Download The Wiskott-Aldrich Syndrome and X-Linked

From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
The Wiskott-Aldrich Syndrome and X-Linked Congenital Thrombocytopenia
Are Caused by Mutations of the Same Gene
By Qili Zhu, Min Zhang, R. Michael Blaese, Jonathan M.J. Derry, Anne Junker, Uta Francke,
Shi-Han Chen, and Hans D. Ochs
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessivedisordercharacterizedbythrombocytopenia,small
platelets,eczema,recurrentinfections,andimmunodeficiency. Besidesthe classic WAS phenotype,there is a group
of patients with congenitalX-linkedthrombocytopenia
(XLT) who have small platelets but only transient eczema,
if any, and minimal immune deficiency. Because the gene
responsible for WAS has been sequenced, it was possible
to correlatethe WAS phenotypeswith WAS gene mutations.
Using a fingerprinting screening technique, we determined
the approximate location of the mutation in 13 unrelated
WAS patients with mild to severe clinical symptoms. Direct
sequence analysis of cDNA and genomic
DNA obtainedfrom
patient-derived cell lines showed 12 unique mutations distributed throughout the WAS gene, including insertions, deletions, and point mutations resulting in amino acid substiOf 4
tutions, termination, exon skipping, or splicing defects.
unrelated patients with the XLT phenotype,3 had missense
mutations affecting exon2 and 1 had a splice-sitemutation
affecting exon 9. Patients with classic WAS had more complex mutations, resultingin termination codons, frameshift,
andearly termination. Thesefindingsprovidedirectevidence that XLT and WAS are caused by mutations of the
same gene and suggestthat severe clinical phenotypes are
associated with complex mutations.
0 1995 by The American Societyof Hematology.
T
severity of WAS-associated symptoms was scored from 1 to 5 , based
on the following criteria. A score of 1 was given to patients with
thrombocytopenia and small sized platelets, but without any other
symptoms or clinical findings. Patients with platelet abnormalities
and a history of mild, transient, eczema, with or without minor
infections, received a score of 2. Patients with persistent but manageable eczema, with recurrent infections, or with both received a score
of 3. Those patients with persistent and difficult to control eczema
associated with frequent potentially life-threatening infections were
scored as 4. A score of 5 was assigned if patients presenting with
eczema and/or frequent infections developed autoimmune disease.
Seven of the 13 families reported more than 1 affected male patient.
Of the 13 unrelated WAS patients included in this study, 1 infant
(BM), diagnosed prenatally by linkage analysis and receiving a transplant at 1 month of age with bone marrow from an HLA-matched
sibling had not been observed long enough to be scored; up to the
time of marrowtransplantation, BM‘s only findings were thrombocytopenia, small platelets, and petechiae. However, his older brother,
who died of meningitis at 6 years of age, had severe clinical symptoms and was assigned a score of 4.Patient AR, who presented with
congenital thrombocytopenia and persistent small platelet size, but
HE WISKOTT-ALDFUCH syndrome (WAS) is an Xlinked recessive disorder characterized by thrombocytopenia, small platelet volume, eczema, recurrent bacterial
and viral infections, autoimmune diseases, increased risk of
maligancies, and abnormal B- and T-cell f~ncti0n.l.~
Patients
with WAS respond poorly to immunization with polysaccharides and certain protein antigen^,^.^ and lymphocyte proliferation in response to mitogens and antigens is variably dep r e ~ s e d Distinguishable
.~
from this classic WAS phenotype
are boys with X-linked thrombocytopenia (XLT) whose
platelets are small and who either are free of eczema6”’ or
have a history of only mild and transient
Most
patients with XLT have normal susceptibility to infections
and normal immune functions. The gene for XLT has been
mapped to Xpll.22, the same band to which the classic
WAS phenotype had been mapped?~”~12
The gene responsible for WAS has been isolated, cloned,
and ~equenced.’~
Encoded by 12 exons and composed of
1,823 bp, the cDNA encodes a 502-amino acid, proline-rich
protein (WAS protein) of unknown function that appears to
be of central importance for the function of hematopoietic
cell lineages.13.14 WASgene transcript was absent in 2 of 5
Epstein-Barr virus (EBV)-transformed B-lymphoblastoid
cell lines (B-LCL) derived from affected male patients, and
sequence analysis of genomic DNA showed three independent mutations, strongly suggesting that the newly identified
gene was the WAS gene.”
To determine the mutation pattern of the WAS gene and
to correlate location and type of mutations with clinical phenotypes, we studied 13 additional unrelated WAS families
whose affected members presented with mild to severe disease. Twelve unique mutations, which were distributed
throughout the WAS gene, were identified and included insertions, deletions, and point mutations resulting in stop codons, amino acid substitutions, exon skipping, or splicing
defects.
