Abstract

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immune deficiency disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. X-linked thrombocytopenia (XLT) is an allelic variant of WAS which presents with a milder phenotype, generally limited to thrombocytopenia. WAS and XLT are caused by mutations of the Wiskott-Aldrich syndrome protein (WASP) gene which encodes a 502-amino acid protein, named WASP. WASP is thought to play a role in actin cytoskeleton organization and cell signaling. Here, we report the identification of 141 unique mutations, 71 not previously reported, from 227 WAS/XLT families with a total of 262 affected members. When possible we studied the effects of these mutations on transcription, RNA splicing, and protein expression. By analyzing a large number of patients with WAS/XLT at the molecular level we identified 5 mutational hotspots in the WASP gene and have been able to establish a strong association between genotype and phenotype. (Blood. 2004;104:4010-4019)

Introduction

The Wiskott-Aldrich syndrome (WAS) (Online Mendelian Inheritance in Man [OMIM] 301000) is a rare X-linked recessive immunodeficiency disorder characterized by thrombocytopenia and small-sized platelets, eczema, recurrent bacterial and viral infections, a high incidence of autoimmunity, and an increased risk of malignancies.1,2  Immunologic abnormalities characteristic for patients with WAS involve both B- and T-cell function3  and include defective monocyte chemotaxis4  as well as abnormal morphology of stimulated dendritic cells.5  Most affected infants have normal numbers of circulating lymphocytes but often develop lymphopenia by age 6 to 8 years or earlier3  possibly because of increased apoptosis.6,7  Patients with the classic WAS phenotype fail to respond to polysaccharide and to selected protein antigens.3  X-linked thrombocytopenia (XLT) (OMIM 313900) is a relatively benign disorder characterized by congenital thrombocytopenia and small platelets, easy bruising, sometimes associated with transient eczema. The data presented here indicate that the prevalence of XLT equals that of WAS. The incidence of WAS/XLT, estimated to be 10 in 1 million live births,8  has to be updated when new data from the US and European registries become available. The gene responsible for WAS, the Wiskott-Aldrich Syndrome protein gene (WASP), was cloned and sequenced in l994.9  The WASP gene has 12 exons containing 1823 base pairs and encodes a 502-amino acid protein which is predominantly expressed in hematopoietic cells.9  It was subsequently shown that mutations of the WASP gene can cause X-linked thrombocytopenia, indicating that XLT is a mild allelic variant of WAS.10,11  More recently, it was recognized that mutations within the Cdc42 binding site cause congenital X-linked neutropenia, without the platelet defect or any of the other findings characteristic for WAS/XLT.12 

WASP contains several unique domains, including a pleckstrin homology (PH) domain, a guanosine triphosphatase (GTPase) binding domain (GBD), a proline-rich region with SH3 binding motifs, a verprolin and a cofilin homology domain, and the C-terminal acidic region which is involved in the reorganization of the actin cytoskeleton by activating Arp2/3 complex-mediated actin polymerization.13,14  Considering the unique functions of these domains, WASP appears to play critical roles in signal transduction and in the regulation of cytoskeletal reorganization.

Because of the complex structure and multifunctional design of WASP, and the variations in the clinical phenotype resulting from mutations of the WASP gene, a correlation between phenotype and genotype was examined by a number of investigators. A strong association, reported by some15-19  but not by others,2,20,21  may have implications on long-term prognosis and on the selection of therapies with variable risks.

In this analysis, we have examined the molecular defect in 227 unrelated WAS/XLT families studied in 2 centers, Seattle, Washington, and Brescia, Italy. We identified 141 unique mutations, of which 71 had not been previously reported. WASP mutations consisted of single nucleotide substitutions, splice site mutations, insertions, deletions, and complex mutations. A scoring system was designed to identify the clinical phenotype, and the effect of the mutations on transcription and protein expression was determined. Five hotspots, 2 preferentially associated with WAS and 3 with XLT, were identified, and a strong relationship between clinical phenotype and the expression of WASP was established.

Patients, materials, and methods

Patients

Of the 262 patients from 227 families included in this report, 184 (from 161 families) were studied molecularly at the University of Washington, Seattle, and included patients from the United States and Canada (n = 163), Central and South America (n = 5), Europe (n = 4), New Zealand (1), and South East Asia (n = 11), and the remaining 78 patients (from 68 families) were analyzed at the University of Brescia, Italy, and included patients from Europe (n = 68), the Middle East (n = 8), and New Zealand (n = 2) (Table 1). A total of 59 families listed in Table 1 were reported previously10,15,18,22,23  (see footnotes of Table 1). The clinical diagnoses of WAS or XLT were based on typical clinical and laboratory findings, including thrombocytopenia and small platelets, eczema, recurrent frequent or severe infections, and abnormal immune function. When possible, patients were assigned a score based on the clinical severity of their disease, using a previously described scoring system.11,24  Briefly, a score of 1 was assigned to patients with isolated thrombocytopenia and small platelets; a score of 0 to 1 (0.5) if thrombocytopenia was intermittent.23  A score of 2 was assigned to patients with microthrombocytopenia who had a history of localized eczema that responded promptly to standard therapy and/or occasionally suffered from uncomplicated upper respiratory infections. A score of 2 to 3 (2.5) was assigned to patients with microthrombocytopenia, persistent but therapy-responsive eczema, or frequent infections that may be severe enough to require intermittent antibiotic therapy; a score of 3 was given when both criteria, therapy-responsive eczema and frequent infections requiring intermittent antibiotics, were present. A score of 4 was assigned to patients with microthrombocytopenia, persistent and difficult-to-treat eczema requiring continuous treatment with steroid ointment and occasionally oral antibiotics for superinfection of the eczema, and/or severe life-threatening infections that may include abscesses, pneumonia (including Pneumocystis carinii pneumonia), meningitis, sepsis, and recurrent herpes simplex infection. A score of 5 was assigned to patients with WAS/XLT who developed autoimmunity or malignancy (most patients progressing to a score of 5 had classic WAS, some were originally diagnosed as XLT).

Table 1.

WASP mutations in 262 patients from 227 families


Patient
 

Origin
 

Age, y
 

Score
 

Exon
 

Mutation type
 

gDNA mutation
 

cDNA mutation (if different from gDNA)
 

