Bruno Varet, Michael Snyder, Samir K. Ballas, Béatrice Ducot, Marie-Claude Babron,
Gordon W. Stewart, Paolo Gasparini, Achille Iolascon, and Jean Delaunay
Dehydrated hereditary stomatocytosis (DHS)
is a rare genetic disorder of red cell permeability to cations, leading to a well-compensated hemolytic anemia. DHS was shown
previously to be associated in some families
with a particular form of perinatal edema,
which resolves in the weeks following birth
and, in addition, with pseudohyperkalemia
in one kindred. The latter condition was
hitherto regarded as the separate entity,
“familial pseudohyperkalemia.” DHS and familial pseudohyperkalemia are thought to
stem from the same gene, mapping to 16q23q24. This study screened 8 French and 2
American families with DHS. DHS appeared
to be part of a pleiotropic syndrome in some
families: DHS ⴙ perinatal edema, DHS ⴙ
pseudohyperkalemia, or DHS ⴙ perinatal
edema ⴙ pseudohyperkalemia. If adequately
attended to, the perinatal edema resolved
spontaneously after birth. Logistic regression showed that increased mean corpuscular volume and mean corpuscular hemoglobin concentration were the parameters best
related to DHS. In patients in whom cation
fluxes were investigated, the temperature
dependence of the monovalent cation leak
exhibited comparable curves. Specific recombination events consistently suggested
that the responsible gene lies between markers D16S402 and D16S3037 (16q23-q24). The
95% confidence limits (Zmax > 3.02) spanned
almost the complete 9-cM interval between
these 2 markers. (Blood. 2000;96:2599-2605)
© 2000 by The American Society of Hematology
Introduction
Genetic disorders of the red cell membrane permeability to Na⫹ and K⫹
account for a number of rare hereditary hemolytic anemias, characterized by an increased passive leak of the monovalent cations Na⫹ and
K⫹. The intraerythrocytic cation concentrations can be altered to various
extents, as well as cell hydration and morphology.1 Hereditary dehydrated stomatocytosis (DHS) (OMIM 194380),2-3 or hereditary xerocytosis, usually appears as a well-compensated chronic hemolysis. Familial pseudohyperkalemia (OMIM 177720) is characterized by an
increase of the K⫹ concentration in plasma when freshly drawn
blood is allowed to stand at temperatures lower than 37°C. It is
devoid of hematologic symptoms.4-9 DHS and pseudohyperkalemia
have a dominant inheritance pattern.
Dehydrated hereditary stomatocytosis has long been regarded
as a purely hematologic condition.2,3,10-17 The osmotic resistance is
increased and the osmotic gradient ektacytometric curve shows a
leftward shift.1,18,19 Splenectomy dramatically increases the thromboembolic risk.20 Recently, DHS was shown to be part of a broader
syndrome (OMIM 603528) in some families where it was associated with a particular form of perinatal edema,21,22 which will be
designated PEDHS. Pseudohyperkalemia22 could also be present.
Although DHS and pseudohyperkalemia are lifelong manifestations, PEDHS resolves within weeks or months after birth (if not
before). We hypothesized22 that the present pleiotropic syndrome
would stem from the same gene. That the genes presumably
responsible for both DHS and familial pseudohyperkalemia were
found to map to 16q23-qter23,24 supported the one-gene hypothesis.
In 10 families, we reassessed the hematologic presentation of DHS.
We compiled the various combinations of symptoms within the
pleiotropic syndrome and evaluated the phenotypical variations that
occurred within some kindreds. The temperature dependence of the
monovalent cation leak was found comparable under distinct clinical
presentations. Specific recombination events consistently suggested that
the responsible gene lay between markers D16S402 and D16S3037
(16q23-q24) in a 9-cM interval.