MATERIALS AND METHODS
Patients and Their Phenotypes
Affected members of 13 unrelated WAS families with variable
phenotypes were included in this analysis (Table 1). All patients
had in common thrombocytopenia and small platelet volume. The
Blood, Vol 86, No 10 (November 15). 1995: pp 3797-3804
From the Department of Pediatrics,University of Washington
School of Medicine, Seattle, WA;the Clinical Gene Therapy Branch,
National Centerfor Human Genome Research, National Institutes of
Health, Bethesda. MD; Howard Hughes Medical Institute, Beckman
Center for Molecular and Genetic Medicine, Stanford University
Medical Center,Stanford, CA; the Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada.
Submitted June 12, 1995; accepted July 18, 1995.
Supported bygrantsfrom
the National lnstitutes of Health
(H017427 [H.D.O.]),the March of Dimes and Birth Defects Foundation (6-0116 [H.D.O.] and 6-0694 [S.-H.C.]), and the Howard
Hughes Medical Institute (Investigator [U.F.] and Associate
[J.M.J.D.]),and in part by B.C. Medical Services Foundation 5 S4511 (A.J.). A portion of this work was conducted through the
Clinical Research Center of the University of Washington (RR-37).
Address reprint requests to Hans D. Ochs, MD, Department of
Pediatrics, RD-20, University of Washington School of Medicine,
Seattle, WA 98195.
The publication costsof this article were defrayedin part by page
chargepayment. 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.
0006-4971/95/8610-0013$3.00/0
3797
mic
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
3798
ZHU ET AL
Table 1. Clinical Phenotypes and Mutations of the WASP Gene Observed in 12 Unrelated Patients With WAS
Other Affected
Mutation
Members
Family
Patient
Score* Transcriptt(Score*)
AR
CS
JS
BG
Exon
1
0
ND
2
2
2
1 (2)
++
0
(+)
(+)
2
4
2 (2, 2)
2
2
9
5
1 (5)
0
++
++
TH
5
4
3 (4. 4, 2)
(+)
1
10
RM
5
0
(-)
3
MB
4
5 (all 4)
(+)
9
MS
DM
BM*
-
1 (4)
+
EA
PP
3
4
0
2 (4. 4)
(+)
1
++
10/11
CR
5
0
(+)
10
DNA
C290T
ArgE6Cys
C290T
C174A
Ala47Asp
A953G (ATG
bp
gtg)
G407A
C71T
C995T
C329T
11
cDNA
+
13
t + c at +2nt of
-
intron 9
t c at +2nt of
intron 11
61-65 i n s C
9 bp del, 11 bp ins
intron 10
del1301-1305
G
Mutation
Expected Effect
C290T
C290T
C174A
del
Arg86Cys
Frameshiftrer
G407A
C71T
C995T
Skipping of exon 3
(87 bpK329T
965 ins 114 bp
Gly125Arg
Argl3Ter
Arg32lTer
Truncated protein
Skipping exon 11
(115 bp)
61-65 ins C
1372 ins 13 bp
del 38 aa, frameshift
del1301-1305
G
ins 38 aa
Frameshimer
Frameshimer
Frameshimer
* Scoring system is described in text.
t Determined by Northernblot(Fig 2); ++ and + indicate normal amounts of transcript, (+) indicates severely reduced but detectable
transcript, and (-) indicates transcript that is undetectable by Northern blot but detectable by RT-PCR.
When marrow transplantation was performed at 1 month of age, BM had no symptoms other than thrombocytopenia, small platelets, and
petechiae.
*
without eczema or increased susceptibility to infections, was evaluated carefully and found to be immunologically normal; he was given
a score of 1. However, shortly after splenectomy, AR developed
pneumococcal sepsis, suggesting that hemay have mild immune
deficiency. All of the remaining 11 patients had additional symptoms, including various degrees of immune abnormalities. Three
patients (CS, JS, and BG) with transient, mild eczema and infrequent
infections responding well to antibiotics were given scores of 2. One
patient (EA) whose persistent eczema and infections were wellcontrolled by standard therapy was given a score of 3. The remaining
7 patients had severe clinical symptoms and were given a score of
4 if without and a score of 5 if with symptoms of an autoimmune
disorder.
Cell Lines
B-LCLs established by inoculating peripheral blood mononuclear
cells (PBMCs) with supernatants from the marmoset cell line B95.8I5
were available from all but 1 WAS patient. From patient AR, mRNA
and genomic DNA were isolated from frozen spleen cells that had
been obtained during splenectomy.
Selection of Primers
Primers for amplification of cDNA and genomic DNA and for
dideoxynucleotide fingerprinting were selected from theknown
cDNA and genomic DNA sequences of the WAS gene'3 and designed to identify the mutations of our patient population (Table 2).