Predicted protein change
 

Western blot
 
1-S   US   18   3   1   Insertion   41 insA   —††  fs stop aa 37   Absent  
2-B   Sweden   6 (dec.)   4   1   Insertion   41 insG   —   fs stop aa 37   ND  
3-S   US   4   5   1   Deletion   56-57delG   —   fs stop aa 44   Absent  
4*-S   US   ?   3   1   Insertion   62-64insC   —   fs stop aa 37   Absent  
5-S   US   0.2   3   1   Insertion   65-69insG   —   fs stop aa 37   ND  
6*-S   US   5.0 (dec.)   5   1   Nonsense   71C>T   —   R13X   Absent  
7a*/b-S   US   1.9/<2   4/3**  1   Nonsense   71C>T   —   R13X   Absent  
8-B   Italy   1.6 (dec.)   4   1   Nonsense   71C>T   —   R13X   ND  
9-B   Italy   8   4   1   Nonsense   74G>T   —   G14X   ND  
10-S   US   5   3-4   1   Missense   104T>C   —   S24P   Reduced  
11-S   Thailand   12   2   1   Missense   105C>T   ND   S24F   ND  
12§-B   Italy   1.9 (dec.)   5   1   Deletion   115-116delC   —   fs stop aa 44   Absent  
13*-S   US   12   2   1   Missense   113C>T   —   L27F   Reduced  
14-S   US   7.5   3-4   1   Deletion   122-124del   —   Inframe   ND  
15-S   US   1   1   1   Missense   125G>A   —   E31K   Absent  
16-S   Canada   2   2   1   Missense   125G>A   —   E31K   Reduced  
17-B   Italy   7   2-3   1   Missense   125G>A   —   E31K   Absent  
18-S   Chile   0.3   1   1   Missense   125G>A   —   E31K   Reduced  
19-S   US   0.4   ND   1   Missense   125G>A   —   E31K   Absent  
20-S   US   0.5   1   1   Nonsense   134C>T   —   R34X   ND  
21-S   US   1.1   3   1   Nonsense   ND   134C>T   R34X   Absent  
22—S   US   0.2   5   1   Nonsense   134C>T   —   R34X   Absent  
23-B   Turkey   5   2   1   Missense   138T>A   —   L35H   ND  
24*-S   US   2.1   5   1   Missense   138,139TC>AT   —   L35H   Reduced  
25-S   Greece   0.3   1   1   Deletion   140-142delTT   —   fs stop aa 36   Absent  
26*-S   US   4   4   1   Deletion   140-142delTT   —   fs stop aa 36   Absent  
27-B   Italy   2.3 (dec)   5   1   Deletion   140-142delTT   —   fs stop aa 36   ND  
28-S   US   0.4   ND   1   Insertion   140-142insT   —   fs stop aa 37   Absent  
29*-S   US   8   2   1   Missense   150T>C   —   L39P   Reduced  
30a*/b*-S   US   16/10   2/2   1   Missense   150T>C   —   L39P   Reduced  
31 a/b-B   Italy   14/11   2/2-3   1   Missense   150T>C   —   L39P   Reduced  
32§-B   Italy   6 (dec.)   5   1   Nonsense   155C>T   —   R41X   Absent  
33§-B   Italy   0.7 (dec.)   5   1   Nonsense   155C>T   —   R41X   ND  
34-B   Italy   9   4   1   Nonsense   155C>T   —   R41X   ND  
35-S   US   0.2   2   1   Nonsense   155C>T   —   R41X   Absent  
36-S   US   8   2   2   Missense   168C>T   —   T45M   Reduced  
37-S   US   0.3   1-2   2   Missense   168C>T   —   T45M   Absent  
38a*/b*-S   Canada   8/2   2/2   2   Missense   168C>T   —   T45M   Reduced  
39*-S   US   8   1   2   Missense   168C>T   —   T45M   Reduced  
40-S   US   ?   ND   2   Missense   168C>T   —   T45M   Reduced  
41-B   Croatia   6   2   2   Missense   168C>T   —   T45M   Reduced  
42-S   US   2.5   1   2   Missense   168C>T   —   T45M   Reduced  
43*-S   Polynesia   8   2   2   Missense   174C>A   —   A47D   Reduced  
44*-S   US   22   2   2   Missense   177C>T   —   T48I   Reduced  
45-S   US   6   4   2   Deletion   186-196del   —   fs stop aa 58   Absent  
46-S   US   9   1   2   Missense   201C>T   —   A56V   Reduced  
47-S   US   2.7   1   2   Missense   201C>T   —   A56V   ND  
48*-S   US   ?   1   2   Missense   201C>T   —   A56V   Reduced  
49-B   Italy   16   1   2   Missense   201C>T   —   A56V   Reduced  
50a/b-S   US   75/3   1/2   2   Missense   201C>T   —   A56V   Reduced  
51-S   US   1   ND   2   Deletion   201-213del   —   fs stop aa 71   ND  
52-S   US   ?   2   2   Missense   206C>G   —   P58A   Reduced  
53a/b/c∥-B   Italy   9/7/32   0.5/0.5/0.5   2   Missense   207C>G   —   P58R   Normal  
54*-S   Canada   5 (dec.)   5   2   Deletion   206-210delC   —   fs stop aa 75   ND  
55a*/b*-S   US   5/20   5/5   2   Deletion   211 delT   —   fs stop aa 75   Absent  
56-B   Nicaragua   1.5   4   2   Complex (2 populations)   211 delT   —   fs stop aa 75   ND  
       211T>C    P59P   
57-B   Italy   12 (dec.)   4   2   Missense   224T>C   —   W64R   ND  
58-S   US   13 (dec.)   5   2   Deletion   del 237-46   —   fs stop aa 72   Absent  
59-S   US   1.2   2-3   2   Missense   252G>A   —   C73Y   Reduced  
60-B   Italy   8   3   2   Missense   252G>A   —   C73Y   ND  
61-B   Russia   1.8   2-3   2   Missense   255T>C   —   F74S   ND  
62-S   India   5   2   2   Missense   257G>A   —   V75M   Reduced  
63a*/b*/c*-S   US   5/4/17   2/2/2   2   Missense   257G>A   —   V75M   Reduced  
64a/b-B   Italy   15/24   1/2   2   Missense   257G>A   —   V75M   Reduced  
65-S   US   3   1   2   Missense   257G>A   —   V75M   Normal  
68-S   US   6   2   2   Missense   261A>C   —   K76T   ND  
69-B   Italy   9   2   2   Missense   263G>C   —   D77H   Reduced  
70a/b-B   Italy   7/3   2/1   2   Missense   264A>G   —   D77G   Reduced  
71-S   US   1   2   2   Missense   278T>C   —   S82P   Reduced  
72-B   Serbia   9   5   2   Deletion   279-280delC   —   fs stop aa 126   ND  
73a/b-B   New Zealand   14/9   2/2   2   Missense   290C>A   —   R86S   ND  
74-S   US   11   2   2   Missense   290C>G   —   R86G   Reduced  
75-S   US, African American   19   2   2   Missense   290C>T   —   R86C   Absent  
76-S   US   9   2-3   2   Missense   290C>T   —   R86C   Reduced  
77-S   US   4   2   2   Missense   290C>T   —   R86C   Reduced  
78-S   US   5   2   2   Missense   290C>T   —   R86C   Reduced  
79a*/b*-S   US   ?   2/2   2   Missense   290C>T   —   R86C   Reduced  
80*-S   US   ?   1   2   Missense   290C>T   —   R86C   ND  
81-B   Israel   8   1   2   Missense   290C>T   —   R86C   Reduced  
82-S   US   2   2   2   Missense   290C>T   —   R86C   Reduced  
83-B   Turkey   13   2   2   Missense   290C>T   —   R86C   ND  
84-S   Malaysia   6   1   2   Missense   290C>T   —   R86C   Reduced  
85-S   US   15   1-2   2   Missense   291G>A   —   R86H   Absent  
86*-S   US   10   2   2   Missense   291G>A   —   R86H   Reduced  
87-S   US   0.5   2-3**  2   Missense   291G>A   —   R86H   Reduced  
88-B   Russia   17.5   3   2   Missense   291G>A   —   R86H   Reduced  
89-S   US   5.5   2   2   Missense   291G>T   —   R86L   Absent  
90*-S   Hungary   3.5   4   3   Deletion   312-313delGT   ND   ND   ND  
91-S   US   5   5   3   Nonsense   325G>A   —   W97X   Absent  
92-S   US   1.3   5   3   Nonsense   325G>A   —   W97X   Absent  
93-S   US   1.5   4   3   Nonsense   329C>T   ND   Q99X   Absent  
94*-S   US   ?   5   3   Complex   329C>T   329C>T/exon3del   Q99X/inframe Exon3del   Absent  
95-B   Finland   2.5   1-2   3   Missense   334G>T   —   E100D   ND  
96-B   Italy   5   2   3   Missense   348T>C   —   L105P   Reduced  
97*-S   US   <2   2**  3   Missense   354A>G   —   Y107C   Reduced  
98-S   US   <2   2**  3   Deletion   360-364delC   —   fs stop aa 126   Absent  
99-S   US   0.5   4   3   Deletion   360-364delC   —   fs stop aa 126   Absent  
100-B   Italy   7   4   3   Insertion   360-364insC   —   fs stop aa 121   Absent  
101-S   US   ?   2   3   Insertion   360-364insC   —   fs stop aa 121   ND  
102-S   US   2.1   5   3   Deletion   366-370delC   —   fs stop aa 126   Absent  
103-S   US   0.3   5   3   Deletion   371-372delT   —   fs stop aa 126   Absent  
104-S   US   0.9   1-2   3   Missense   390G>A   —   G119E   Reduced  
105a/b-S   Malaysia   4/0.2   4/1   4   Nonsense   400C>A   Multiple products   C122X   Absent  
106*-S    ?   5   4   Missense   407G>A   —   G125R   Absent  
107-S   Greece   1   5   4   Missense   411T>C   —   L126P   ND  
108-S   US   0.3   5   4   Missense   416T>C   —   F128L   Absent  
109-S   US   ?   5   4   Missense   416T>C   —   F128L   ND  
110*-S   US   9   4   4   Missense   417T>C   —   F128S   ND  
111*-S   US   8   3   4   Complex   425G>A,431G>A   —   E131K, E133K   Absent  
112a*/b*-S   US   14 (dec.)/3   5/4   4   Complex   425G>A,290C>T   —   E131K,R86C   Absent  
113-S   Chile   2   3   4   Missense   431G>A   —   E133K   ND  
114-B   Italy   3.5 (dec.)   5   4   Missense   431G>A   —   E133K   ND  
115-S   US   12   2   4   Missense   433G>T   —   E133D   Reduced  
116-S   US   0.7   1   4   Missense   433G>T   —   E133D   Reduced  
117*-S   US   15   4   4   Missense   435C>T   —   A134V   Reduced  
118-S   US   17   2   4   Insertion   C435 ins 6bp   —   134 DE ins   ND  
119-S   US   2   2   4   Complex   447G>A, IVS11-1g>a   447G>A, exon 11del   R149Q fs stop aa 543   Reduced  
120-B   Italy   3.5 (dec.)   4   4   Deletion   451-458del   —   fs stop aa 165   ND  
121-B   Italy   13 (dec.)   4   4   Insertion   471-476insA   ND   fs stop aa 168   ND  
122#-S   US   13   2   4   Insertion   471-476insA   —   fs stop aa 168   Absent  
123-B   Italy   6 (dec.)   5   4   Deletion   485-486delAG   —   fs stop aa 167   Absent  
124a/b‡-B   Serbia   17/21   5/5   5   Insertion   512-516insC   —   fs stop aa 168   ND  
125-S   US   4   5   5   Insertion   512-516insC   —   fs stop aa 168   Absent  
126-B   Italy   22   5   6   Deletion   570delT   —   fs stop aa 260   ND  
127-S   US   ?   2   6   Deletion   597-597delC   ND   fs stop aa 260   ND  
128-B   Italy   3   4   7   Deletion   644delA   —   fs stop aa 260   Absent  
129-S   US   0.5 (dec.)   2-3**  7   Nonsense   665C>T   —   R211X   Absent  
130a/b-S   US   1.5/0.8   4/3   7   Nonsense   665C>T   —   R211X   Absent  
131-S   US   1   5   7   Nonsense   665C>T   —   R211X   Absent  
132-S   US   ?   3-4   7   Nonsense   665C>T   —   R211X   Absent  
133-S   US Latino   <2   2**  7   Nonsense   665C>T   —   R211X   Absent  
134*-S   US   14   4   7   Nonsense   665C>T   —   R211X   Absent  
135*-S   US   11   4   7   Nonsense   665C>T   —   R211X   Absent  
136-B   Italy   5   4   7   Nonsense   665C>T   —   R211X   ND  
137-B   Italy   6   4   7   Nonsense   665C>T   —   R211X   ND  
138-B   Italy   2.1 (dec.)   4   7   Nonsense   665C>T   —   R211X   ND  
139*-S   US   1.9   4   7   Splice   705A>G   705-768 del   fs stop aa 239   Absent  
140-S   US   >5   5   7   Splice   705A>G   705-768 del   fs stop aa 239   Absent  
141-B   Sweden   <2   2**  7   Nonsense   717C>G   —   S228X   ND  
142-S   US   4   3-4   7   Deletion   731delA   —   fs stop aa 260   Absent  
143‡B   Italy   15   1   7   Missense   741C>G   —   A236G   Reduced  
144-S   US   0.7   ND   7   Complex   Exon 7(−5 to IVS7 +22) (del 27 bp)   764-768 del. IVS7 +22 to 71 (ins 49 bp)   fs stop aa 257   Absent  
145§-B   England   4 (dec.)   5   9   Nonsense   887G>T   —   E285X   ND  
146-B   Italy   10   2   9   Deletion   907delC   —   fs stop aa 291   ND  
147*-S   US   2.5   5   9   Nonsense   923C>T   —   Q297X   Absent  
148-S   Turkey   1.2   3   9   Nonsense   950G>T   —   E306X   Reduced  
149a*/b*†-S   US   ?/1.0   2/2   9   Missense   953A>G   —   M307V   ND  
150a*/b*-S   India   3/1   3/2**  10   Nonsense   995C>T   —   R321X   Absent  
151a*/b*-S   US   ?   5/5   10   Nonsense   995C>T   —   R321X   Absent  
152-B   Italy   7   5   10   Nonsense   995C>T   —   R321X   ND  
153-S   US   ?   ND   10   Nonsense   995C>T   —   R321X   Absent  
154a/b-S   US Latino   7.0/0.2   ND   10   Nonsense   995C>T   —   R321X   Absent  
155-S   US   1   2   10   Deletion   1017-1018delC   Multiple products   Multiple products   Absent  
156a*/b-S   US   3/0.5   2/2   10   Insertion   1025insA   ND   fs stop aa 335   Absent  
157-B   Italy   3 (dec.)   5   10   Deletion   1028delG   ND   fs stop aa 444   Reduced  
158*-S   US   ?   2   10   Complex   1029insT   del 966-1121 (50%)   inframe del 52aa   Red/trunc  
        del exon 10 (25%)   fs stop aa358   
         fs stop aa335   
        1029 insT (25%)    
159*-S   US   10   4   10   Deletion   1030-1035delG   —   fs stop aa 444   Absent  
160-S   US   ?   ND   10   Deletion   1030-1035delG   —   fs stop aa 444   Absent  
161-S   Brasil   2.2   ND   10   Deletion   1030-1035delG   —   fs stop aa 444   ND  
162a/b-S   US   7.0/5   4/4   10   Deletion   1088-1092delC   Multiple products   Multiple products, fs stop aa 444   Absent  
163-S   US   4   5   10   Deletion   1088-1092delC   ND   Multiple products, fs stop aa 444   ND  
164-S   New Zealand   10   2   10   Deletion   1107-1108delGA   Multiple products   Multiple products, fs stop aa 493   Red/trunc  
165*-S   US   8   5   10   Deletion   1109-1113delC   —   fs stop aa 444   Absent  
166*-S   US   6   4   10   Complex   1109C>A, 1110-1113delC   —   P359T, fs stop aa 444   Red/trunc  
167-B   Turkey   5   4   10   Missense   1115C>A   —   P361T   ND  
168-S   Malaysia   9   5   10   Deletion   1115-1119delC   —   fs stop aa 444   ND  
169§-B   Italy   <2   2**  10   Complex   1115-1119insC   —   fs stop aa 494   ND  
      (2 populat.)   1110-1182del    infr.del24aa   
170-B   Turkey   12   5   10   Nonsense   1124C>T   —   R364X   ND  
171-S   US   2   3   10   Deletion   1145-1149delC   Exon 10 del   fs stop aa 444   Absent  
172-B   Italy   10   5   10   Deletion   1184-1185delC   —   fs stop aa 444   Absent  
173-S   Canada   0.5   3   10   Insertion   1187-1191 insC   —   fs stop aa 494   ND  
174-B
 
