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The Molecular Genetic Basis of Glanzmann’s Thrombasthenia in a Gypsy
Population in France: Identification of a New Mutation on the &IIb Gene
By Nicole Schlegel, Odile Gayet, Marie-Christine Morel-Kopp, Beat Wyler, Marie-FranGoise Hurtaud-Roux,
Cecile Kaplan, and John MCGregor
to a premature stop codon and the synthesis of a severely
Glanzmann’sthrombastheniaisa
rare inherited bleeding
truncated form of aIL.Genomic DNA study showed a G --t
disordercaused by a qualitative or quantitative defect of
A substitution, the Gypsy mutation, at the splice donor site
platelet all,,pa. We describe here anew mutation that is the
of intron 15. This mutation results in an abnormal splicing
molecular genetic basisof Glanzmann‘s thrombasthenia in
occurring at an alternative donor site located8 bp upstream
two gypsy families. Our investigation was focused on the
from the mutation. Based onthose results, an allele-specific
(Yllb gene as a result of biochemical and immunologic analysis
PCR analysis was developedto allow a rapid identification of
of patients‘platelets showing undetectable(Yllb but residual
the mutation in patients and potential carriers of the gypsy
p3 levels. The entire allbcDNA was polymerase chain reaccommunity. This PCR analysis can also be used for genetic
tion (PCR) amplified using patients platelet RNA. Sequence
counseling and antenatal diagnosis.
analysis showed an &bp deletion located at the 3‘ end of
0 1995 by The American Societyof Hematology.
exon 15. This deletion causes a reading-frame shift leading
G
LYCOPROTEIN (GP) IIb-IIIa, a calcium-dependent
heterodimeric complex, is a major surface adhesive
protein receptor that plays a vital role in platelet function.’
Sequence determination from cloned cDNAs has shown that
GPIIb-IIIa, also known as uImP3, belongs to the integrin
The genes for aIb4and
are distinct but physically linked within 260 kb on the long arm of chromosome
17 at q21 ”*
Platelets of patients with Glanzmann’s
thrombasthenia, a rare autosomal recessive disorder, show
an absent, severely reduced or dysfunctional aIbp3.9Such
(Yllb p3 defects result in patients showing an extended bleeding time, lack of clot retraction and an absence of platelet
aggregation. This congenital platelet disorder favored by
high frequency of consanguinity, is characterized by its uneven geographic distribution.” Besides isolated cases, three
cluster areas have already been reported, one in France,“~”
one in Israel and J ~ r d a n , ’ and
~ ” ~one in South India.16 Several
molecular genetic defects at the origin of Glanzmann’s
thrombasthenia have already been characterized on the (YIIb
and p3 genes. Among the different genetic anomalies identionly one was reported for a cohesive
fied on the all,,
community.17
Glanzmann’s thrombasthenia is one of the most preoccupying hemorrhagic disorders in the gypsy population, mainly
represented in France by the Manouche tribe. Because of
the seriousness of this disease, which is considered as a
scourge by this population, we focused our research to find
the molecular basis of this genetic defect. Previous characterization of these patients pointed to a probable localization
of the anomaly on the (Yllb gene as suggested by the two
following observations: (1) undetectable albbut residual p3
levels on the patients’ platelet membrane and (2) presence
of both human platelet antigen-l (HPA-1) alloantigens associated with an absence of one of the two HPA-3 alloantigens
as shown by platelet phenotyping of 10 gypsy obligate carriemz6This study has identified a new mutation on the aIIb
gene. We show how this point mutation, a G to A substitution
at position 9,263, leads to a splicing defect and a premature
a l I b chain termination. Moreover, we describe an allele-specific polymerase chain reaction (PCR)analysis method that
can be used for carrier status detection, genetic counseling,
and antenatal diagnosis in the studied population.
MATERIALS AND METHODS
Patients. Informed consent was obtained from patients and their
families for drawing blood. The group under investigation consisted
Blood, Vol 86, No 3 (August l), 1995: pp 977-982
of two families (I and 11) belonging to the French gypsy community.