RNA Isolation and Reverse Transcriptase-Polymerase
Chain Reaction (RT-PCR)
RNA isolation andRT-PCR were performed as previously described.16 Briefly, total RNA was isolated from B-LCLs or spleen
cells using a single-step method and Trizol (GIBCO BRL, Gaithersburg, MD). The first-strand cDNA was synthesized by incubating
2.5 pg of total RNA with oligo (dT) as primer using the Superscrip
Preamplification System kit (GIBCO BRL), as recommended by the
manufacturer. The WAS protein cDNA was amplifed by PCR in
two overlapping fragments. For reaction 1 (Table 2), PCR underwent
35 cycles in20 mmol/L Tris-HC1 (pH 8.4). 50 mmol/L KCI, 1.5
mmoULMgCI,, 0.2 mmol/L dNTP, 0.5 pmol/L primers W-2 and
5.24, 5 pL first-strand cDNA products, and 2.5 U Taq polymerase
(GIBCO BRL) in a total volume of 100 pL at 94°C for 1 minute,
57°C for 1 minute, 72°C for 1.5 minutes, and a final extension step
at 72°Cfor 10 minutes. For reaction 2 (Table 2), PCR wasperformed
in20 mmom Tris-HC1 (pH 8.8). 10 mmol/L KC], 10 mmol/L
(NH&S04, 2 mmol/L MgSO,, 0.1% Triton X-100, 0.1 mg/mL bovine serum albumin (BSA), 0.2 mmom dNTP, 0.5 pmol/L primers
W-906 and W-l708c, 5 pL of the first-strand cDNA products, 2.5
U of the Taq Extender PCR additive (Stratagene, La Jolla, CA), and
2.5 U Taq polymerase in a total volume of 100 p L at 94°C for 1
minute, 57°C for 1 minute, 72°C for 1.5 minutes, and a final extension step at 7 2 T for 5 minutes for 35 cycles. The PCR products
were purified by agarose gel electrophoresis.
Dideoxynuckotide Fingerprinting (ddF) and Direct
Sequencing of cDNA
To screen the amplified WAS protein cDNA for mutations, we
used the modified ddF method originally described by Sarkar et a1"
and the fmol cycle sequencing kit (Promega, Madison, WI). Briefly,
50 to 75 ngof amplified cDNA, 2 pL ddC/dC mixture, 0.25 p L
(0.25 pmol) "P end-labeled primer, 1.25 pL 5X sequence buffer,
0.25 pL Taq polymerase, and distilled H20 to a total volume of 6
pL was incubated in an automatic DNA thermal cycler (PerkinElmer, Norwalk, CT) at 95°C for 30 seconds, 60°C for 30 seconds,
and 72°C for 1 minute for 30 cycles. The resulting products were
electrophoresed in 5% polyacrylamide nondenaturing gels at 4°C.
The mutations suggested by ddF were confirmed by direct sequenc-
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
WASP GENE MUTATIONS
X-THROMBOCYTOPENIA
IN WAS AND
3799
Table 2. Primw PainUsed for Fingerprinting and MutationAnalysis of cDNA and GenomkDNA in 12 WAS Families
Primers
Reaction
W3'P
Fragment
Size
Positiont
W-2*
5.24
GCCTCGCCAGAGAAGACAAG
GCAATCCCCAAAGGTACAGG
2-2 1
1064-1083
W-906$
W-1708cS
ACGACTTCATTGAGGACCAG
TGAGTGTGAGGACCAGGCAG
906-925
1688-1707
W-2
W-l5 6 ~
W-215
W-i2c
W-325
W-496c
GCCTCGCCAGAGAAGACAAG
CGTCCAAGCATCTCAAAGAG
GCTGAGCACTGGACCAAGGA
ACTGGCTTGCAAGTCCAGTC
GGAACAGGAGCTGTACTCAC
TCCACmGCCTCTGATTCC
2-21
137-156
215-234
Intron 2 ( + l 8 to +37)
325-344
477-496
6
W-i2
W-i4c
GTGCCTCAGTGCCACTGTGC
CTCACCTCTGCCCAACTTCC
Intron 2 ( --23 to -42)
Intron 4 (.t25 to +44)
7
5.28
5.24
CAAGAGGmCACTATGAAGG
GCAATCCCCAAAGGTACAGG
Intron 7 ( --51 to -71)
1064-1083
W-1157*
W-1472~
ACTGGACGTTCTGGACCACTG
CTCTGCTTCTCTTCTGCATC
1157-1177
1453-1472
5.30
W-illc
GGGAGAAATGCTCCmCC
ACGAGGCTGACACAAGAlTC
Intron 10 (-40 to -58)
Intron 11 (+l19 to
+138)
1
2
3
4
5
Exon 1:
Exon 2-9:
118 Exon 10:
165
Exon 9:
Exon 10-11:
220 Exon 12:
155
Exon 1:
799
60
522
93
Exon 2:
Intron 2:
Exon 3:
Intron 3:
Exon 4:
Intron 2:
Exon 3:
Intron 3:
Exon 4:
Intron 4
Intron 7:
Exon 8:
Intron 8:
Exon 9:
Intron 9:
Exon 1 0
Exon 10:
Intron 10:
Exon 11:
Intron 10:
37
70
100
102
42
87
100
103
44
71
43
201
154
205
118
216
251
100
58
Exon 11:
Intron 11:
115
128
Primers listed for reactions 1 and 2 were used to amplify WAS protein cDNA in two overlapping fragments. The other primers (reactions 3
through 9) were used to amplify genomic DNA of fragment size listed.