Bosnia
 
4
 
5
 
10
 
Deletion
 
1192delT
 

 
fs stop aa 444
 
Absent
 
175-S   Puerto Rico   1   4   10   Insertion   1286 ins4bp   —   fs stop aa 495   Absent  
176*-S   US   1.8   4   10   Insertion   1301-1305 insG   —   fs stop aa 494   Red/trunc  
177*-S   US Latino   8   5   10   Insertion   1301-1305 insG   —   fs stop aa 494   Red/trunc  
178*-S   US   14   5   10   Deletion   1301-1305 insG   —   fs stop aa 494   Red/trunc  
179∥-B   Italy   9   0.5   11   Missense   1476T>A   —   1481N   normal  
180-B   Italy   7   5   11   Insertion   1486-1487insTC   —   fs lack of term. codon   ND  
181-B   Russia   6   5   12   Deletion   1519delT   —   fs lack of term. codon   Absent  
182-S   US   2.5   1   12   Missense   1542G>C   —   X503S   Absent  
183-B   Turkey   8.5   2   12   Missense   1542G>C   —   X503S   ND  
184-S   US   <2   3-4**  5′UTR-Intron 2   Deletion   del 5′UTR -719 to IVS2+1   No product   No product   Absent  
185-B   Russia   6   4   Intron 1   Splice   IVSI-2a>g   ND   ND   ND  
186-S   US   2.1   5   Intron 2   Splice   IVS2+1g>a   ND   ND   Absent  
187-S   US   13   2   Intron 2   Splice   IVS2+1g>t   ND   ND   Absent  
188-S   US   0.5   ND   Intron 2   Splice   IVS2-1 g>a   Exon 3 del   Inframe del 52 aa   ND  
189*-S   US   4.0   3   Intron 3   Splice   IVS3-1 g>a   ins intron 3   fs stop aa 201   Absent  
190a/b-B   Russia   5/7   3/3   Intron 4   Splice   IVS4-7 t>g   ND   ND   ND  
191-B   Croatia   8   5   Intron 6   Splice   INS6+2t>g   ND   ND   ND  
192*-S   US   7   1   Intron 6   Splice   IVS6+5 g>a   70% ins38nt intron 6/30% normal   fs stop aa 190/normal   Reduced  
193-S   US   14.0   2   Intron 6   Splice   IVS6+5 g>a   ins 38nt intron 6/normal   fs stop aa 190/normal   Reduced  
194-S   US   1.4   2   Intron 6   Splice   IVS6+5 g>a   ins 38nt intron 6/normal   fs stop aa 190/normal   Reduced  
195a/b/c/d-S   US   6/18/7/9   1/1/1/1   Intron 6   Splice   IVS6+5 g>a   ins 38nt intron 6/normal   fs stop aa 190/normal   Reduced  
196a¶/b-S   US   43 (dec.)/55   5/1   Intron 6   Splice   IVS6+5 g>a   ins 38nt intron 6/normal   fs stop aa 190/normal   Reduced  
197-S   US African American   12   2   Intron 6   Splice   IVS6+5 g>a   ins 38nt intron 6/normal   fs stop aa 190/normal   Reduced  
198a/b-B   Italy   4/1.8 (dec.)   5/5   Intron 7   Splice   IVS7+1g>t   ND   ND   Absent  
199-B   Sweden   10 (dec.)   4   Intron 7   Splice   IVS7+1g>a   ND   ND   ND  
200-S   US   <3   2   Intron 8   Splice   IVS8+1g>a   Exon 8 del   fs stop aa 246   Absent  
201-S   US   1   4   Intron 8   Splice   IVS8+1g>a   Exon 8 del   fs stop aa 246   Absent  
202-S   US American Indian   10   3-4   Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   Absent  
203-S   US   <2   2   Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   Absent  
204-B   Italy   7.9   2-3**  Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   ND  
205-B   Italy   1.5 (dec.)   5   Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   Absent  
206-B   Sweden   6   2   Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   ND  
207-S   US   0.1   ND   Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   Absent  
208a/b/c/d-S   US Latino   12/5/1.5/0.5   4**/3**/3**/2   Intron 8   Splice   IVS8+1g>c   ND   fs stop aa 246   Absent  
209-S   Nepal   2   ND   Intron 8   Splice   IVS8+1g>1   Exon 8 del   ND   ND  
210-S   US   0.8   3   Intron 8   Splice   IVS8+1 to+6 del gtga   Exon 8 del   fs stop aa 246   Absent  
211-S   Thailand   9   4   Intron 8   Splice   IVS8+1 to+6 del gtga   Exon 8 del   fs stop aa 246   Absent  
212-S   US   0.5   2   Intron 8   Splice   IVS8+1 to+6 del gtga   Exon 8 del   fs stop aa 246   Absent  
213-S   US   1.0   3   Intron 8   Splice   IVS8+1 to+6 del gtga   Exon 8 del   fs stop aa 246   Reduced, normal size  
214-S   US   2.0   ND   Intron 8   Splice   IVS8+2 to3 del tg   Exon 8 del   fs stop aa 246   Absent  
215a§/b-B   Italy   11/2.1   3/2   Intron 8   Splice   IVS8+3insT   ND   ND   ND  
216-S   US   8   4   Intron 8   Splice   IVS8+5g>a   Exon 8 del   fs stop aa 246   Absent  
217-S   US   3.0   2   Intron 8   Splice   IVS8-2a>g   Intron 8ins   fs stop aa 328   Absent  
218-S   US   1   3   Intron 9   Splice   IVS9+1g>a   ND   ND   Absent  
219-B   Italy   22   4   Intron 9   Splice   IVS9+2del tgag   ND   ND   ND  
220a*/b*-S   US   2/<2   4/2**  Intron 9   Splice   IVS9+2t>c   80% ins 114nt of Intron 9 20% Intron9ins   fs stop aa 326   Absent  
221-B   Sweden   22   5   Intron 9   Splice   IVS9+2t>g   ND   ND   ND  
222*-S   US Greek   1   4   Intron 10   Complex   IVS10del 9nt, ins 11 nt   1372 ins 13nt   fs stop aa 498   Red/trunc  
223§-B   Italy   <2   2-3**  Intron 10   Splice   IVS10-3delC   ND   ND   ND  
224§-B   Italy   <2   1**  Intron 10   Splice   IVS10-2a>t   ND   ND   Reduced  
225-B   Turkey   7   4   Intron 10   Splice   IVS10-2a>c   ND   ND   ND  
226*-S   Canada   4   4   Intron 11   Splice   IVS11+2t>c   Exon 11 del   fs stop aa 543   Absent  
227*-S
 
US
 
?
 