From the INSERM U473, Le Kremlin-Bicêtre, France; Laboratoire d’Hématologie,
d’Immunologie et de Cytogénétique, Hôpital de Bicêtre, Assistance PubliqueHôpitaux de Paris, Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre, France;
Laboratoire de Biochimie I, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France;
Laboratoire de Biologie Spécialisée, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France;
Laboratoire de Biologie Moléculaire, Faculté de Pharmacie, Université Paris V,
Paris, France; Service de Pédiatrie Générale, Hôpital de Bicêtre, Le Kremlin-Bicêtre,
France; INSERM U 409, Faculté de Médecine Xavier-Bichat, Paris, France; Service
d’Hématologie, Hôpital Michallon, Grenoble, France; Service d’Hématologie, Hôpital
Necker-Enfants-Malades, Paris, France; Hospital Laboratories and Clinical Pathology,
University of Massachusetts Medical Center, Worcester, MA; Department of
Medicine, Cardeza Foundation for Hematologic Research, Philadelphia, PA; INSERM
U 292, Le Kremlin-Bicêtre, France; INSERM U 535, Le Kremlin-Bicêtre, France;
Department of Medicine, University College, London, UK; Servizio di Genetica
Medica, IRCCS-Casa Sollievo Soflerenza, San Giovanni Rotondo, Italy; Dipartimento
di Biomedicina dell’ Età Evolutiva, Università degli Studi di Bari, Bari, Italy.
Submitted December 2, 1999; accepted May 25, 2000.
BLOOD, 1 OCTOBER 2000 䡠 VOLUME 96, NUMBER 7
Patients, materials, and methods
Kindreds
The genealogic trees of the 10 studied kindreds (8 French, 2 American)
are presented in Figure 1. There were respectively 4, 3, 2, and 1 families
Supported by the Institut National de la Santé et de la Recherche Médicale
(Unité 473), the Fonds d’Etudes et de Recherche du Corps Médical de l’Assistance
Publique-Hôpitaux de Paris, the Délégation à la Recherche Clinique de l’Assistance
Publique-Hôpitaux de Paris (CRC96082), the Fondation pour la Recherche
Médicale, the Faculté de Médecine Paris-Sud (France), the Telethon Projects E-645
and E-743, the MURST, the Ministero Italiano delle Sanità (Italy), and by Action
Research and the WELLCOME Trust (UK).
Reprints: Sabine Grootenboer, INSERM U 473, 84 rue du Général-Leclerc,
94276 Le Kremlin-Bicêtre, France; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2000 by The American Society of Hematology
2599
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2600
GROOTENBOER et al
BLOOD, 1 OCTOBER 2000 䡠 VOLUME 96, NUMBER 7
Figure 1. Genealogic trees and microsatellite analysis. Genealogic trees. The names of the families were given after the town where the diagnosis was first made or where
one or several patients are living (AR, Arras; BI, Bicêtre; CL, Clichy; DA, Dax; GR, Grenoble; PH, Philadelphia, PA; TR, Troyes; VA, Vanves; VE, Vesoul; WO, Worcester, MA).
Arrow indicates the proband; black, fully expressed hematologic picture; gray, weakly expressed hematologic picture (on the basis of red cell parameters and osmotic
gradient ektacytometry); triangle, pseudohyperkalemia; asterisk, history of perinatal edema; S, splenectomized patient; small circle, stillborn baby with nonimmune
hydrops fetalis; oblique bars, deceased persons; ne, nonexamined persons. Specific information on family GR is provided in the text. Microsatellite analysis. The
shaded areas designate chromosomal segments assumed to carry the responsible gene. D16S3061 was tested when D16S3037 was not informative. In member II.4 of family
GR, it could not be formally established whether the crossover event on the DHS chromosome had taken place between markers D16S402 and D16S3061 (the most likely
possibility owing to the length of the interval, as indicated in the figure), or markers D16S3061 and D16S3037. Inset A shows assumed arrangement of markers and
genetic distances between them on chromosome 16 (for references, see text). Inset B shows the occurrence of crossover events on either side of the responsible gene,
assumed to lie in the D16S402-D16S3037 interval. Recombination events telomeric to the responsible gene, not reported before to our knowledge, left the gene on the
centromeric subhaplotype side (D16S511-D16S402); those centromeric to the gene left it on the telomeric subhaplotype side (D16S3061-D16S303
7-D16S520-D16S498-D16S3074).
with DHS alone, DHS ⫹ pseudohyperkalemia, DHS ⫹ PEDHS, and all 3
manifestations (Figure 2). None of these kindreds had been genetically
studied before. Altogether, 37 patients and 18 unaffected members were
investigated. Signed informed consent was obtained from all individuals
investigated. Parents signed for minors.
