The Wiskott–Aldrich syndrome is an X-linked hereditarydisorder associated with combined immunodeficiency, thrombocytopenia,small platelets, eczema, and increased susceptibility to autoimmunedisorders and cancers. It is caused by mutations in the gene(WAS) for the Wiskott–Aldrich syndrome protein (WASP).We investigated family members of the patients originally describedby Wiskott in 1937 and identified a new frame shift mutationin exon 1 of WAS. This mutation is likely to be the hypothesizedgenotype that caused the severe form of the Wiskott–Aldrichsyndrome in the three brothers described by Wiskott.
In 1937, Alfred Wiskott, a German pediatrician, described threebrothers who presented shortly after birth with thrombocytopenia,bloody diarrhea, eczema, and recurrent ear infections; all threedied early in life from intestinal bleeding and sepsis. Wiskottcommented that "the origin of the hemorrhagic diathesis is adysfunction in the line of the platelets."1 The observationthat all three brothers were affected, whereas their sistersshowed no symptoms, led Wiskott to propose that the syndromeis due to a "hereditary thrombopathia."1 In 1954, Aldrich etal. traced six generations of a family and found that 16 of40 males, but no females, died of the syndrome first describedby Wiskott, thus clearly showing an X-linked mode of inheritance.2
The Wiskott–Aldrich syndrome is now known as an X-linkedhereditary disorder associated with combined immunodeficiency,thrombocytopenia, small platelets, eczema, and an increasedrisk of autoimmune disorders and cancers. It has a broad rangeof phenotypes (Online Mendelian Inheritance in Man no. 301000
[OMIM]
).3,4,5,6
The severe form of the Wiskott–Aldrich syndrome and itsmilder manifestations — X-linked thrombocytopenia andX-linked neutropenia — are caused by mutations in thegene for the Wiskott–Aldrich syndrome protein (WAS), locatedat Xp11.22–p11.23 and cloned in 1994.7,8,9,10 WAS andseveral related proteins are involved in the reorganizationof the actin cytoskeleton by activating the actin-related protein2/3 complex that mediates actin polymerization in all cellsof the hematopoietic system.3,11,12,13,14,15 Mutations in theWAS gene result in truncated or absent WASP in these cells,but there is no strict correlation between the mutant genotypeand the expression of WASP or the phenotype of the syndrome.11,15,16,17,18The disorder can be cured through hematopoietic stem-cell transplantation.19
We recruited members of the family described by Wiskott in 1937in order to identify the hypothesized mutation in WAS that causedthe severe phenotype of the Wiskott–Aldrich syndrome inthe three brothers. Genetic testing for the mutation was carriedout in three generations of the kindred.
Methods
After obtaining written informed consent, we performed mutationanalyses in Subjects III-6, III-8, III-9, III-10, IV-2, IV-3,V-1, and V-2 (Figure 1). Genomic DNA was extracted from peripheralwhite cells. After amplification, WAS exons 1 through 12 wereanalyzed with the single-strand conformation polymorphism methodand the aberrant fragment of exon 1 was investigated by meansof double-strand sequencing. All other WAS exons from SubjectsIII-10 and V-1 were reanalyzed through direct sequencing (withthe use of a kit from Applied Biosystems). The primer sequencesused for amplification and the methods used for the screeningof mutations have been described previously.18 A WAS mutationwas ruled out in 400 controls (200 men and 200 women) by meansof denaturing high-performance liquid chromatography with theWave system (Transgenomics); the aberrant fragment was observedin DNA from a female carrier, analyzed with the use of bufferB (on a gradient of 53 to 61%) at a running temperature of 64°C.20,21To study the expression of the mutant allele, RNA was isolatedfrom an obligate female carrier and transcribed into complementaryDNA (cDNA) with the use of a first-strand cDNA synthesis kit(Amersham Biosciences). The cDNA was sequenced with the useof a pair of primers: one located in the 5' untranslated region(1cF: 5'TCGCCAGAGAAGAC-AAGGGC3') and one in exon 3 (3cR: 5'CATCT-CCAGCGAAGGTGTGG3').
Figure 1. Pedigree of the Three Brothers (Subjects III-3, III-4, and III-5) Described by Wiskott in 1937.
Open symbols represent unaffected family members, solid squares affected men, circles with solid dots obligate female carriers, the circle with an open dot a female carrier identified by gene sequencing, the triangle an aborted fetus, symbols with a slash deceased affected family members, asterisks family members who were genetically tested, a minus sign family members with a negative result on genetic testing for the mutation, and a plus sign family members with a positive result. A positive result indicated that the subject was a carrier of the 73–74delAC mutation in the coding sequence. The age at death is indicated under the symbols representing the three brothers who were originally described by Wiskott.