5
 
Intron 11
 
Splice
 
IVS11+2t>g
 
Exon 11 del
 
fs stop aa 543
 
Absent
 

Patient
 

Origin
 

Age, y
 

Score
 

Exon
 

Mutation type
 

gDNA mutation
 

cDNA mutation (if different from gDNA)
 

Predicted protein change
 

Western blot
 
1-S   US   18   3   1   Insertion   41 insA   —††  fs stop aa 37   Absent  
2-B   Sweden   6 (dec.)   4   1   Insertion   41 insG   —   fs stop aa 37   ND  
3-S   US   4   5   1   Deletion   56-57delG   —   fs stop aa 44   Absent  
4*-S   US   ?   3   1   Insertion   62-64insC   —   fs stop aa 37   Absent  
5-S   US   0.2   3   1   Insertion   65-69insG   —   fs stop aa 37   ND  
6*-S   US   5.0 (dec.)   5   1   Nonsense   71C>T   —   R13X   Absent  
7a*/b-S   US   1.9/<2   4/3**  1   Nonsense   71C>T   —   R13X   Absent  
8-B   Italy   1.6 (dec.)   4   1   Nonsense   71C>T   —   R13X   ND  
9-B   Italy   8   4   1   Nonsense   74G>T   —   G14X   ND  
10-S   US   5   3-4   1   Missense   104T>C   —   S24P   Reduced  
11-S   Thailand   12   2   1   Missense   105C>T   ND   S24F   ND  
12§-B   Italy   1.9 (dec.)   5   1   Deletion   115-116delC   —   fs stop aa 44   Absent  
13*-S   US   12   2   1   Missense   113C>T   —   L27F   Reduced  
14-S   US   7.5   3-4   1   Deletion   122-124del   —   Inframe   ND  
15-S   US   1   1   1   Missense   125G>A   —   E31K   Absent  
16-S   Canada   2   2   1   Missense   125G>A   —   E31K   Reduced  
17-B   Italy   7   2-3   1   Missense   125G>A   —   E31K   Absent  
18-S   Chile   0.3   1   1   Missense   125G>A   —   E31K   Reduced  
19-S   US   0.4   ND   1   Missense   125G>A   —   E31K   Absent  
20-S   US   0.5   1   1   Nonsense   134C>T   —   R34X   ND  
21-S   US   1.1   3   1   Nonsense   ND   134C>T   R34X   Absent  
22—S   US   0.2   5   1   Nonsense   134C>T   —   R34X   Absent  
23-B   Turkey   5   2   1   Missense   138T>A   —   L35H   ND  
24*-S   US   2.1   5   1   Missense   138,139TC>AT   —   L35H   Reduced  
25-S   Greece   0.3   1   1   Deletion   140-142delTT   —   fs stop aa 36   Absent  
26*-S   US   4   4   1   Deletion   140-142delTT   —   fs stop aa 36   Absent  
27-B   Italy   2.3 (dec)   5   1   Deletion   140-142delTT   —   fs stop aa 36   ND  
28-S   US   0.4   ND   1   Insertion   140-142insT   —   fs stop aa 37   Absent  
29*-S   US   8   2   1   Missense   150T>C   —   L39P   Reduced  
30a*/b*-S   US   16/10   2/2   1   Missense   150T>C   —   L39P   Reduced  
31 a/b-B   Italy   14/11   2/2-3   1   Missense   150T>C   —   L39P   Reduced  
32§-B   Italy   6 (dec.)   5   1   Nonsense   155C>T   —   R41X   Absent  
33§-B   Italy   0.7 (dec.)   5   1   Nonsense   155C>T   —   R41X   ND  
34-B   Italy   9   4   1   Nonsense   155C>T   —   R41X   ND  
35-S   US   0.2   2   1   Nonsense   155C>T   —   R41X   Absent  
36-S   US   8   2   2   Missense   168C>T   —   T45M   Reduced  
37-S   US   0.3   1-2   2   Missense   168C>T   —   T45M   Absent  
38a*/b*-S   Canada   8/2   2/2   2   Missense   168C>T   —   T45M   Reduced  
39*-S   US   8   1   2   Missense   168C>T   —   T45M   Reduced  
40-S   US   ?   ND   2   Missense   168C>T   —   T45M   Reduced  
41-B   Croatia   6   2   2   Missense   168C>T   —   T45M   Reduced  
42-S   US   2.5   1   2   Missense   168C>T   —   T45M   Reduced  
43*-S   Polynesia   8   2   2   Missense   174C>A   —   A47D   Reduced  
44*-S   US   22   2   2   Missense   177C>T   —   T48I   Reduced  
45-S   US   6   4   2   Deletion   186-196del   —   fs stop aa 58   Absent  
46-S   US   9   1   2   Missense   201C>T   —   A56V   Reduced  
47-S   US   2.7   1   2   Missense   201C>T   —   A56V   ND  
48*-S   US   ?   1   2   Missense   201C>T   —   A56V   Reduced  
49-B   Italy   16   1   2   Missense   201C>T   —   A56V   Reduced  
50a/b-S   US   75/3   1/2   2   Missense   201C>T   —   A56V   Reduced  
51-S   US   1   ND   2   Deletion   201-213del   —   fs stop aa 71   ND  
52-S   US   ?   2   2   Missense   206C>G   —   P58A   Reduced  
53a/b/c∥-B   Italy   9/7/32   0.5/0.5/0.5   2   Missense   207C>G   —   P58R   Normal  
54*-S   Canada   5 (dec.)   5   2   Deletion   206-210delC   —   fs stop aa 75   ND  
55a*/b*-S   US   5/20   5/5   2   Deletion   211 delT   —   fs stop aa 75   Absent  
56-B   Nicaragua   1.5   4   2   Complex (2 populations)   211 delT   —   fs stop aa 75   ND  
       211T>C    P59P   
57-B   Italy   12 (dec.)   4   2   Missense   224T>C   —   W64R   ND  
58-S   US   13 (dec.)   5   2   Deletion   del 237-46   —   fs stop aa 72   Absent  
59-S   US   1.2   2-3   2   Missense   252G>A   —   C73Y   Reduced  
60-B   Italy   8   3   2   Missense   252G>A   —   C73Y   ND  
61-B   Russia   1.8   2-3   2   Missense   255T>C   —   F74S   ND  
62-S   India   5   2   2   Missense   257G>A   —   V75M   Reduced  
63a*/b*/c*-S   US   5/4/17   2/2/2   2   Missense   257G>A   —   V75M   Reduced  
64a/b-B   Italy   15/24   1/2   2   Missense   257G>A   —   V75M   Reduced  
65-S   US   3   1   2   Missense   257G>A   —   V75M   Normal  
68-S   US   6   2   2   Missense   261A>C   —   K76T   ND  
69-B   Italy   9   2   2   Missense   263G>C   —   D77H   Reduced  
70a/b-B   Italy   7/3   2/1   2   Missense   264A>G   —   D77G   Reduced  
71-S   US   1   2   2   Missense   278T>C   —   S82P   Reduced  
72-B   Serbia   9   5   2   Deletion   279-280delC   —   fs stop aa 126   ND  
73a/b-B   New Zealand   14/9   2/2   2   Missense   290C>A   —   R86S   ND  
74-S   US   11   2   2   Missense   290C>G   —   R86G   Reduced  
75-S   US, African American   19   2   2   Missense   290C>T   —   R86C   Absent  
76-S   US   9   2-3   2   Missense   290C>T   —   R86C   Reduced  
77-S   US   4   2   2   Missense   290C>T   —   R86C   Reduced  
78-S   US   5   2   2   Missense   290C>T   —   R86C   Reduced  
79a*/b*-S   US   ?   2/2   2   Missense   290C>T   —   R86C   Reduced  
80*-S   US   ?   1   2   Missense   290C>T   —   R86C   ND  
81-B   Israel   8   1   2   Missense   290C>T   —   R86C   Reduced  
82-S   US   2   2   2   Missense   290C>T   —   R86C   Reduced  
83-B   Turkey   13   2   2   Missense   290C>T   —   R86C   ND  
84-S   Malaysia   6   1   2   Missense   290C>T   —   R86C   Reduced  
85-S   US   15   1-2   2   Missense   291G>A   —   R86H   Absent  
86*-S   US   10   2   2   Missense   291G>A   —   R86H   Reduced  
87-S   US   0.5   2-3**  2   Missense   291G>A   —   R86H   Reduced  
88-B   Russia   17.5   3   2   Missense   291G>A   —   R86H   Reduced  
89-S   US   5.5   2   2   Missense   291G>T   —   R86L   Absent  
90*-S   Hungary   3.5   4   3   Deletion   312-313delGT   ND   ND   ND  
91-S   US   5   5   3   Nonsense   325G>A   —   W97X   Absent  
92-S   US   1.3   5   3   Nonsense   325G>A   —   W97X   Absent  