The classification of kindreds was established based on the maximum
number of manifestations recorded (Figure 2). We are aware of the fact that
we could underclassify some families for having missed, for example, the
only member with pseudohyperkalemia in one kindred. PEDHS could be
overlooked, had it been restricted to a mild edema and to the prenatal
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BLOOD, 1 OCTOBER 2000 䡠 VOLUME 96, NUMBER 7
DEHYDRATED HEREDITARY STOMATOCYTOSIS
2601
determined using 86Rb⫹ as a tracer for K⫹ in a NaCl/MOPS/glucose
solution, containing (concentration expressed in mmol/L): Na⫹, 145; K⫹, 5;
Cl⫺, 150; MOPS-Na⫹, 15 (pH 7.4 at room temperature); glucose, 5; and
ouabain and/or bumetanide, 0.1 each, if required (ouabain and bumetanide
inhibit the Na⫹ pump and the Na⫹, K⫹ cotransporter, respectively). The
temperature dependence of the K⫹ leak (eg, the monovalent cation passive
leak) was established by measurement of the ouabain ⫹ bumetanideresistant 86Rb⫹ influx as a function of temperature in the same solution.31
Microsatellite analysis
Figure 2. Diagram accounting for the various combinations of DHS (as a pure
hematologic picture), pseudohyperkalemia, and PEDHS. a indicates DHS with
variable expression. b indicates pseudohyperkalemia with variable expression.
Except for family BI, PEDHS was inconstant, as far as we could assess. Family WO
was provisionally considered, by default, as devoid of pseudohyperkalemia (see text).
period, before ultrasound was available. No case of PEDHS could be found in
7 families. In each of the other 3 families, there was 1 stillborn fetus and 1
or 2 living members with antecedents of PEDHS.
Red cell indices and reticulocyte counts
Red cell indices and reticulocyte counts were measured in a H3 Blood Cell
Counter (Bayer Diagnostics, Tarrytown, NY). Osmotic gradient ektacytometry25 was carried out as previously described.26 Logistic regression was
performed with the Stata software (Stata Corporation, College Station, TX).
The subjects were distributed in 2 groups (DHS and normal), based on their
clinical and hematologic phenotypes. Red cell membrane proteins were
analyzed, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE),27,28 with some modifications.29 Usually, most if not all
affected and unaffected members of each family were investigated in
this respect.
Assessment of pseudohyperkalemia
Freshly drawn blood (lithium heparin tubes) was immediately placed in a
water bath at 20° and 4°C for 6 hours (in duplicate).22 Aliquots, secured at
t ⫽ 0 and t ⫽ 6 hours, were centrifuged (4°C, 2500 rpm, 10 minutes). The
supernatant was again centrifuged (4°C, 5000 rpm, 7 minutes). Plasma
potassium concentration was determined according to standard automatic
procedures. Hemoglobinemia was measured spectrophotometrically followed by a double-derivative calculation to rule out any interference with
bilirubin.30
Some families were seen in our department and blood incubations for
kalemia determination were started on site. When field trips were necessary,
whole blood incubations were performed locally. At the end of the
incubations (as described above), the recovered plasma aliquots were
temporarily secured at 4°C (4-8 hours) until they reached our department.
Under such conditions, kalemia could undergo no further change because
sera and erythrocytes had been separated. The 2 American families were not
included in this protocol. Other methods (not shown) pleaded against
pseudohyperkalemia in family PH. No tests were available as regards
family WO.
Analyses were performed using microsatellites slightly different from those
used before23: D16S511, D16S402, D16S3061 (optional), D16S3037,
D16S520, D16S498, and D16S3074. Their order from centromere to
telomere and the genetic distances between them were derived from several
online databases.32 In some cases, marker D16S3037 did not bring any
information because family members were homozygotes. We then used
marker D16S3061, centromeric yet close (0.1 cM) to marker D16S3037
(Figure 1, Inset A). We were not aware of other microsatellites in the
D16S402-D16S3037 interval. The polymerase chain reaction (PCR) products were monitored in a ABI Prism 377 DNA Sequencer (Applied
Biosystems, Foster City, CA). Results were processed through Genescan
Software. The PCR was performed twice and fragment lengths measured on
different gels.