The unusually high frequency of Glanzmann’s disease within this
population is related to a high consanguinity level as illustrated for
family I (Fig 1). These families have been followed by us for a period
of over fifteen years for medical counseling related to prevention and
treatment of hemorrhagic episodes. Previous functional, biochemical, and immunologic studies have characterized 52 individuals (23
females, 29 males) into 1 1 homozygous patients (4 females, 7 males)
with type I Glanzmann’s thrombasthenia, 32 heterozygotes (15
females, 17 males), and 9 unaffected subjects (4 females, 5
~nales).~”~’
Controls. After informed consent, 10 normal subjects were the
source of control DNAs. Two of them were also selected as sources
of platelet control RNA.
Ampl8cation and analysis of platelet allbmRNA. Total platelet
RNA was isolated from patients and controls using a modified version of the technique of Chomczynski and Sacchi” as described by
Djaffar et d?’First-strand cDNA was synthetized from platelet total
RNAusing Moloney murine leukemia virus reverse transcriptase
(GIBCO Laboratories, Grand Island, NY) and either random hexanucleotides [p(dN)6] or oligo dT (Boehringer-Mannheim, GmbH,
Germany). PCR amplifications were performed using several sets of
oligonucleotides derived from the normal aIIbcDNA sequence,’*
designed to cover the entire am coding sequence in overlapping
fragments. An alIbcDNA (kindly provided by Mortimer Poncz, The
Children’s Hospital of Philadelphia, Philadelphia, PA) was used as
a control for PCR amplifications. Thirty rounds of amplification
were performed under the following conditions: denaturation 94°C
for 60 seconds, annealing 70°C for 90 seconds, and extension 72°C
for 120 seconds, with a IO-second increase of the extension time
per cycle for the 15 last cycles, and with a final elongation step of
7 minutes at 72°C. The blunt-ended fragments were cloned into the
H i n d site of pUC 18 and sequenced in both directions using either
From the Service d’Hdmatologie Biologique, Hbpital Robert Debrdandthe Service d’lmmunologie Plaquettaire, INTS, Paris and
theINSERM U 331, Lyon, France.
Submitted October 14, 1994; accepted March 7, 1995.
Address reprint requests to Dr Nicole Schlegel, Service d’Hdmatologie Biologique, Hbpital Robert Debri, 48 Boulevard Sincrier,
75019 Paris, France.
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.
0 1995 by The American Society of Hematology.
0006-4971/95/8603-0$3.00/0
977
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SCHLEGEL ET AL
*.
.
"'*
""
-+
VI
VI1
the M13 universal or reverse sequencing primers (New England
Riolabs, Beverly, MA).
Anlp/ifccr!iorl m e / e m r / u i . s of c y I I , , gene. High molecular weight
genomic DNA was extracted from peripheral blood lymphocytes
after the standard phenol-chloroform technique." DNA aliquots (100
ng) were amplified. using the above described PCR conditions, with
primers derived from the normal all,,gene sequence" (see Results)
and designed to flank the allhcDNA defective region. The PCR
products were subcloned and sequenced as previously indicated.
A//e/r-.~prcific
PCR cmo/wis. Two different PCR amplifications
encompassing the defective region were designed for each genomic
DNA sample, using a common antisense primer derived from the
normal sequence" and either of two sense primers derived from the
normal or mutant allhsequence. Thirty rounds of amplification were
performed on each 100 ng aliquot of genomic DNA under the following conditions: denaturation 94OC for 60 seconds, annealing 66°C
for 120 seconds and extension 72°C for 120 seconds. The PCR
products were analyzed after electrophoresis in a 3% Nu Sieve agarose gel (FMC Bio Products. Rockland. ME).
