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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 699-701
CORRESPONDENCE
A Case of Wiskott-Aldrich Syndrome With Dual Mutations in Exon 10 of the WASP Gene: An Additional De Novo One-Base Insertion, Which
Restores Frame Shift Due to an Inherent One-Base Deletion, Detected in
the Major Population of the Patient's Peripheral Blood
Lymphocytes
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LETTER |
To the Editor:
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder
characterized clinically by the triad of thrombocytopenia, recurrent
infections due to defects in the immune system, and severe eczema. The
gene responsible for WAS (WASP gene) was recently cloned.1 Since then, large numbers of mutations have been
reported in WAS patients with variable clinical
phenotypes,2 and it has also been shown that X-linked
thrombocytopenia (XLT) resulted from a mutation of the same
gene.3
We performed mutation analysis of a boy with WAS and found dual
mutations in exon 10 of the WASP gene. The patient died before the present study; thus, only genomic DNA derived from his peripheral blood lymphocytes was available. Genomic DNA was purified, and each
exon of the WASP, including flanking introns, was amplified by
polymerase chain reaction (PCR). Primers used and each PCR condition
were described elsewhere.4 Amplified fragments were purified and directly sequenced using an ABI PRIZM Dye Terminator Cycle
Sequencing Ready Reaction Kit (Perkin Elmer, Foster City, CA) and an
automated ABI 373A DNA sequencer. The conditions used for the
sequencing reaction were also described before.5 To avoid
PCR-generated sequence errors, we performed sequencing at least twice
using a different set of all PCR products in both strands.
The patient studied had two elder brothers who had been diagnosed as
WAS based on their clinical and laboratory findings. They died from
severe pneumonia and septicemia with bleeding episodes at 10 months of
age and 47 months of age, respectively. At 4 years of age, the patient
died from unpredictable intracranial hemorrhage; meanwhile, we obtained
his peripheral blood lymphocytes and kept them frozen. After the
patient died, the couple had a daughter. Genomic DNAs from his mother
and his sister were then obtained to determine their carrier status of
the disease after informed consent.
Mutation analysis of the patient's WASP gene showed two
mutations in exon 10. One mutation was a one-base insertion (A+) at nucleotide number 1099 or 1100. The other was a one-base deletion (G ) from 5 consecutive Gs of nucleotide number 1127 to 1131. When we
made a careful observation of the patient's sequencing results, we
found small-tide ambiguous waves after the A+ site in both the
directions (data not shown), as if it contained a small amount of the
fragments without A+. To confirm this, the fragments of the different
PCR including the mutations were cloned into the TA vector
PCRII (Invitrogen Corp, Carlsbad, CA), and the sequence of
each clone was examined. Most of the clones possessed the dual
mutations (A+ and G); however, we could find some clones with the
single mutation, all of which had only the G mutation (Table
1). Sequencing studies of the PCR fragments
of the patient's mother and sister showed that both were carriers for
the disease. They had the mutant WASP allele together with the
normal allele, and the mutant allele included only the G mutation.
The results of the cloning and sequencing experiment of their PCR
fragments were shown in Table 1.
From these results, we speculated the origin of the patient's dual
mutations; the one-base deletion mutation (G) was derived from his
mother, and the other one-base insertion mutation (A+) was generated de
novo. The results of the cloning studies suggested that de novo
mutation occured after fertilization, possibly at some level of
hematological progenitor. It was shown that the patient's clones
consisted mostly of the dual mutations, with very minor clones having
only the G mutation. PCR-generated artifacts were very unlikely,
because 6 different PCR products showed the same results.
Deduced amino acid sequence in both the mutants were shown in Fig
1. In comparison with the single mutation
G, the changes of amino acid in the dual mutation were restricted to
the position of 356-365 (Fig 1). The area involved 10 amino acids, and
actually 7 amino acids were replaced. Thus, although we could not
evaluate the two mutant WASP protein functions, the defect in the case of the dual mutations was considered to be milder than that of the
single mutation. A similar case with dual mutations in the WASP
gene was recently reported as mild clinical phenotype,6 however, the origin of the dual mutations was not discussed.
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