Linkage analysis was performed on the basis of an autosomal dominant
disease with a penetrance of 95%. This value was chosen in consideration
of the 2 symptomless individuals (members I.1 and III.6 from family GR)
thought to carry the DHS-associated haplotype (see below), and the 37
patients. Family BI was noninformative. The disease-gene frequency was
set to 0.0012 as previously described,23 and the marker alleles frequencies
were calculated from the parental allele repartition. Multipoint linkage was
performed by use of the GENEHUNTER software package.33 The approximate 95% confidence limits for the maximum recombination fraction (␪max)
at Zmax were calculated by the 1-lod-down method.34
Results
The hematologic picture
The term DHS will refer solely to the hematologic manifestations,
including those evidenced by ektacytometry, and irrespective of the
presence or absence of other manifestations. In general, hemoglobin was nonsignificantly diminished, whereas the reticulocytes
were markedly increased, indicating a well-compensated anemia
(Figure 3). There was a definite tendency toward macrocytosis. The
Determination of intraerythrocytic cation concentrations and
cation flux rates across the membrane
Blood samples (10 mL, citrate-phosphate-dextrose [CPD]-adenine tubes,
4°C) were shipped to London immediately after venisection. Intracellular
electrolytes were measured as described.31 A travel control was included
with samples from VE and VA. Travel controls showed a rise in [Na⫹] and a
fall in [K⫹] of 3 to 5 mmol/L cells during overnight storage on ice in CPD
(Chetty and Stewart, unpublished observations). Isotopic fluxes were
Figure 3. Red cell parameters in DHS, whether nonhematologic manifestations
were present or not. The means between patients and controls were compared
using the nonpaired Student test. Œ, patients; ‚, symptomless family members. (A)
Hemoglobin was nonsignificantly decreased (P ⫽ .39). (B) Reticulocytes were
significantly increased (P ⬍ .001). (C) MCV was significantly increased (P ⬍ .001).
(D) MCHC was significantly increased (P ⬍ .001).
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BLOOD, 1 OCTOBER 2000 䡠 VOLUME 96, NUMBER 7
GROOTENBOER et al
Figure 4. Osmotic gradient ektacytometry. The deformability index (DI) was
measured for increasing osmolality of the red cells containing medium. Full line
indicates control; dotted line, a patient whose curve was shifted leftward. The Omin
point corresponds to the osmolality at which the red cells are maximally swollen. The
O’ point corresponds to the osmolality at which DI has half its maximal value.
mean corpuscular hemoglobin concentration (MCHC) was significantly increased; however, hyperdense cells (MCHC ⬎ 41g/dL)
failed to appear as significantly increased (threshold, 4%) (not
shown). Logistic regression showed that MCHC and mean corpuscular volume (MCV) were the most significantly linked to DHS
(odds ratios: 5.5 and 1.3, respectively, with P ⫽ .003 and P ⫽ .004,
respectively). SDS-PAGE showed a normal profile in all the
affected members who were investigated within each family
(not shown).
The osmotic gradient ektacytometric curve was shifted to the
left (Figure 4). The deformability index remained within the
normal range. The leftward displacement of the O⬘ point indicated
cellular dehydration. The leftward displacement of Omin witnessed
an increase of the red cell surface area/volume ratio. It appeared as
the most consistent parameter in DHS.
Four affected patients underwent splenectomy as a treatment for
their hemolytic disease. Long-term follow-up revealed the occurrence of thromboembolic complications in all of them. Patient II.2
from family AR, splenectomized at the age of 15, sustained a deep
venous thrombosis at the age of 24. Patient II.2 from family DA,
also splenectomized at the age of 15, developed a portal vein
thrombosis 8 days later.35 Patient I.1 from family TR (who
probably had DHS) died at the age of 32 of pulmonary thrombotic
complications in the postsplenectomy period. Patient I.1 from
family VE, splenectomized at the age of 44, suffered from recurrent
episodes of superficial and deep venous thrombosis (once a year for
11 years and then less frequently), leading to severe venous
ulceration in the lower limbs. These manifestations are consistent
with the report by Stewart and coworkers20 and confirm that
splenectomy is hazardous in DHS.
symptoms, PEDHS or pseudohyperkalemia, could vary on an
intrafamilial basis, as detailed in the following.
The hematologic symptoms were missing or strongly reduced in
some members even though they carried the DHS-associated
haplotype. In family DA, member II.2 had conspicuously disturbed
red cell parameters (and thrombotic antecedents), whereas only
MCV, Omin, and O’ were slightly altered in member I.1. In family
GR, member III.1, the proband, had an unmistakable DHS.
(Member IV.1 [unmistakable PEDHS] is not described here in
detail.) Other members had strongly reduced hematologic symptoms yet they displayed, at the very least, a leftward shift of the
Omin point at the ektacytometer. On the other hand, members I.1
and III.6 failed to even exhibit this feature. Member III.4 was in the
same situation but, instead, she had had a perinatal edema (see
above) offering a distinct facet of the pleiotropic syndrome.