RESULTS
Analysis ($platelet ail,, mRNA clnd genomic DNA. The
full-length cDNA of patient V. 17 (patient I7 from the
fifth generation in family I ) (Fig 1) and one control were
amplified by PCR. Such amplifications resulted in the generation of overlapping fragments that, when separated by agarose gel electrophoresis, were similar in size and intensity
to controls (data not shown). Such results suggested that the
transcription of the patient's all,,mRNAmightbenormal
and that no major insertion or deletion was responsible for
the Glanzmann's thrombasthenia. However, subcloning and
sequence analysis showed an 8-bp deletion localized at posi-
(Yllh
Fig 1. Generation tree of gypsy family 1. Roman
numbers refer to generations and arabic numbers
refer to individuals In = 42). Different members of
this family had their platelets characterized by clinical, functional, biochemical,andimmunochemical
studies. Among the 42 individuals in generations IV
through VII, 10 were characterized as type I thrombasthenic homozygotes (6 males and 4 females), 27
as heterozygotes (15 males and 12 females), and 5
as normals (3 males and 2 females). Nineteen of the
42 individuals were available for genetic molecular
studies (indicated with arrows). 8, individuals not
available for the study; t, deceased individuals;0 or
0,normals; half-filled circles or half-filled squares,
heterozygotes;0 or B,homozygotes; (-4, putative
but not proven links to the family.
tions 1,538 to 1,545 on the (Yllh cDNA (Fig 2, left panel).
This deletion corresponds to exon 15/intron 15 boundary on
the genome. To further analyze whether the deletion was
caused by splicing errors, the flanking sequence running
from position 9.192 to 9,284 was then amplified from genomic DNA using a couple of two 25-mer primers (sense : 5'GGTGAAGGCCTCTGTCCAGCTACTG-3' and antisense :
S'-GTGCCCAGTGGCTCCAACACACATC-3'). A fragment of 214 bp was obtained that, when sequenced, showed
a G -, A substitution at the GT splice donor site of intron
15 junction (Fig 2, right panel). This mutation leads to splicing at an alternative, normally cryptic, GT splice donor site.
This abnormal splicing site, located 8 nucleotides upstream
fromthenormal site (Fig 3). results in the 8-bp deletion
observed on the cDNA. Moreover, this 8-bp deletion causes
a reading-frame shift responsible for a premature TGA stop
codon at positions 1,756 to 1,758 on the cDNA (Fig 3). The
deduced polypeptide sequence would be a truncated form of
the (Y chain of (Yllh (Fig 4) with a normal amino terminal
sequence containing the signal peptide and the four putative
calcium-binding domains. However, this truncated polypeptide. ending with 70 aberrant amino acids, would lack the
last half of the extra-cytoplasmic domain and both the transmembrane and cytoplasmic domains. The entire p chain of
allh
would be absent.
Allele-specific PCR nttnlysis. This analysis was designed
to establish the homozygosity of patient V.17 for the G
A mutation and to examine the inheritance pattern of this
mutation in the gypsy population. PCR amplifications were
performed from genomic DNAof the patient, his mother,
+
ENIA
EW
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A
THE
MUTATION ON
979
(11lb
GENE
-
GATC
POSITION Y263
1st BASE. IKTRON
"""
e-
+SILENT
\l['TATION
A
PATIENT
cDNA
PATIENT
GENOMIC DNA
CONTROL
GENOMIC DNA
B
R g 2. Identification of the molecular genetic anomaly of gypsy Glanzmann's thrombasthenia on urnmRNA and an,gene. (A) Sequencing
patient V.17 full-length am cDNA showed an 8-bp deletion corresponding t o nucleotides 1,538 through 1,545; (B) sequence analysis of a 214bp sequence, amplified by PCR from patientV.17 urngene, showed a G--t A point mutation affecting the
splice donor site of intron
15. A silent
mutation can be observed on the controlsequence.
primer. These data confirm that the patient is homozygous
for the mutation. Moreover, a 278-bp DNA fragment was
obtained using the DNA from the patient's mother (individual IV. 15) with both the normal and the mutant sense primers, confirming that the mother is heterozygous for the mutation. The allele-specific PCR analysis was then extended to
all the other available members of the two gypsy families.