Results
Genetic testing for the mutation revealed a deletion of twonucleotides at positions 73 and 74 in WAS exon 1 (coding sequence,73–74delAC22,23; the first nucleotide is the A of theATG translation-initiation codon). This mutation is not listedin WASPbase,24an Internet-based database of WAS mutations. Thedeletion results in a frame shift that starts with amino acid25; the shifted reading frame is open for another 11 amino acidsbefore it results in a stop codon (protein sequence, Thr25ProfsX1222,23)(Figure 2).
Figure 2. Chromatograms Showing the Mutation in Subject V-1 (Panel A) and the Wild-Type Sequence in the Same Subject after Stem-Cell Transplantation (Panel B).
In Panel A, the black boxes delineate nucleotide triplets upstream of the mutation; the arrow and dashed black line indicate the two nucleotides (AC) in positions 73 and 74 that are deleted in affected persons, causing a frame shift; and the red boxes delineate the nucleotide triplets in the new open reading frame. Panel B shows the position of the nucleotides in the wild type (outlined by dashed box).
To further characterize the 73–74delAC mutation in thecoding sequence, we first sought it in 400 normal subjects (200men and 200 women) serving as controls; none carried the mutation.This result makes it improbable that the mutation is a polymorphicvariant in the normal population. Next, we performed RNA analysisin a female carrier (Subject IV-2). She was heterozygous forthe aberrant variant in exon 1. However, after the sequencingof exons 1 through 3 of the generated cDNA, we observed monoallelicexpression, which indicates the decay of nonsense-mediated messengerRNA (mRNA) (the destruction of mRNA with a premature stop codon)(Figure 3). This observation is a sign that the 73–74delACmutation in the coding sequence is likely to result in the completeabsence of WASP in affected men.25
Figure 3. Nonsense-Mediated Decay of Mutant mRNA, as Shown in Genomic DNA (Panel A) and cDNA (Panel B) in a Female Carrier (Subject IV-2).
Panel A shows a chromatogram of genomic DNA in exon 1, revealing a heterozygous frame-shift deletion. The arrow indicates the site of the deletion. Panel B shows a chromatogram of the cDNA, revealing that WAS mRNA is expressed from only one allele.
We identified this mutation in three generations of the pedigree:Subjects III-10, IV-2, and V-1. Subject V-1 had presented withsymptoms compatible with the severe form of the Wiskott–Aldrichsyndrome: bloody diarrhea, severe infections, eczema, and thrombocytopeniawith small platelets. He is alive and well after receiving ahematopoietic stem-cell transplant from an HLA-matched unrelateddonor (Figure 2B). Subject III-10, a sister of the three affectedbrothers described by Wiskott (Subjects III-3, III-4, and III-5),was identified as a carrier of the mutation, but three of hersisters do not carry the mutation. As expected, Subject IV-2,the mother of Subject V-1, also carries the X-linked mutation(Figure 1).
Discussion
Our analysis indicates that Subjects II-2 and II-4 were obligatecarriers of the 73–74delAC mutation in the coding sequenceafter they inherited either a germ-line WAS mutation from oneof their parents (Subjects I-1 and I-2) or the mutation fromtheir mother (Subject I-2, the grandmother of Wiskott's patients),who could have been a silent carrier of an ancestral mutation.Since Subjects III-10 and IV-2 are carriers of the mutation,Subject III-2 must be an obligate female carrier.
Our findings indicate the improbability of a spontaneous mutationin Subject V-1 and provide strong evidence that the three affectedbrothers (Subjects III-3, III-4, and III-5) also had the 73–74delACmutation in the coding sequence of WAS. Almost 70 years afterWiskott's initial clinical description of the Wiskott–Aldrichsyndrome in three brothers, we found that a mutation in theX-linked WAS gene caused the severe phenotype.
Supported by Deutsche Krebshilfe.
No potential conflict of interest relevant to this article wasreported.
We are indebted to Drs. W. Friedrich and K. Schwarz, Departmentof Pediatrics, University Hospital Ulm, Ulm, Germany, for providinginformation about the molecular analysis and stem-cell transplantationperformed in Subject V-1, as well as for providing blood samplesfor additional analysis.
Source Information
From the Departments of Hematology and Oncology (V.B., M.H.A.) and Immunology and Infectious Diseases (B.H.B.), Dr. von Haunersches Children's Hospital, Ludwig Maximilians University; and the Department of Obstetrics and Gynecology, Klinikum rechts der Isar, Technical University Munich (M.B., A.M.) — both in Munich; and the Department of Pediatrics, Municipal Hospital Dresden–Neustadt, Academic Section of the Technical University Dresden, Dresden, Germany (M.K.).
Address reprint requests to Dr. Meindl, Department of Obstetrics and Gynecology, Klinikum rechts der Isar, Technical University Munich, Ismaninger Straße 22, D-81675 Munich, Germany, or at alfons.meindl{at}lrz.tu-muenchen.de.
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