93-S   US   1.5   4   3   Nonsense   329C>T   ND   Q99X   Absent  
94*-S   US   ?   5   3   Complex   329C>T   329C>T/exon3del   Q99X/inframe Exon3del   Absent  
95-B   Finland   2.5   1-2   3   Missense   334G>T   —   E100D   ND  
96-B   Italy   5   2   3   Missense   348T>C   —   L105P   Reduced  
97*-S   US   <2   2**  3   Missense   354A>G   —   Y107C   Reduced  
98-S   US   <2   2**  3   Deletion   360-364delC   —   fs stop aa 126   Absent  
99-S   US   0.5   4   3   Deletion   360-364delC   —   fs stop aa 126   Absent  
100-B   Italy   7   4   3   Insertion   360-364insC   —   fs stop aa 121   Absent  
101-S   US   ?   2   3   Insertion   360-364insC   —   fs stop aa 121   ND  
102-S   US   2.1   5   3   Deletion   366-370delC   —   fs stop aa 126   Absent  
103-S   US   0.3   5   3   Deletion   371-372delT   —   fs stop aa 126   Absent  
104-S   US   0.9   1-2   3   Missense   390G>A   —   G119E   Reduced  
105a/b-S   Malaysia   4/0.2   4/1   4   Nonsense   400C>A   Multiple products   C122X   Absent  
106*-S    ?   5   4   Missense   407G>A   —   G125R   Absent  
107-S   Greece   1   5   4   Missense   411T>C   —   L126P   ND  
108-S   US   0.3   5   4   Missense   416T>C   —   F128L   Absent  
109-S   US   ?   5   4   Missense   416T>C   —   F128L   ND  
110*-S   US   9   4   4   Missense   417T>C   —   F128S   ND  
111*-S   US   8   3   4   Complex   425G>A,431G>A   —   E131K, E133K   Absent  
112a*/b*-S   US   14 (dec.)/3   5/4   4   Complex   425G>A,290C>T   —   E131K,R86C   Absent  
113-S   Chile   2   3   4   Missense   431G>A   —   E133K   ND  
114-B   Italy   3.5 (dec.)   5   4   Missense   431G>A   —   E133K   ND  
115-S   US   12   2   4   Missense   433G>T   —   E133D   Reduced  
116-S   US   0.7   1   4   Missense   433G>T   —   E133D   Reduced  
117*-S   US   15   4   4   Missense   435C>T   —   A134V   Reduced  
118-S   US   17   2   4   Insertion   C435 ins 6bp   —   134 DE ins   ND  
119-S   US   2   2   4   Complex   447G>A, IVS11-1g>a   447G>A, exon 11del   R149Q fs stop aa 543   Reduced  
120-B   Italy   3.5 (dec.)   4   4   Deletion   451-458del   —   fs stop aa 165   ND  
121-B   Italy   13 (dec.)   4   4   Insertion   471-476insA   ND   fs stop aa 168   ND  
122#-S   US   13   2   4   Insertion   471-476insA   —   fs stop aa 168   Absent  
123-B   Italy   6 (dec.)   5   4   Deletion   485-486delAG   —   fs stop aa 167   Absent  
124a/b‡-B   Serbia   17/21   5/5   5   Insertion   512-516insC   —   fs stop aa 168   ND  
125-S   US   4   5   5   Insertion   512-516insC   —   fs stop aa 168   Absent  
126-B   Italy   22   5   6   Deletion   570delT   —   fs stop aa 260   ND  
127-S   US   ?   2   6   Deletion   597-597delC   ND   fs stop aa 260   ND  
128-B   Italy   3   4   7   Deletion   644delA   —   fs stop aa 260   Absent  
129-S   US   0.5 (dec.)   2-3**  7   Nonsense   665C>T   —   R211X   Absent  
130a/b-S   US   1.5/0.8   4/3   7   Nonsense   665C>T   —   R211X   Absent  
131-S   US   1   5   7   Nonsense   665C>T   —   R211X   Absent  
132-S   US   ?   3-4   7   Nonsense   665C>T   —   R211X   Absent  
133-S   US Latino   <2   2**  7   Nonsense   665C>T   —   R211X   Absent  
134*-S   US   14   4   7   Nonsense   665C>T   —   R211X   Absent  
135*-S   US   11   4   7   Nonsense   665C>T   —   R211X   Absent  
136-B   Italy   5   4   7   Nonsense   665C>T   —   R211X   ND  
137-B   Italy   6   4   7   Nonsense   665C>T   —   R211X   ND  
138-B   Italy   2.1 (dec.)   4   7   Nonsense   665C>T   —   R211X   ND  
139*-S   US   1.9   4   7   Splice   705A>G   705-768 del   fs stop aa 239   Absent  
140-S   US   >5   5   7   Splice   705A>G   705-768 del   fs stop aa 239   Absent  
141-B   Sweden   <2   2**  7   Nonsense   717C>G   —   S228X   ND  
142-S   US   4   3-4   7   Deletion   731delA   —   fs stop aa 260   Absent  
143‡B   Italy   15   1   7   Missense   741C>G   —   A236G   Reduced  
144-S   US   0.7   ND   7   Complex   Exon 7(−5 to IVS7 +22) (del 27 bp)   764-768 del. IVS7 +22 to 71 (ins 49 bp)   fs stop aa 257   Absent  
145§-B   England   4 (dec.)   5   9   Nonsense   887G>T   —   E285X   ND  
146-B   Italy   10   2   9   Deletion   907delC   —   fs stop aa 291   ND  
147*-S   US   2.5   5   9   Nonsense   923C>T   —   Q297X   Absent  
148-S   Turkey   1.2   3   9   Nonsense   950G>T   —   E306X   Reduced  
149a*/b*†-S   US   ?/1.0   2/2   9   Missense   953A>G   —   M307V   ND  
150a*/b*-S   India   3/1   3/2**  10   Nonsense   995C>T   —   R321X   Absent  
151a*/b*-S   US   ?   5/5   10   Nonsense   995C>T   —   R321X   Absent  
152-B   Italy   7   5   10   Nonsense   995C>T   —   R321X   ND  
153-S   US   ?   ND   10   Nonsense   995C>T   —   R321X   Absent  
154a/b-S   US Latino   7.0/0.2   ND   10   Nonsense   995C>T   —   R321X   Absent  
155-S   US   1   2   10   Deletion   1017-1018delC   Multiple products   Multiple products   Absent  
156a*/b-S   US   3/0.5   2/2   10   Insertion   1025insA   ND   fs stop aa 335   Absent  
157-B   Italy   3 (dec.)   5   10   Deletion   1028delG   ND   fs stop aa 444   Reduced  
158*-S   US   ?   2   10   Complex   1029insT   del 966-1121 (50%)   inframe del 52aa   Red/trunc  
        del exon 10 (25%)   fs stop aa358   
         fs stop aa335   
        1029 insT (25%)    
159*-S   US   10   4   10   Deletion   1030-1035delG   —   fs stop aa 444   Absent  
160-S   US   ?   ND   10   Deletion   1030-1035delG   —   fs stop aa 444   Absent  
161-S   Brasil   2.2   ND   10   Deletion   1030-1035delG   —   fs stop aa 444   ND  
162a/b-S   US   7.0/5   4/4   10   Deletion   1088-1092delC   Multiple products   Multiple products, fs stop aa 444   Absent  
163-S   US   4   5   10   Deletion   1088-1092delC   ND   Multiple products, fs stop aa 444   ND  
164-S   New Zealand   10   2   10   Deletion   1107-1108delGA   Multiple products   Multiple products, fs stop aa 493   Red/trunc  
165*-S   US   8   5   10   Deletion   1109-1113delC   —   fs stop aa 444   Absent  
166*-S   US   6   4   10   Complex   1109C>A, 1110-1113delC   —   P359T, fs stop aa 444   Red/trunc  
167-B   Turkey   5   4   10   Missense   1115C>A   —   P361T   ND  
168-S   Malaysia   9   5   10   Deletion   1115-1119delC   —   fs stop aa 444   ND  
169§-B   Italy   <2   2**  10   Complex   1115-1119insC   —   fs stop aa 494   ND  
      (2 populat.)   1110-1182del    infr.del24aa   
170-B   Turkey   12   5   10   Nonsense   1124C>T   —   R364X   ND  
171-S   US   2   3   10   Deletion   1145-1149delC   Exon 10 del   fs stop aa 444   Absent  
172-B   Italy   10   5   10   Deletion   1184-1185delC   —   fs stop aa 444   Absent  
173-S   Canada   0.5   3   10   Insertion   1187-1191 insC   —   fs stop aa 494   ND  
174-B
 