The same point mutation, designed as the Gypsy mutation,
was found in all affected members and allowed the identification of 4 homozygote, 19 heterozygote, and 6 normal individuals (Fig 5). The data are consistent with results obtained
and unrelated healthy donors using a common 18-mer
antisense oligonucleotide primer (5"CGGTCCAGCTGCAGCTCG-3'). This antisense primer was combined to one
of two 19-mer sense oligonucleotide primers (5"CAAGACACCCGTGAGCTGG-3' or 5"CAAGACACCCGTGAGCTGA-3') that correspond to the normal or mutant 5' end
sequence of intron 15. A DNA fragment with the expected
size (278 bp) was obtained using control genomic DNA with
the normal, but not the mutant sense primer. In contrast,
a 278-bp DNA fragment was obtained using patient V.17
genomic DNA with the mutant, but not the normal sense
GENOMIC DNA
Fig 3. Analysis ofthe
ullb
mRNA and gene anomalies in
gypsy Glanzmann's thrombasthenia. The G -t A (position
9,263) substitutionatthe
GT
splice donor site of intron15 r e
sults in the presence and use of
a cryptic GT donor site. This new
GT site is located 8 nucleotides
NORMAL
upstream from its normal position (within exon 151. This abnormal
splicing
mechanism
leads to the deletion of the last ClffY
8 bp of exon 15. In addition, it
produces a reading-frame shift
and a premature TGA stop codon
(positions
1,756 through
1,758).
cDNA
I538
Is45
1756 1758
4 ACC. TCC.
4 TTC. AAC. ATC. .........A. GAT.4 GAG.
4 GCA ....--c
+..CCC. CTC.
d...
CCC. 8 hp DELETION
TT. CAA. CAT. C
t
READING FRAME
SHIFT
.........ACA. TCA
A
PREM~~VRE
STOP CODON
1 7 ~ 6m-
n
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SCHLEGEL
980
AL
Ca++
SIGNAL
PEPTIDE
BINDING
HPA-3
,DOMAINS
*
*
31
243
+
TRANSMEMRRANE CYTOPLASMIC
*
437
ABNO$MAL
*
1*
S12
5x2
4
-
-
STOP
Fig 4. Consequences of the G A substitution at the5' end of intron15 of allbgene on the putative(Yllb truncated polypeptide. The G A
substitution at position 9,263 of the patient V.17 genomic DNA leads t o a putative truncated allbmolecule (582 amino acids v 1,0391 from
which the last 70 amino acids are aberrant. This putative truncated protein would contain thesignal peptide 131 first amino acids), and the
four putative calcium binding domains. However, the last half of the extracytoplasmic domain (containing the HPA-3 antigen sequence at
missing.
amino acid 8 4 3 1 and both thetransmembrane and the cytoplasmic domains would be
viafunctional,biochemical,
andimmunologic assays. Resultsobtained in the present study confirmthe autosomal
recessiveinheritancepatternofthe
Gypsy mutation in the
investigated families. The Gypsy mutation was not detected
in the genomic DNAs of IO controls.
DISCUSSION
This study shows a new genetic defect present in individuals from two apparently unrelated gypsy families suffering
fromtype I Glanzmann's thrombasthenia. These results,
which are the molecular basisof Glanzrnann's thrombasthenia in the French gypsy community. follow the work of
Newman et all7 performed on two other cohesive communities: the Iraqi-Jewish andtheArab
populationsliving in
Israel. The migration of gypsies from India to Europe took
two roads: a northern one, throughCentralEurope,and
a
southern one through the Middle East. Because preliminary
studies of the gypsy patients indicated
a probable location
ofthe defect on the all,,
it was of interest to verify
if theArab mutation, an 18-bpdeletionfoundon
the (Yllh
cDNA by Newman et aI.l7 was present in the gypsy population. The sequencing of the entire (Yllh cDNA allowed us to
eliminate this hypothesis as well as the eight other defects
previously
described
on
the
(Yllh
Among these
eight
thrombasthenic defects, three point mutations lead to single
amino acid substitutions affecting the calcium-binding domains of the (Yllh mo~ecule.1y~."3.'J
Another anomaly is the
result of a deletion inversion mechanism in exon 25 leading
to the substitution of IO amino acids by 8 aberrant amino
acids.23 The four other mutations are responsible for gross
3
4
N M N M N MN MN M
1
2
-""
-
7
c N MN M
"Z
Fig 5. Identification of the GWm A mutation in the gypsy population byallele-specific PCR analysis. Genomic DNA aliquots, isolated from
patient V.17 (family 11, his mother, and a control, were PCR amplified using a common antisense primer (derived from the normalallb gene
sequence of exon 171 and either oftwo sense primers corresponding to the normal
(primer NI or mutant (primerM1 5' end sequence of intron
15. A single fragment is obtained with primerN. but not primer M, for control DNA (lane 11. In contrast, a single fragment is obtained with
primer M, but not primer N, for the patient's DNA (lane 21. Fragments are obtained with both primers N and M for the mother's DNA (lane
31. Lanes 4 t o 6 show results obtainedwith three additional gypsy heterozygotes. Lane 7 corresponds t o a normal gypsy individual.