t The numbers represent the cDNA sequence position of exons or, if indicated, the position within introns. + indicates the position counted
from the 5' splice site of an intron; - indicates the position counted from the 3' splice site.
Used also for dideoxynucleotide fingerprinting, together with primers W-249 (starting at cDNA nucleotide 249) (5"GTGCTTCGTGAAGGATAACC), W-516 (5'-ACCACCAACACCAGCCAATG),W-756 (5'-CCAGTGGATTCAAGCATGTC), and W-l406 (5'-CCACCACCTCAGAGCTCAGA).
*
ing using selected primers and the fmol cycle sequencing kit (Promega) according to the manufacturer's recommendation.
DNA Pur$cation and Sequencing of Genomic DNA
resed through agaroselformaldehyde gel and transferred to a nylon
membrane (Magna NT, MSI, Westboro, MA). The radiolabeled
WAS protein cDNA clone M5.5, a 750-bp cDNA fragment o f the
WAS gene, was used as probe for hybridization, as previously de~cribed.'~ After
enrichment for Poly A (+) mRNA, the larger of the
two transcripts originally describedr3was no longer present, leaving
only the 2.0-kb transcript.
DNA was extracted from B-LCLs or spleen cells as previously
described.I6 Purified genomic DNA samples were amplified with
primer pairs (Table 2) designed to span the suspected mutation sites.
The PCR conditions used were as follows. For reactions 3 through
7 (Table 2), the samples were incubated for 5 minutes at94°C
followed by 30 cycles at 94°C for 1 minute, 55°C for 1 minute, and
72°C for 1 minute. The conditions for reactions 8 and 9 (Table 2)
were identical to those for reaction 2, which have been already
described. The amplifiedDNA fragments were separated by agarose
gel electrophoresis and electroelution. Direct sequencing was perfomed by the dideoxynucleotide chain termination method," using
either the sequenase DNA sequencing kit (US Biochemical Corp,
Cleveland, OH)or the fmol cycle sequencing kit (Promega) as recommended by the manufacturers.
To identify theapproximate location of mutations, we
screened the entire coding region and part of the noncoding
region of WASprotein cDNA withddF. This procedure
showed abnormal sequences in all 13 patients reported here
and allowed us to predict the approximate location of each
defect. AdditionalWASpatientsneedto
be studiedin a
similar manner to determine the overall effectiveness of this
method in detecting mutations.
Northern Blot Analysis
Direct Sequencing of cDNA and Genomic DNA
Poly A (+) mRNA samples isolated from 30 p g total RNA with
the PolyATtract mRNA isolation system (Promega) were electropho-
OnlyafterintroducingtheTaqExtender
PCR additive
provided by Stratagene were we able to consistently obtain
RESULTS
Screening for Mutations by ddF
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
ZHU ET AL
3800
61-65ins
c
C329T(stop)
Skipping of exon3
t + c,+2 n.t.,
ins of 114 bD
1372 ins 13 bp
I
/
/
/
/
+
/
exon 1
2
3
4 5 6
7
8
9
10
l1
12
C201T”
21l del T’
/\
G”
G291A’512-516ins
C741
C”
G2911T’
Fig 1. Schematic representation of the WAS gene showing the location of the mutations and nucleotide changes. *Patients reported in
Derry et al.” “Patients reported in Villa et al.”
PCR products from exons 10 and 11. Single nucleotide substitutions (point mutations), observed in the genomic DNA
of 10 patients withninenovelmutations,werethemost
frequent mutations; in the remaining 3 patients, we found
insertions or deletions of nucleotides within the genomic
DNA (Table I and Fig 1).