Bosnia
 
4
 
5
 
10
 
Deletion
 
1192delT
 

 
fs stop aa 444
 
Absent
 
175-S   Puerto Rico   1   4   10   Insertion   1286 ins4bp   —   fs stop aa 495   Absent  
176*-S   US   1.8   4   10   Insertion   1301-1305 insG   —   fs stop aa 494   Red/trunc  
177*-S   US Latino   8   5   10   Insertion   1301-1305 insG   —   fs stop aa 494   Red/trunc  
178*-S   US   14   5   10   Deletion   1301-1305 insG   —   fs stop aa 494   Red/trunc  
179∥-B   Italy   9   0.5   11   Missense   1476T>A   —   1481N   normal  
180-B   Italy   7   5   11   Insertion   1486-1487insTC   —   fs lack of term. codon   ND  
181-B   Russia   6   5   12   Deletion   1519delT   —   fs lack of term. codon   Absent  
182-S   US   2.5   1   12   Missense   1542G>C   —   X503S   Absent  
183-B   Turkey   8.5   2   12   Missense   1542G>C   —   X503S   ND  
184-S   US   <2   3-4**  5′UTR-Intron 2   Deletion   del 5′UTR -719 to IVS2+1   No product   No product   Absent  
185-B   Russia   6   4   Intron 1   Splice   IVSI-2a>g   ND   ND   ND  
186-S   US   2.1   5   Intron 2   Splice   IVS2+1g>a   ND   ND   Absent  
187-S   US   13   2   Intron 2   Splice   IVS2+1g>t   ND   ND   Absent  
188-S   US   0.5   ND   Intron 2   Splice   IVS2-1 g>a   Exon 3 del   Inframe del 52 aa   ND  
189*-S   US   4.0   3   Intron 3   Splice   IVS3-1 g>a   ins intron 3   fs stop aa 201   Absent  
190a/b-B   Russia   5/7   3/3   Intron 4   Splice   IVS4-7 t>g   ND   ND   ND  
191-B   Croatia   8   5   Intron 6   Splice   INS6+2t>g   ND   ND   ND  
192*-S   US   7   1   Intron 6   Splice   IVS6+5 g>a   70% ins38nt intron 6/30% normal   fs stop aa 190/normal   Reduced  
193-S   US   14.0   2   Intron 6   Splice   IVS6+5 g>a   ins 38nt intron 6/normal   fs stop aa 190/normal   Reduced  
194-S   US   1.4   2   Intron 6   Splice   IVS6+5 g>a   ins 38nt intron 6/normal   fs stop aa 190/normal   Reduced  
195a/b/c/d-S   US   6/18/7/9   1/1/1/1   Intron 6   Splice   IVS6+5 g>a   ins 38nt intron 6/normal   fs stop aa 190/normal   Reduced  
196a¶/b-S   US   43 (dec.)/55   5/1   Intron 6   Splice   IVS6+5 g>a   ins 38nt intron 6/normal   fs stop aa 190/normal   Reduced  
197-S   US African American   12   2   Intron 6   Splice   IVS6+5 g>a   ins 38nt intron 6/normal   fs stop aa 190/normal   Reduced  
198a/b-B   Italy   4/1.8 (dec.)   5/5   Intron 7   Splice   IVS7+1g>t   ND   ND   Absent  
199-B   Sweden   10 (dec.)   4   Intron 7   Splice   IVS7+1g>a   ND   ND   ND  
200-S   US   <3   2   Intron 8   Splice   IVS8+1g>a   Exon 8 del   fs stop aa 246   Absent  
201-S   US   1   4   Intron 8   Splice   IVS8+1g>a   Exon 8 del   fs stop aa 246   Absent  
202-S   US American Indian   10   3-4   Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   Absent  
203-S   US   <2   2   Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   Absent  
204-B   Italy   7.9   2-3**  Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   ND  
205-B   Italy   1.5 (dec.)   5   Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   Absent  
206-B   Sweden   6   2   Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   ND  
207-S   US   0.1   ND   Intron 8   Splice   IVS8+1g>a   ND   fs stop aa 246   Absent  
208a/b/c/d-S   US Latino   12/5/1.5/0.5   4**/3**/3**/2   Intron 8   Splice   IVS8+1g>c   ND   fs stop aa 246   Absent  
209-S   Nepal   2   ND   Intron 8   Splice   IVS8+1g>1   Exon 8 del   ND   ND  
210-S   US   0.8   3   Intron 8   Splice   IVS8+1 to+6 del gtga   Exon 8 del   fs stop aa 246   Absent  
211-S   Thailand   9   4   Intron 8   Splice   IVS8+1 to+6 del gtga   Exon 8 del   fs stop aa 246   Absent  
212-S   US   0.5   2   Intron 8   Splice   IVS8+1 to+6 del gtga   Exon 8 del   fs stop aa 246   Absent  
213-S   US   1.0   3   Intron 8   Splice   IVS8+1 to+6 del gtga   Exon 8 del   fs stop aa 246   Reduced, normal size  
214-S   US   2.0   ND   Intron 8   Splice   IVS8+2 to3 del tg   Exon 8 del   fs stop aa 246   Absent  
215a§/b-B   Italy   11/2.1   3/2   Intron 8   Splice   IVS8+3insT   ND   ND   ND  
216-S   US   8   4   Intron 8   Splice   IVS8+5g>a   Exon 8 del   fs stop aa 246   Absent  
217-S   US   3.0   2   Intron 8   Splice   IVS8-2a>g   Intron 8ins   fs stop aa 328   Absent  
218-S   US   1   3   Intron 9   Splice   IVS9+1g>a   ND   ND   Absent  
219-B   Italy   22   4   Intron 9   Splice   IVS9+2del tgag   ND   ND   ND  
220a*/b*-S   US   2/<2   4/2**  Intron 9   Splice   IVS9+2t>c   80% ins 114nt of Intron 9 20% Intron9ins   fs stop aa 326   Absent  
221-B   Sweden   22   5   Intron 9   Splice   IVS9+2t>g   ND   ND   ND  
222*-S   US Greek   1   4   Intron 10   Complex   IVS10del 9nt, ins 11 nt   1372 ins 13nt   fs stop aa 498   Red/trunc  
223§-B   Italy   <2   2-3**  Intron 10   Splice   IVS10-3delC   ND   ND   ND  
224§-B   Italy   <2   1**  Intron 10   Splice   IVS10-2a>t   ND   ND   Reduced  
225-B   Turkey   7   4   Intron 10   Splice   IVS10-2a>c   ND   ND   ND  
226*-S   Canada   4   4   Intron 11   Splice   IVS11+2t>c   Exon 11 del   fs stop aa 543   Absent  
227*-S
 
US
 
?
 
5
 
Intron 11
 
Splice
 
IVS11+2t>g
 
Exon 11 del
 
fs stop aa 543
 
Absent
 

The “B” associated with the patient number identifies Brescia, the “S” identifies Seattle as the study center. a/b/c/d indicates individuals from the same family; ND, not determined; Red/trunc, reduced in quantity and truncated (small protein size); and dec., deceased.

*

Reported in Zhu et al,15  1997.

Reported by Kwan et al,22  1995.

Reported by Villa et al,10  1995.

§

Reported by Wengler et al,18  1995.

Reported by Notarangelo et al,23  2002.

This patient had a score of 1, at age 43 developed fatal lymphoma (score 5).

#

Remutation occurred in an early lymphocyte population.

**

This patient underwent BMT before 2 years of age.

††

The cDNA mutation was the same as the gDNA mutation.

PBMC isolation and cell lines

After informed consent was obtained to sequence DNA and set up cell lines for biochemical and immunologic analysis, heparinized blood was collected by venipuncture and shipped overnight or transported on the same day at room temperature to 1 of the 2 centers. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ) density gradient centrifugation and washed twice with phosphate-buffered saline (PBS). B-lymphoblastoid cell lines (B-LCLs) were established by infecting PBMCs with Epstein-Barr virus (EBV) containing supernatant as previously described.25  T-cell lines were established as follows: isolated PBMCs were cultured at a concentration of 2 × 106/mL in the presence of phytohemagglutinin (PHA; 3-5 μg added at weekly intervals). During the 2nd and the 3rd week, interleukin 2 (IL-2) at 500 U/mL was added. After 2 to 4 weeks in culture the cells were lysed and studied for the presence of WASP by Western blot analysis.

Nucleic acid isolation and cDNA synthesis

Genomic DNA (gDNA) was extracted from whole blood or buffy coat using the QIAamp DNA Blood mini kit (Qiagen, Valencia, CA). Total RNA was isolated from PBMCs or from established lymphoblastoid cell lines with Trizol (Life Technologies, Grand Island, NY) and submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) using the Thermoscript RT-PCR kit (Life Technologies) according to manufacturers' instructions.

PCR reaction and sequence analysis

cDNA (1 μg) was amplified in 2 overlapping fragments using primers and conditions previously reported.26  Direct sequencing was performed using the ABI Prism Big Dye Terminator Cycle Sequencing System (Perkin Elmer, Chatsworth, CA), and chromatograms were generated on an Applied Biosystems Model 373 or 310 DNA sequencer according to manufacturers' instructions. gDNA (0.2-1 μg) was amplified using intronic primer pairs that flanked 1 to 4 exons, including all intron/exon junctions. For the patients analyzed at the University of Washington, we selected primer pairs reported previously by Derry et al27 ; for the patients analyzed at the University of Brescia, the primers were previously reported by Giliani et al.28 

Western blot analysis

B-LCLs or IL-2-dependent T-cell lines from patients and from a WASP-positive (healthy individual) and a WAS-negative (WAS patient with a nonsense mutation in exon 1) control subject were lysed and submitted to Western blot analysis as previously described.15,23  From each sample, 20 μg total protein was electrophoresed through a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene diflouride (PVDF) Immobilon-P membrane (Millipore, Bedford, MA). For the experiments performed in Seattle, the membrane was incubated with the polyclonal anti-WASP antibody 503, and for those carried out in Brescia, the anti-WASP monoclonal antibody 3F3-A529  was used. The membrane was then washed 4 times, and results were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).15 

Results

Clinical scores

Sufficient information was available to clinically score 248 patients with WAS/XLT (Table 1). A score of 0 to 1 (0.5 in Table 1) was given to 4 patients with intermittent XLT,23  a score of 1 (including 4 patients with a score of 1-2) was given to 33 patients, a score of 2 was given to 73 patients (including 9 patients with a score of 2-3), a score of 3 was given to 29 patients (including 5 patients with a score of 3-4), and a score of 4 was given to 47 patients; 53 patients progressed to a score of 5.

Mutation analysis

We identified and characterized at total of 141 unique WASP mutations in 262 patients with WAS/XLT from 227 unrelated families on the gDNA, mRNA, and/or protein level (Figure 1; Table 1).

Figure 1.

A schematic illustration of WASP representing the 12 exons and the major functional domains. The mutations of WASP listed in Table 1 are visualized according to their location in the exons and the exon/intron junctions. Each symbol represents a single family with a WASP mutation. Missense mutations are located mostly in exons 1 to 4; deletions and insertions are distributed throughout the WASP gene, and splice site mutations are found predominantly in introns 6, 8, 9, and 10. PH indicates pleckstrin hemology; WH1, WAS homology 1; GBD, GTPase binding domain; VD, verprolin homology domain; and CD, cofilin homology domain.

Figure 1.

A schematic illustration of WASP representing the 12 exons and the major functional domains. The mutations of WASP listed in Table 1 are visualized according to their location in the exons and the exon/intron junctions. Each symbol represents a single family with a WASP mutation. Missense mutations are located mostly in exons 1 to 4; deletions and insertions are distributed throughout the WASP gene, and splice site mutations are found predominantly in introns 6, 8, 9, and 10. PH indicates pleckstrin hemology; WH1, WAS homology 1; GBD, GTPase binding domain; VD, verprolin homology domain; and CD, cofilin homology domain.