In families BI, the occurrence of PEDHS was constant, whereas it
was inconstant in families GR and VE as far as we could document.
Plasma K⫹ values (after 6 hours of incubation at 20°C) were above
normal only in all DHS members from families BI and VA, but only
in some within kindreds AR and CL (Figure 5).
The cation leak
Measurements of intracellular Na⫹ and K⫹ concentrations and of
flux rates are shown in Table 1. All values obtained were consistent
with previously reported results for DHS.22,23 Individual GR III.1,
however, showed normal intracellular Na⫹ and K⫹ concentrations,
even after storage in the cold, but the isotopic flux rates showed a
clearly increased ouabain ⫹ bumetanide-resistant K⫹ leak, and a
strikingly elevated Na⫹, K⫹ pump rate at about 6 times normal,
which can explain why the intracellular Na⫹ concentration was not
high. In the temperature studies, all kindreds tested (CL, GR, VA,
VE) showed increased fluxes at 37°C, and the temperature profiles
fell within a narrow envelope that is essentially parallel to normal
in these cases (Figure 6).
Microsatellite analysis
In the 10 investigated families, the location of the responsible gene
was consistent with 16q23-q24 (Figure 1). Some crossover
events repeatedly took place between microsatellites D16S402
and D16S3037. When some uncertainty remained about segregation of D16S3037, marker D16S3061, 0.1 cM centromeric to
marker D16S3037, was used. For example, in family VE, one could
not decide which of the 2 D16S3037 alleles (210 bp/210 bp) had
been transmitted from the father (member I.1) to one of his sons
(member II.3). Use of marker D16S3061 showed that it was
DHS as part of a pleiotropic syndrome; intrafamilial
phenotypical variations in some kindreds
PEDHS has occurred in at least one living member of families BI,
GR, and VE, and stillborn antecedents were present in each of these
kindreds. Pseudohyperkalemia appeared in 4 of 8 tested families
(families AR, BI,22 CL, and VA) (Figure 2). The presentation of the
pleiotropic syndrome was not immutable within all kindreds. In
some of them, we observed that any manifestation, the hematologic
Figure 5. Pseudohyperkalemia in DHS. Upon blood incubation (6 hours, 20°C),
kalemia raised in all or only in some DHS members from families AR, BI, CL, and VA.
Kalemia was raised in none of the DHS members from other kindreds. Œ, families
DHS ⫹ pseudohyperkalemia; , families DHS alone; ‚, healthy members.
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BLOOD, 1 OCTOBER 2000 䡠 VOLUME 96, NUMBER 7
DEHYDRATED HEREDITARY STOMATOCYTOSIS
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Table 1. Intracellular Naⴙ and Kⴙ concentrations
Intracellular electrolytes
(mmol/L cells)
Isotopic K⫹ influx (5 mmol/L external [K⫹]) (mmol/L cells 䡠 h)
[Na⫹]
[K⫹]
pump
ouabain-sensitive
Na⫹, K⫹ cotransport
bumetanide-sensitive
Residual leak
CL II.7
—
—
3.360
0.690
0.139
CL II.3*
—
—
3.455
0.889
0.186
CL III.1
—
—
3.049
0.318
0.201
CL II.4*
—
—
3.750
0.700
0.145
Control
—
—
2.070
0.510
0.066
GR III.1
9.2
95.9
12.650
2.033
0.220
VA II.2*
21.1
77.8
3.081
2.233
0.144
VE II.5†
21.7
82.2
4.965
2.007
0.204
Travel control
15.1
96.9
—
—
—
Normal
5-11
85-105
1-2
0-1
0.06-0.10
Donor
Na⫹,
K⫹
Blood was always stored for at least 18 hours on ice prior to analysis. Intracellular electrolyte concentrations of family BI members, provided elsewhere,22 were consistent
with values obtained in VA II.2 and VE II.5.
* DHS ⫹ pseudohyperkalemia.
† DHS ⫹ (personal) history of PEDHS.
subhaplotype D16S3061-D16S3037-D16S520-D16S498-D16S3074
(249-210-183-220-193 bp) that had been passed on en bloc.