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A NEW THROMBASTHENIA MUTATION ON THE
C Y I ~GENE
~
deletions in the am molecule. A nonsense opal mutation
in exon 17 leading to a premature chain termination was
Two different splicing defects are responsible
for a 42 or 34 amino acids deletion as a result of a skipping
process of exon 26” or exon 28,25 respectively. A 4.5-kb
deletion in the am gene encompassing exons 2 through 9
was also de~cribed.‘~
The identified defect on the am gene in the gypsy population is a G + A substitution at the splice donor site of intron
15. This mutation induces an abnormal splicing mechanism
leading to a premature stop codon. The encoded polypeptide
would correspond to a severely shortened protein consisting
of 582 amino acids (v 1,039 in normal a I ~This
) . sequence
represents a truncated form of aIblacking the last half part
of the extra-cytoplasmic domain and both the transmembrane
and cytoplasmic domains. Deletion
using truncated forms of aIband p3 showed that the cytoplasmic and
transmembrane domains are not required for the assembly
of the complex. The N-terminal sequence alone is able to
assemble to p3 in a soluble form of the complex.”36 However, we showed in previous results that the
complex
is not detected in the patients’ platelets as assayed by crossed
immunoelectrophoresis.z7~z9
The above results suggest that
the deletion is too drastic to allow a normal folding of the
molecule, impairing either the complex assembly or its recognition by antibodies. Nevertheless, because the nonsense
mutation on exon 17 of aIIb
described by Kat0 et al” leads
to a truncated protein unable to assemble to P3, one can
hypothesize that the gypsy nonsense mutation on intron 15
would also prevent assembly to p3 and that the two subunits
would thus be rapidly degraded.
The point mutation detected on the patient’s aIb gene
allowed us to design oligonucleotide primers to be used in
an allele-specific PCR analysis. The specificity of this simple
and rapid test was shown by analyzing previously characterized thrombasthenic individuals compared with unrelated
controls. Thus, initial diagnosis based on functional, immunologic, and biochemical data could be confirmed for four
homozygote patients from both families, and the heterozygosity of 19 individuals of the gypsy community could be
established. This allele-specific PCR analysis provides a useful tool for the screening of potentially affected individuals
and carriers in this large population not only present in
France, but also widespread throughout the world. We are
now able to offer genetic counseling and antenatal diagnosis,
which is highly demanded, to the gypsy population. Because
of the high hemorrhagic risks of blood sampling from thrombasthenic fetuses, an antenatal diagnosis could be adapted
to amniocytes or to DNA extracted from chorionic villous
trophoblasts during early gestation.
ACKNOWLEDGMENT
We are grateful to Peter Newman for proposing the PCR amplification strategy; Mortimer Poncz for providing a,,,,
cDNA probes;
and Ckile Chahi for secretarial assistance.
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1995 86: 977-982
The molecular genetic basis of Glanzmann's thrombasthenia in a
gypsy population in France: identification of a new mutation on the
alpha IIb gene
N Schlegel, O Gayet, MC Morel-Kopp, B Wyler, MF Hurtaud-Roux, C Kaplan and J Mc Gregor
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