As shown in Table 2, the most common mutations were missense mutations (n = 80 families) followed by splice site mutations (n = 44), deletions (n = 40), and nonsense mutations (n = 35). Insertions and complex mutations made up less than 13% of the mutations identified.

Table 2.

Distribution of mutations in 227 WAS/XLT families


Mutation type
 

Families affected (%)
 
Missense   80 (35.3)  
Splice   44 (19.4)  
Deletion   40 (17.6)  
Nonsense   35 (15.4)  
Insertion   18 (7.9)  
Complex   10 (4.4)  
Total
 
227 (100)
 

Mutation type
 

Families affected (%)
 
Missense   80 (35.3)  
Splice   44 (19.4)  
Deletion   40 (17.6)  
Nonsense   35 (15.4)  
Insertion   18 (7.9)  
Complex   10 (4.4)  
Total
 
227 (100)
 

Protein expression

We were able to directly determine the quantity and size of WASP in cell lysates generated from B-LCL or T-cell lines established from 187 individuals belonging to 160 unrelated families. The 2 anti-WASP antibodies used gave identical results. Mutated WASP of normal size was present, although often at reduced amounts, in affected males from 69 (43%) families which were classified as “WASP positive” (Table 3). In contrast, WASP was absent, reduced in quantity and size (truncated) in affected members of the remaining 91 families (57%), which were classified as “WASP negative.”

Table 3.

Types of mutations correlate with the expression of WASP


Mutation type
 

WASP+ families affected (%)
 

WASP families affected (%)
 
Missense   51 (73.9)   10 (11)  
Splice   8 (l* = 1, V* = 7) (11.6)   23 (l* = 19, V* = 4) (25.3)  
Complex   4 (5.8)   4 (4.4)  
Deletion/insertion   5 (7.2)   32 (35.1)  
Nonsense   1 (1.5)   22 (24.2)  
Totals
 
69 (100)
 
91 (100)
 

Mutation type
 

WASP+ families affected (%)
 

WASP families affected (%)
 
Missense   51 (73.9)   10 (11)  
Splice   8 (l* = 1, V* = 7) (11.6)   23 (l* = 19, V* = 4) (25.3)  
Complex   4 (5.8)   4 (4.4)  
Deletion/insertion   5 (7.2)   32 (35.1)  
Nonsense   1 (1.5)   22 (24.2)  
Totals
 
69 (100)
 
91 (100)
 

The distribution of mutations observed in WASP+ and WASP families is significantly different (ϰ2 = 72.3, 4 df, P < .001). The association was also highly significant, using the r × c version of the Fisher exact test (P < .001). l* indicates invariant sites; V*, variant sites.

Most WASP+ patients had scores of 1 of 2.5 (2-3), characteristic of the XLT phenotype (Table 1). The majority of the WASP+ families had missense mutations (n = 51, 73.9%) followed by a much smaller percentage who had splice site mutations (n = 8, 11.6%) (Table 3). Of the 8 WASP+ families with splice site mutations, 7 had mutations affecting a variant splice site and only 1 affected an invariant splice site. The opposite is true for WASP- families with splice site mutations: 19 had mutations affecting an invariant splice site and only 4 had a variant splice site mutation.

Most patients with deletions or insertions affecting exon 10 that resulted in frameshift and stop at either amino acid (aa) 444 (n = 13) or at aa 493/494 (n = 9) failed to express WASP; those whose lymphocytes expressed WASP (n = 7) had a reduced quantity of truncated, presumably nonfunctional, WASP. Of the 20 patients that could be scored, 16 had a WAS phenotype; of the 4 patients with the XLT phenotype, 2 were 2 years of age or younger (Table 1).

The majority of the WASP-negative patients had scores of 3 or greater (Table 1). Mutations associated with absence of WASP were predominantly deletions/insertions, nonsense mutations, or splice site mutations accounting for 85% of the WASP- families (Table 3). The rest were missense and complex mutations. An exception was WASP-negative infants who were scored when aged 2 years or younger and who had mutations that were expected to result in a severe WAS phenotype (eg, nonsense mutations, deletions, insertions, splice site, complex mutations, and missense mutations in exon 4 that resulted in failure to express WASP). Of 49 infants with these characteristics, 32 (65%) had scores of 3 to 5, whereas 17 (35%) had scores of 1 to 2.5. In contrast, of 69 WASP- patients older than 2 years when the scores were obtained, and with similar mutations, 64 (93%) had scores of 3 to 5 and only 5 (7%) had scores of 1 to 2.5 (χ2 = 14.23, 1 degree of freedom [df], P = .0002), suggesting that it may take up to several years for a WASP- infant to present with the severe WAS phenotype expected from mutations that result in lack of WASP. It may, therefore, be prudent to consider scores of 1 to 2.5 as indeterminate when observed in infants aged 2 years or younger with mutations that predict absence of WASP and a severe WAS phenotype.

Mutational hotspots

In the 227 unrelated families studied we found 5 mutational “hotspots” which were defined as occurring in 6 or more unrelated families (> 2.5%) (Table 4).

Table 4.

WASP hotspot mutations and clinical phenotype


Mutation
 

Affected families (no. patients)
 

% of total families
 

Score 1-2.5*, no. patients
 

Score 3-5, no. patients
 
168C > T (T45M)   7 (8†)   (3.1)   7   0  
290C > N/291G > N (R86S/G/C/H/L)   17 (19)   (7.5)   19   0  
IVS6 + 5g > a fs stop aa 190/normal   6 (10)   (2.6)   9   1  
665C > T (R211X)   10 (11)   (4.4)   1   10  
IVS8 + 1g > a/c/t, fs stop aa246   10 (13†)   (4.4)   5   6  
Totals
 
50 (61)
 
(22)
 
41
 
17
 

Mutation
 

Affected families (no. patients)
 

% of total families
 

Score 1-2.5*, no. patients
 

Score 3-5, no. patients
 
168C > T (T45M)   7 (8†)   (3.1)   7   0  
290C > N/291G > N (R86S/G/C/H/L)   17 (19)   (7.5)   19   0  
IVS6 + 5g > a fs stop aa 190/normal   6 (10)   (2.6)   9   1  
665C > T (R211X)   10 (11)   (4.4)   1   10  
IVS8 + 1g > a/c/t, fs stop aa246   10 (13†)   (4.4)   5   6  
Totals
 
50 (61)
 
(22)
 
41
 
17
 

Three of the hotspot mutations (T45M, R86N, IVS6 + 5g > a) are associated with low scores, and 2 hotspot mutations (R211X, IVS8 + 1g > a) are associated with high scores (ϰ2 = 36.11, 4 df, P < .001). Fisher exact test for r × c tables, P < .001.

*

A score of 2 to 3 is listed as a score of 2.5; scores 1 to 2.5 are considered XLT; scores of 3 to 5 represent the WAS phenotype.

One patient with the 168C > T missense mutation and 2 patients with the IVS8 + 1g > a spalice site mutation could not be scored because of insufficient clinical data.

The 168C>T mutation, found in 7 families, results in the substitution of threonine with methionine at position 45 and the 290C>N/291G>N mutations, observed in 17 unrelated families, result in the substitution of arginine at position 86 with either a serine, glycine, cysteine, histidine, or leucine. The IVS6+5g>a mutation, present in 6 unrelated families, results in both abnormal and normal splicing products. The 665C>T mutation, which converts arginine at codon 211 to a stop codon, was found in 10 families, and the IVS8+1g>n mutation, identified in 10 families, results in the deletion of exon 8, leading to frameshift and premature stop of translation. These 5 mutations account for 22% of all families included in this study. Three of these 5 mutations (168C>T, 290C>N/291G>N, and IVS6+5g>a) were consistently found in WASP+ patients with a mild phenotype (XLT) who had a low score, whereas the other 2 (665C>T and IVS8+1g>n) were predominantly WASP- and had a high score (P < .001).

Remutation

Patient 122-S was found to have gDNA with a single nucleotide insertion (471-476insA) which results in frameshift and premature stop at aa 168. This mutation is expected to cause severe WAS. However, at 12 years of age, patient 122-S has mild disease with a score of 2. His cDNA, generated from mRNA isolated from PBMCs, consists of 2 populations: the majority has the 471-476insA mutation, and a small fraction has a normal sequence. Western blot analysis revealed a small amount of WASP in extracts from both EBV-induced B lymphoblasts and IL-2-dependent T lymphoblasts, suggesting that the remutation occurred in an early lymphoid precursor population. A remutation (211T>C, resulting in the silent mutation P59P) was also observed in patient 56-B.

Discussion

Mutations of the WASP gene result in 3 distinct phenotypes: the classic WAS triad of thrombocytopenia/small platelets, recurrent infections as a result of immunodeficiency, and eczema1,2 ; the milder XLT variant, characterized predominantly by thrombocytopenia/small platelets10,11 ; and congenital neutropenia without the clinical findings characteristic for WAS/XLT.12 

To investigate a possible phenotype/genotype association we (1) developed a scoring system that allowed the differentiation of WAS and XLT, (2) analyzed genomic DNA and cDNA from each subject for mutations of WASP, and (3) determined the effect of the mutation on WASP expression by cultured lymphoblasts.

Of the 141 unique WASP mutations identified in our series of 227 unrelated families, 71 had not been previously reported.9-11,15-23,27-44  Most mutations consisted of single nucleotide substitutions, small insertions and deletions, and splice site mutations and were distributed throughout the coding region and the intron-exon junctions. Except for a large deletion involving the 5′ untranslated region (UTR) and exons 1 and 2 (del5′UTR [-719]-intron2+1), mutations were not found outside the coding region, and none of the WAS/XLT patients were found to lack WASP expression without having a demonstrable mutation affecting either the exons or the exon-intron junctions.