In family GR, the fact that DHS had no penetrance in one
member of the oldest generation (member I.1 or I.2) rendered
difficult the interpretation of the haplotypes. Backing up from
generation III (member III.1: overt DHS; member III.4: PEDHS)
through generation II (DHS weakly expressed), a highly plausible
haplotype pattern including crossover events was found. It implied
that members I.1 and III.6 would carry the DHS-associated
haplotype. These 2 extreme and puzzling cases led us to equal the
penetrance of the condition to 95% in the study.
Crossover events occurring in the D16S402-D16S3037 (9-cM)
interval appeared consistently either centromeric (family VE) or
telomeric (families GR and TR) to the gene (Figure 1, Inset B).
Significant lod scores (Zmax ⱖ 3.02) were obtained between these 2
markers (Figure 7). The telomeric region was completely excluded
from microsatellite D16S520. The 95% confidence limits spanned
almost the complete 9-cM interval between markers D16S402D16S3037.
Discussion
Spurred by initial observations by Entezami and colleagues21 and
ourselves,22 the aims of this work were to (1) revisit the hematologic picture of DHS, (2) define how DHS was part of a broader
syndrome including PEDHS or pseudohyperkalemia and assess the
Figure 6. The (ouabain ⴙ bumetanide-resistant) monovalent cation leak as a
function of the temperature. In family CL, the data reflect the mean ⫾ SEM of 4
affected members (2 with DHS ⫹ pseudohyperkalemia, 2 with DHS alone). Data bear
on single individuals in families GR (DHS alone), VA (DHS ⫹ pseudohyperkalemia),
and VE (DHS ⫹ history of PEDHS). Normal represents the mean of 6 control laboratory
volunteers. F, family CL; f, six controls; Œ, family GR; E, family VA; 䡺, family VE.
Figure 7. Lod score. The 95% confidence limits (Zmax ⱖ 3.02) for the location of the
responsible DHS locus spanned almost the complete 9-cM interval between markers
D16S402-D16S3037 (16q23-q24).
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GROOTENBOER et al
phenotypical variations observed within some kindreds, (3) evaluate the alteration of the cation leak, and (4) determine whether the
responsible gene would in all kindreds map to 16q23-q24.23
The hematologic picture
We confirmed that red cell parameters can give a substantial hint at
the diagnosis: well-compensated hyperhemolysis, high reticulocyte
count, increased MCV and MCHC, but a variable increase in the
percentage of hyperdense cells. Specifically, logistic regression
showed that MCHC and MCV were the parameters most significantly linked to DHS. Osmotic gradient ektacytometry yielded the
most consistent information through the leftward shift of Omin
point. In the absence of available ektacytometry, increase in the
osmotic resistance, measured on fresh blood, would be the best
diagnostic test to detect DHS. The Omin point (osmolality at which
the red cells are maximally swollen in ektacytometry) corresponds
to the 50% hemolysis point in the fragility osmotic testing.
DHS as part of a pleiotropic syndrome; intrafamilial
phenotypical variations in some kindreds
The presentation of the pleiotropic syndrome was not immutable within
all kindreds. We observed that any manifestation, the hematologic
symptoms, PEDHS and pseudohyperkalemia, could vary on an intrafamilial basis. The most compelling case was provided by family GR.
Member III.1 presented with typical hematologic symptoms but no
known history of PEDHS, and her cousin (member III.4) had had a plain
history of PEDHS but displayed no hematologic manifestations.
PEDHS deserves special comments. It would be a mistake to
infer a cause-to-effect relationship between fetal edema and fetal
anemia (by analogy with immune hydrops fetalis that follows
severe ␣-thalassemia, for instance). Rather, DHS and PEDHS share a
relationship of coexistence with regard to the same genetic cause.
Treating anemia in utero was of no use to cure the edema.21 The
transient character of the edema, when present, remains to be
accounted for.
Homogeneity of red cell cation abnormalities
All kindreds tested showed typical abnormalities of intracellular
Na⫹ concentrations and flux rates, as defined for DHS,23 with the
exception of family GR. Family GR showed near normal intracellular Na⫹ and K⫹ concentrations, whereas the flux rates indicated a
significant cation leak with a more than usually obvious increase in
the Na⫹, K⫹ pump rate. The explanation for this is unclear. This
illustrates that it is not possible to exclude the presence of “leaky
red cells” on the basis of measurement of intracellular Na⫹ and K⫹
concentrations alone, without recourse to flux rate measurement as
well. As for the temperature dependence of the cation leak, all
members tested exhibited a comparable curve that was simply
shifted up the y-axis compared to normal, as seen previously in an
Irish family with DHS.23,36 Altogether, despite some variations, it
appeared that the cation leak essentially showed similar patterns
whatever the status of DHS, alone or part of a broader syndrome.