Pointmutations resulting in XLT. Two unrelatedpatients (AR and CS) with a mild phenotype (scores of l and
2) had the same missense mutation, a C * T transition at
position 290 in exon 2, resulting in the substitution of arginine with cystein (Arg86Cys). This mutation had no effect
on the amount of mRNA (Fig 2, lane 2). Patient JS had a
C A transversion at position 174 in exon 2, resulting in
the substitution of alanine with aspartic acid (Ala47Asp) and
a markedreduction in theamount of transcript (datanot
shown). cDNA analysis of patient BG showed a deletion of
the last 13 bp of exon 9 (bp953-965) due to an A + G
transition at position953 of genomic DNA. Instead ofchanging the normal codon 307 from ATG (Met) to GTG (Val),
this mutation activates a cryptic splice site at 13 nucleotides
5‘ to the normal splice site (5’-GAGgtgagg-3’; Fig 3). The
deletion of 13 nucleotides results in frameshift and premature
termination 137 triplets downstream in exon IO. Northern
blot analysis of BG’s B-LCLs indicates markedly reduced
transcript (Fig 2, lane 8).
Point mutations resulting in classic WAS. Patient MS
with a severe form of WAS hada G + A transition at position
407 in exon 4, resulting in the substitution of glycine with
arginine (Glyl25Arg). This missense mutation did not interfere with transcription (Fig 2, lane 3). Two patients (DM and
TH), both with severe WAS, were found to have nonsense
mutations due to single nucleotide substitutions resulting in
termination codons. DM had a C * T transition at position
71 in exon 1 (Arg13Ter) and normal amount of transcript
(Fig 2, lane 4). TH had a C T transition at position 995
in exon 10 (Arg321Ter) and reduced transcript (Fig 2, lane
5). The PCR-amplified cDNA obtained from a B-LCLof
patient RM consisted of a strong band that migrated faster
than the normal product and
a second, much fainter band
(<25%) of normalsize.Direct
sequence analysis of the
faster migrating, dominant band of RM’s amplified cDNA
showed a deletion of 87 bp, representing exon 3. Sequence
1 2 3 4 5 6 7 8 9 l0111213
-
+
+
Fig 2. Northern blot analysis of WAS gene expression. PolyA (+I
mRNA was electrophoresed and transferred to a nylon membrane,
and the blot was probed with the radiolabeled WAS protein cDNA
clone M5.5. Lane 1 represents a B-LCL from a normal control. Lanes
2 through 13 are WAS patients CS (lane 21, M S (3). DM (4). TH (5).
R M (6). M B (71, BG 181, B M (91, EA (101, PP (111, and CR (12). In lane
13, mRNA from patient JNl.l.l, known to have a markedly reduced
tran~cript,’~
is shown as a negative control. A strong single band of
2.0 kbcould be identified in the normal control andin the lanes
representingfive WAS patients. A control hybridizationwith pactin
cDNA (2.0 kbl is provided in each lane.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
GENE
WASP
MUTATIONS IN WAS AND X-THROMBOCYTOPENIA
:\
3801
A
1
c
c
G
A
G
G
Fig3.Sequenceanalysis
of
cDNA and genomic DNA derived
from Datient BG shows a deletion of 13 bp involving nucleotides 953 to 965 at the 3' end of
exon 9 caused by an A G transition at position 953. This mutation generated a new splice site
(gtg) within exon 9. The deleted
segment is boxed.
-
\
cDNA
Normal
BG
G
T
\l
A
G
G
A
c
G
G
analysis of genomic DNA showed that the skipping of exon
3 was the direct consequence of a nonsense mutation, a
C 4T transition, affecting nucleotide 329 in exon 3 (Fig 4);
this would result in the formation of a termination codon
(Gln99Ter). To explore the possibility of alternative splicing,
we sequenced individual clones of RM's cDNA after PCR
amplification. Ten clones were found to have exon 3 deleted
and 3 clones were of normal size and contained the termination codon. Northern blot analysis failed to detect transcript
(Fig 2, lane 6), suggesting that both mRNA populations are
poorly transcribed or unstable, although definitively present
as shown by RT-PCR. The t -P c transition at position +2
of the 5' splice site of intron 9 of patient MB resulted in the
activation of a cryptic splice site and the insertion of the 5'
segment of intron 9 consisting of 114 bp into this patient's
cDNA. This insertion caused frameshift and a termination
codon within the inserted segment. On Northern blot, the
amount of mRNA detected was severely reduced and appeared to migrate slower (Fig 2, lane 7). In patient BM, the
t 4 c transition at position + 2 of the 5' splice site of intron
cDNA
\
\
/
Normal
RM
GATC GATC
1g
G
1 1 resulted in the deletion of exon 11 (1 15 bp) due to loss
of the donor splice site. The resulting mRNA is abundant
and, as expected, of smaller size (Fig 2, lane 9). A protein
made from this transcript would lack38 amino acids encoded
by exon 11 and, because of frameshift, would be unstable.