As was observed in smaller series reported previously,9-11,15-22,30,35,39,43  the predominant mutations of WASP were missense mutations which were typically located in exons 1, 2, 3, and 4. Only 4 missense mutations, 1 each in exon 7, 9, 10, and 11, were observed downstream of exon 4. One of those, P361T in exon 10, is the only missense mutation identified to date affecting the polyproline region of WASP. In addition, 2 unrelated families, both with the XLT phenotype, had a point mutation affecting the termination codon of exon 12 (codon 503X>S), resulting in the absence of WASP.

The second most common WASP mutation affected splice sites and occurred predominantly in the downstream half (introns 6-11) of the WASP gene, similar to previous reports.15,17-19,30  Mutations affecting variant splice sites resulted in multiple splicing products, and often included small amounts of normal WASP. Insertions and deletions, with 2 exceptions, resulted in frameshift and premature stop of translation. Deletions were more common in areas with a series of cytosine or guanosine nucleotides and typically involved less than 10 nucleotides. Complex mutations were rare and involved double missense mutations, point mutations followed by a deletion, or a combination of deletions and insertions.

Of the 5 “hotspots,” 3 were associated with mild diseases: the substitution of arginine at position 86 with 1 of 5 different amino acids, was observed most frequently (17 families), followed by substitution of threonine with methionine at position 45 (7 families), and the variant splice site mutation IVS6+5g>a that generates simultaneously abnormal and reduced amounts of normal WASP (6 families). The other 2 hotspot mutations, the nonsense mutation Arg211X and the splice site mutation (IVS8+1g>n), both present in 10 families, were preferentially associated with the WAS phenotype. There was no association with ethnicity or geographic origin, and it is unclear why these 5 hotspot mutations occur in more than 20% of WAS/XLT families reported here. It is possible that these sequences are prone to mistakes during DNA replication. The alternative explanation that these hotspots represent naturally occurring polymorphisms is highly unlikely because no other mutations within the WASP gene could be identified in any of these patients with WAS/XLT, and none of these hotspot mutations was observed in more than 300 normal X chromosomes in which WASP was sequenced.

Thrombocytopenia and small-sized platelets were present in all patients with WASP mutations, including the members of 2 families with missense mutations in exon 2 (P58A) and exon 11 (I481N),23  respectively, who had intermittent petechiae and thrombocytopenia, with platelet counts as low as 30 × 109/L alternating with normal platelet counts (100-250 × 109/L) when asymptomatic. No patients with congenital neutropenia were identified, possibly reflecting a selection bias.

To explore a phenotype/genotype correlation, we divided the patients into 2 categories: WASP positive if the mutated protein was of normal size and WASP negative if protein was absent or truncated. Patients with mutations that allowed the expression of normal-sized mutated protein, often in reduced quantity, developed the XLT phenotype, whereas those patients whose lymphocytes could not express WASP or expressed only truncated WASP were more likely to present with the WAS phenotype (P < .001). Progression to a score of 5 because of either autoimmune disease or malignancy was observed in both groups, but far more frequently in WASP-negative patients. Exceptions to this rule exist as shown in Tables 1 and 3 and may make it difficult in individual cases to accurately predict the clinical course based solely on the type of mutation of the WASP gene. There are several explanations for these occasional discrepancies. Splice site mutations, especially if affecting variant intronic nucleotide positions often allow the generation of multiple splicing products, including normally spliced mRNA, resulting in a small quantity of normal WASP. For example, the hotspot mutation IVS6+5g>a, causes the insertion of 38 nucleotides from the proximal end of intron 6, which results in a frameshift and early termination of transcription, but also in the production of a small amount of normal WASP mRNA. Another mechanism that can modify the clinical phenotype is “remutation” because of in vivo (somatic) reversion of the inherited mutation. This mechanism was observed in patient 122-S who was found to have 2 populations of WASP cDNA and protein, mutated and normal. In contrast to the XLT phenotype (score 2) observed in patient122-S, a severe WAS phenotype (score of 4) was present in a nonrelated patient (121-B) with the same mutation. Wada et al31  reported a patient with WAS/XLT with remarkable clinical improvement after puberty when he was found to have undergone remutation. There may be other genes that are capable of modulating the clinical phenotype of patients with WAS/XLT. Disease-modifying genes have been demonstrated in patients with chronic granulomatous disease (CGD) whose clinical phenotypes were strongly influenced by polymorphisms of myeloperoxidase and FcγRIIIb.45  Genes that determine an individual's probability to develop atopic diseases, such as allergies and eczema, or genes that control the effectiveness of host defense against infections may modify the WAS/XLT phenotype. Finally, environmental factors, such as exposure to common or uncommon infectious agents, failure to establish the diagnosis at an early age, or less than optimal care, may affect the severity of the clinical presentation. Scoring before the age of 2 years is unreliable because it often suggests a phenotype that is milder than expected from the type of WASP mutation identified. For this reason, scores of 1 to 2.5 observed in infants aged 2 years should be considered as indeterminate.

Of the 248 patients with clinical information that allowed a score to be established, 53 (mostly nonsense or splice site mutations resulting in the absence of WASP) eventually reached a score of 5, indicating the progression to autoimmune disease or malignancy. Only 6 of the 53 patients with a score of 5 were found to have missense mutations, and all but 1 patient had exon 4 mutations and failed to express WASP. We observed a high rate of autoimmunity or malignancies in patients with splice site mutations that resulted in premature termination at codon 444 or 493. A strikingly higher incidence of lymphoma has recently been reported in patients with WAS/XLT with splice site mutations, nonsense mutations, and mutations resulting in frameshift than in patients with missense mutations.46 

We conclude from this in-depth analysis of 2 large cohorts of families with WASP mutations, one predominantly from North America and the other from Europe, that the clinical phenotype of WAS or XLT, severe or mild, is strongly influenced by the effect of the mutation on protein expression. The frequency and severity of infections, the extent of eczema, and the progression to autoimmune diseases and malignancies correlated, although not always invariably, with the absence of WASP in patient lymphocytes. However, patients with missense mutations allowing expression of mutated WASP and those with splice site mutations resulting in multiple products, including small amounts of normal WASP, present overwhelmingly with the milder XLT phenotype. As expected, there is some overlap, especially in infants aged 2 years or younger, which makes it more difficult for the treating physician to predict the final outcome in a patient with a new diagnosis of WASP mutation. The dismal prognosis for patients with the WAS phenotype justifies the use of procedures such as stem cell transplantation that, by themselves, are associated with considerable morbidity and potential mortality.

Prepublished online as Blood First Edition Paper, July 29, 2004; DOI 10.1182/blood-2003-05-1592.

Supported in part by grants from the National Institutes of Health (HD17427-33), the March of Dimes Birth Defects Foundation (96-0330), the Immunodeficiency Foundation, the Jeffrey Modell Foundation, the DeJoria Wiskott-Aldrich Research Fund (H.D.O.); the MIUR-FIRB (project RBNE01Y3N3_004), MURST (Cofin 2002), the European Union “Quality of Life and Management of Living Resources” (contract QLG1-CT-99-01 090), and the European Union (grant QLGI-CT-2001-01 395); and the Berlucchi Foundation (L.D.N.).

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.

We thank Dr Lianne Sheppard for assistance with the statistical data analysis. The following individuals allowed the Seattle and Brescia laboratories to study their patients and provided material that was invaluable for this investigation: O. Alvarez, N. Amaurilio, J. Anžič, R. Ashok, U. Aysegul, D. Beardsley, V. Berdovicas, L. Bird, M. Blaese, V. Bonagura, W. G. Borges, C. Bottom, L.A. Boxer, L. Bruckner, R. Buckley, J. Bussell, M. Caniglia, F. Candotti, L. Cardinali, J. Casella, B. Casey, H. Chapel, M. Clayton, G. Colon, M.E. Conley, V. Correra, M. Cowan, C. Cunningham-Rundles, C. Curry, N. Day, G. del Toro, G. Eames, M. Elder, G. Elinder, A. Etzioni, A. Fasth, C. Fernandes, L. Filipovitch, C. Frantz, T. Fuengoer, R. Fuleihan, R. Galanello, E. Gelfand, V. Gentile, C. Gibbons, E. Gillan, W. Greg, M. Grimley, P. Harper, T. Hays, W. Hicks, H. Hill, R. Hostoffer, J. Hutter, R. Insel, A.-M. Irani, J. Jazbec, A. Junker, W. Kamchaisatian, M. Kanariou, Z. Karakas, G. Kato, H. Kayserili, B. Kerlin, A. Kheradpour, G. Kleiner, L. Kobrynski, A. Laszlo, C. L. Lee, E. Lichtenberg, J. Lipton, J. Litzman, A. Malta Corea, P. Martin, S. Martino, M. Masi, D. De Mattia, B. Mazer, C. Mendez, C. Mercer, C. Mullen, D. Murphey, D. Nugent, M. Oblender, J. Oleske, K. O'Neill, W. Owen, L. Pachman, S. Pasic, M. Pelidis, M. J. Petruzzi, A. Plebani, V. Poggi, K. Provo, M. Pulsipher, M. Rabusin, U. Ramenghi, I. Reznick, F. Rosen, H. Rosenblatt, C. Rossbach, E. Salman, O. Sanal, J. Sande, R. Schiff, M. Schumacher, S. Scommegna, R. Shapiro, A. Shigeoka, B. Sibly, J. W. Sleasman, D. Steele, C. Stotts, K. Sullivan, F. Tancredi, W. Tcheng, D. Toro, M. T. Trakultivakorn, A. Tsang, S. Turk, E. Tyler, W. Vasconcelos, K. Vettenranta, A. Ventura, M. Wang, K. Weinberg, G. Wilson, J. Winkelstein, L. Wolff, D. Wright, and J. Wu.

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