This pattern is close also to that encountered in chromosome
16-related familial pseudohyperkalaemia.24 On the other hand, it
clearly departed from that found in a variant form of hereditary
stomatocytosis with pseudohyperkalemia.31
The genetic homogeneity
In all kindreds tested, mapping of the responsible gene was
consistent with 16q23-q24. This supports the view that the DHS
pleiotropic syndrome is a monogenic syndrome. Specific crossover
events suggested that the responsible gene would lie between
markers D16S402 and D16S3037 (or D16S3061 if tested). In
previous studies, Carella and coworkers23 and Iolascon and colleagues24 used 2-point linkage analysis. The latter did not allow
assessment of how likely was the presence of a gene over the whole
interval between 2 particular markers. The crossover events
exhibited by the Irish DHS family, referred to as family A,23 were
comparable to those observed in family VE presented here. In the
present study, using a multipoint linkage analysis, the 95%
confidence limits (Zmax ⱖ 3.02) spanned almost the complete 9-cM
interval between markers D16S402 and D16S3037.
The kindreds do not share a common haplotype because they
are unrelated in all likelihood. PEDHS was expressed in member II.5
from family VE (along with DHS) who carries telomeric subhaplotype (D16S402, D16S3061, D16S3037, D16S520, D16S498, and
D16S3074). It was also expressed in member IV.1 (not detailed
here) from family GR who carries the restricted centromeric
subhaplotype (D16S511, D16S402). These 2 cases matched with a
D16S402-D16S3037 interval location of the PEDHS locus, just as
they do for DHS itself. Therefore, it is doubtful whether a second
locus would come into play as for PEDHS.
This study agrees with the works by Innes and associates37 that
excluded a linkage of DHS with the ␣-adducin, ␤-adducin, and
stomatin genes, and by Gallagher and Smith,38 ruling out a linkage
with the hIK1 gene, a Gardos channel candidate. A variant form of
hereditary stomatocytosis was described31 and did not map to
chromosome 16.39
In this work, a complete account of the DHS hematologic
phenotype was provided in 10 families. DHS appeared to be part of
a pleiotropic syndrome, including perinatal edema and pseudohyperkalemia in some kindreds. We further showed intrafamilial
phenotypical variations in some kindreds. The monovalent cation
passive leak displayed comparable patterns whatever the symptoms. Finally, specific recombination events and lod score calculations consistently suggested that the responsible gene would lie
between markers D16S402 and D16S3037 in a 9-cM interval
(16q23-q24) (Zmax ⱕ 3.02). Gene identification is pending.
Acknowledgments
We are grateful to the investigated families for their kind cooperation. We thank Dr M. C. Chetty, Dr F. Clerget, Pr A. Spira, and Pr
M. Vidaud for their critical advice, and also Ms. M. Dehan and Dr
F. Baklouti for their careful reading of the manuscript.
Clinician and laboratory investigators having participated in the
work: Dr C. Barro (Hôpital Michallon, Grenoble, France), Dr J.-P.
Blondel (Laboratoire de Biologie Médicale, Arras, France), Drs B.
Cantelou and F. Lifermann (Centre Hospitalier, Dax, France), Drs
B. Coupe, P. Saladin, and P. Thierry (Centre Hospitalier PaulMorel, Vesoul, France), Dr G. Dine (Centre Hospitalier, Troyes,
France), and Dr R. A. Drachtman (Robert Wood Johnson University Hospital, NJ).
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2000 96: 2599-2605
Pleiotropic syndrome of dehydrated hereditary stomatocytosis,
pseudohyperkalemia, and perinatal edema maps to 16q23-q24
Sabine Grootenboer, Pierre-Olivier Schischmanoff, Ingrid Laurendeau, Thérèsa Cynober, Gil
Tchernia, Jean-Paul Dommergues, Didier Dhermy, Mireille Bost, Bruno Varet, Michael Snyder, Samir
K. Ballas, Béatrice Ducot, Marie-Claude Babron, Gordon W. Stewart, Paolo Gasparini, Achille
Iolascon and Jean Delaunay
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