Insertions and deletions affecting genomic DNA resulting
in classic WAS. In 3 unrelated WAS patients, the mutations
were due to the insertion or the deletion of nucleotides from
genomic DNA. EA was found to have a single nucleotide
(C) insertion between nucleotides 61 and 65 (GCCCG
GCCCCG) of exon I, affecting codon 11 and resulting in
frameshift and premature termination at codon 37; the
amount of mRNA on Northern blot was markedly reduced
(Fig 2, lane 10). A deletion and insertion of small DNA
fragments, observed in genomic DNA of patient PP, resulted
in a new splice site and the addition of 13 bp derived from
intron 10 (Fig 5), causing premature termination close to the
3'end of exon 12. A B-LCL derived from this patient contained abundant mRNA (Fig 2, lane 11). A proteinmade
from this transcript would be 4 amino acids shorter but most
/
/
\
v
C
\:
C
Fig 4. Sequenceanalysis of
cDNA and genomic DNA of the
WAS gene from a normal control
and patient RM. Analysis
of
cDNA obtained from an EBV-induced B-LCL established from
patient RM shows a deletion of
exon 3. Thisdeletion is due to a
C -Ttransition affecting nucleotide 329 within exon 3, resulting
in a termination codon and the
skipping of an exon.
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
ZHU ET AL
3802
A
B
G
G
C
G
A
E\
g
G
T
G
G
C
A
\
T
Genomic DNA
Normal
PP
C
C
C
C
T
G
G
G
i
C
B
A
C
A
C
C
del
r - - - - - - - - - -INormal-ccctctgtgdtgetccctd..
III1IIIIII
I I I I I I I I I I
PP
t
G
C
A
A
a
C
D
c'
Intron 10
Exon 11
c c t g c t g c ~ A C C C C T G G G G C C C C A G A GIIIIIIIIIIIIIIIIIIIIIIIIIIII
I I I I I I I I , I I I I , I I I I J I I I , I , , I ,
-c c ctc 1gtgc at c t g
I
L"""""""1
ACCCCTGGGGCCCCAGAG
Ins
Intron 104-l+
Exon 11 (ins 13 bp)
likely unstable due to frameshift and amino acid substitution
at the carboxy terminal. The deletion of a single nucleotide
(G) between nucleotides 1301 and 1305 (CGGGGGC +
CGGGGC) in exon 10 of patient CR. which was also found
in genomic DNA, resulted in frameshift, premature termination at codon 444, and either reduced transcription or unstable mRNA (Fig 2, lane 12).
DISCUSSION
To explore whether all WAS patients, irrespective of the
clinical phenotype, have mutations of the WAS gene, we
selected for mutation analysis 13 unrelated patients with
clinical and laboratory findings that ranged from isolated
platelet anomalies, characteristic for XLT, to the classic
WAS phenotype.
Using the dideoxy fingerprinting method for rapid screening, followed by sequence analysis of both cDNA and genomic DNA, we were able to identify mutations of the WAS
gene in 9 boys with classic WAS and in4 with thrombocytopenia and small platelets. The most common mutations observed were point mutations of genomic DNA that resulted
in either amino acid substitutions, the generation of termination codons, or the insertion or deletion of segments of
cDNA. Only 3 of the 12 mutations identified were due to
mutations other than single point mutations of genomic
DNA. These mutations include insertions and deletions of
single or multiple base pairs resulting in frameshift and
amino acid substitutions followed by early termination.
-
Fig 5. Sequence analysis of cDNA end genomic
DNA of patient PP shows an insertion of 13 bp in
patient pp,s cDNA ,Al. This
derived
from
intron 10, was due to a complex rearrangement including a deletion and insertion affecting intron 10
(B and Cl.
Of the 18 unique mutations of the WAS gene reported to
dateI3.l9(Fig l), 3 affected arginine 86, suggesting that codon
86 may be a mutation hot spot. Substitution of arginine
with cysteine (2 patients reported here) resulted in a mild
phenotype; substitution with leucine or with histidine" resulted in a classic phenotype.
A simple scoring system to assess disease severity was
designed to clearly identify patients with a mild phenotype
resembling XLT. Three of the four unrelated XLT patients
included in the analysis were found to have point mutations
resulting in amino acid substitutions within exon 2 (Arg86Cys in 2 patients and Ala47Asp in 1 patient). The fourth
patient with XLT had a point mutation within exon 9, resulting in the creation of a new splice site. This new splice
site caused the deletion of 13 nucleotides and resulted in
frameshift and the formation of a truncated and most likely
unstable protein. It is possible that the circulating lymphocytes of the affected members of this family express two
populations of mRNA: one having the deletion and the other
a less detritmental missense mutation, Met307Val. Villa et
all9 recently reported three unique mutations of the WAS
gene in 3 unrelated boys with thrombocytopenia and small
size platelets. Two of their patients had missense mutations
within exon 2 and exon 7, respectively. The third mutation
reported was more complex, consisting of a single nucleotide
insertion, which resulted in frameshift and termination in
exon 5 . At this time, without knowing the function of the
WAS protein, it is difficult to understand why mutations
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
3803
WASPGENE MUTATIONS IN WAS ANDX-THROMBOCYTOPENIA
resulting in truncated proteins can lead to a mild phenotype
affecting only platelets.
Of the 9 patients with classic WAS phenotypes (score 3
to 5), only 1 had a simple missense mutation. Of the remaining 8 patients, 2 had nonsense mutations affecting exons
1 and 10, respectively, and 6 had complex mutations resulting in deletions or insertions, frameshift, and early termination. Sequence analysis of R " s genomic DNA showed
a C + T transition at nucleotide 329, introducing a nonsense
codon. However, the RT-PCR product consisted of two
cDNA populations: one was of normal size and contained
the termination codon and the other was shorter in size due
to a deletion of 87 nucleotides. This deletion was found to
represent the 5' section of the exon described as exon 3 in
our original r e p ~ r t . 'Reexamination
~
of this region showed
that this exon was divided by a 100-nucleotide-long intron
into two exons: the 87-nucleotide-long exon 3 and the 102nucleotide-long exon 4 (see erratum to Deny et al,13 and
Ochs et all4). Sequence analysis of the exodintron junctions
surrounding the deleted exon failed to identify alternative
splice sites that could account for the skipping of exon 3.
Thus, the nonsense mutation at position 329 must be responsible for the deletion of exon 3 in the majority of the gene
transcripts. Skipping of exons containing nonsense mutations
has been observed in the fibrillin gene FBNl, which is responsible for Marfan's syndrome"; in the cystic fibrosis
gene"; and in the gene involved in the complementation
group C of Fanconi anemia:* suggesting that nonsense mutations may alter the selection or the effectiveness of regional
splice site^.'^,^^ The reduced amount of mRNA found in
this patient's B-LCL, suggesting insufficient transcription or
unstable mRNA, shows that the resulting protein is not only
truncated but also diminished, which is compatible with a
severe clinical phenotype.
Exodintron splice junction mutations were observed in
two unrelated families. Although both of these mutations
were t + c transitions at position+2 of the 5' splice site of
intron 9 and 11, respectively, the consequences on transcription were different, depending which intron was affected.
The t + c transition 5' of intron 9 resulted in the activation
of an alternative splice site, the inframe addition of 114
nucleotides derived from intron 9, the generation of a termination codon within the inserted segment and a severely
diminished transcript. The t + c transition 5' of intron 11
resulted in the skipping of exon 11, the loss of 115 nucleotides, an out of frame transcript, a truncated protein, and a
normal amount of transcript. For 5' splice sites, the generation of an alternative splice site is dependent on the presence
of such a site in the vicinity of the mutation site and on a
certain degree of homology between the sequences of the
old and the new splice sitesm In both patients, the consensus
sequence at the exodintron junction, Ggtgag, was mutated
to Ggcgag. In patient MB, a cryptic splice site, located 114
bp 3', with the sequence TGgtcag, was available, whereas
in patient BM, no such cryptic splice site existed, and, as a
consequence, exon 11 is skipped.
The tools are now available to identify mutations within
the WAS gene, to confirm the diagnosis in patients with
atypical phenotypes, to recognize carrier females, and to
perform prenatal diagnosis. However, despite cloning the
WAS gene and progress in identifying unique mutations,
little is known about the function of the WAS protein and
its role in the normal development of T and B lymphocytes
and the production of platelets. Analysis of the effect of
naturally occurring mutations of the WAS gene on cell function should provide new insights into the regulation of the
immune response and platelet homeostasis.
REFERENCES
1. Wiskott A: Familitirer, angeborener Morbus Werlhofii? Monatsschr Kinderheilkd 68:212, 1937
2. Aldrich, RA, Steinberg AG, Campbell DC: Pedigree demonstrating a sex-linked recessive condition characterized by draining
ears, eczematoid dermatitis and bloody diarrhea. Pediatrics 13:133,
1954
3. Cooper MD, Chase HP, Lowman JT, Krivit W, Good RA:
Wiskott-Aldrich syndrome: An immunologic deficiency disease involving the afferent limb of immunity. Am J Med 44:499, 1968
4. Sullivan KE, Mullen CA, Blaese RM, Winkelstein JA: A multiinstitutional survey of the Wiskott-Aldrich syndrome. J Pediatr
125:876, 1994
5. Ochs HD, Slichter SJ, Harker LA, Von Behrens WE, Clark
RA, Wedgwood W:The Wiskott-Aldrich syndrome: studies of lymphocytes, granulocytes, and platelets. Blood 55:243, 1980
6. Canales L, Mauer AM: Sex-linked hereditary thrombocytopenia as a variant of Wiskott-Aldrich syndrome. N Engl JMed
277:899, 1967
7. Chiaro JJ, Dharmkrong-at A, Bloom GE: X-linked thrombocytopenic purpura. I. Clinical and genetic studies of a kindred. Am J
Dis Child 123:565, 1972
8. Stormorken H, Hellum B, Egeland T, Abrahamsen TG,Hovig
T X-linked thrombocytopenia and thrombocytopathia: Attenuated
Wiskott-Aldrich syndrome. Thromb Haemost 65:300, 1991
9. Donnir M, Schwartz M, Carlsson KU, Holmberg L: Hereditary
X-linked thrombocytopenia maps to the same chromosomal region
as the Wiskott-Aldrich syndrome. Blood 72:1849, 1988
10. Notarangelo LD, Parolini 0, Faustini R, Porteri V, Albertini
A, Ugazio AG: Presentation of Wiskott-Aldrich syndrome as isolated
thrombocytopenia. Blood 77: 1125, 1991 (letter)
11. Puck JM, Siminovitch KA, Poncz M, Greenberg CR, Rottem
M, Conley M E : Atypical presentation of Wiskott-Aldrich syndrome:
Diagnosis in two unrelated males based on studies of maternal T
cell X chromosome inactivation. Blood 75:2369, 1990
12.De Saint-Bade G, Schlegel N, Caniglia M, Le Deist F,
Kaplan C, Lecompte T, Piller F, Fischer A, Griscelli C: X-linked
thrombocytopenia and Wiskott-Aldrich syndrome: Similar regional
assignment but distinct X-inactivation pattern in carriers. Ann Hemato1 63:107, 1991
13. Deny JMJ, Ochs HD, Francke U: Isolation of a novel gene
mutated in Wiskott-Aldrich syndrome. Cell 78:635, 1994 (erratum
Cell 79: 1994)
14. Ochs HD, Deny JMJ, Zhu Q, Blaese RM, Francke U: The
Wiskott-Aldrich gene, in Caragol I, EspaiiolT, Fontan G, Matamoros
N (eds): Progress in Immune Deficiency V. Barcelona, Spain,
Springer-Verlag Ibirica, 1995, p 32
15. Levitt D, Ochs HD, Wedgwood RJ: Epstein-Barr virus-induced lymphoblastoid cell lines derived from the peripheral blood
of patients with X-linked agammaglobulinemia can secrete IgM. J
Clin Immunol 4:143, 1984
16. Zhu Q, Zhang M, Rawlings DJ, Vihinen M, Hagemann T,
Saffran DC, Kwan S-P, Nilsson L, Smith CIE, Witte ON, Chen
S-H, Ochs HD: Deletion within the Src homology domain 3of
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
3804
Bruton’s tyrosine kinase resulting in X-linked agammaglobulinemia
(XLA). J Exp Med 180:461, 1994
17. Sarkar G, Yoon H-S, Sommer SS: Dideoxy fingerprinting
(ddF): A rapid and efficient screen for the presence of mutations.
Genomics 13:441, 1992
18. Sanger F, Nicklen S, Coulson AR:DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463,
1977
19. Villa A, Notarangelo L, Macchi P, Mantuano E, Cavagni G,
Brugnoni D, Strina D, Patrosso MC, Ramenghi U, Sacco MG,
Ugazio A, Vezzoni P: X-linked thrombocytopenia and Wiskott-Aldrich syndrome are allelic diseases with mutations in the WASP gene.
Nat Genet 9:414, 1995
20. Dietz HC, Valle D, Francomano CA, Kendzior RJ Jr, Pyeritz
ZHU ET
AL
RE, Cutting GR: The skipping of constitutive exons in vivo induced
by nonsense mutations. Science 259:680, 1993
21. Hull J, Shackleton S, Harris A: The stop mutation R553X in
the C R R gene results in exon slupping. Genomics 19:362, 1994
22. Gibson RA, Hajianpour A, Murer-Orlando M, Buchwald M,
Mathew CG: A nonsense mutation and exon skipping in the Fanconi
anaemia group C gene. Hum Mol Genet 2:797, 1993
23. Belgrader P, Maquat LE: Nonsense but not missense mutations can decrease the abundance of nuclear mRNA for the mouse
major urinary protein, while both types of mutations can facilitate
exon skipping. Mol Cell Biol 14:6326, 1994
24. Cooper DN, Krawczak M: Single basepair substitution, in
Cooper DN, Krawczak M (eds): Human Gene Mutation. Oxford,
UK, Bios Scientific Publishers, 1993, p 109 and 257
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1995 86: 3797-3804
The Wiskott-Aldrich syndrome and X-linked congenital
thrombocytopenia are caused by mutations of the same gene
Q Zhu, M Zhang, RM Blaese, JM Derry, A Junker, U Francke, SH Chen and HD Ochs
Updated information and services can be found at:
http://www.bloodjournal.org/content/86/10/3797.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.