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INTRIGUING COMBINATION OF MUTATIONS IN WAS PATIENT. T. Freiberger 1,2 , B. Ravčuková 1,2 , P. Čižnár 3 1 Molecular Genetics Lab, Centre for Cardiovascular Surgery and Transplantation, Brno, CR 2 Centre for Primary Immunodeficiencies, Masaryk University Brno, CR

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intriguing combination of mutations in was patient

INTRIGUING COMBINATION OF MUTATIONS IN WAS PATIENT

T. Freiberger1,2, B. Ravčuková1,2, P. Čižnár3

1Molecular Genetics Lab, Centre for Cardiovascular Surgery and Transplantation, Brno, CR

2Centre for Primary Immunodeficiencies, Masaryk University Brno, CR

3Dept. Pediatrics, University Hospital, Bratislava, Slovakia

wiskott aldrich syndrome
Wiskott-Aldrich syndrome

X - LINKED

  • eczema
  • thrombocytopenia
  • immunodeficiency
        • Wiskott A, Monatsschr Kinderheilkd, 1937
        • Aldrich RA et al., Pediatrics, 1954
wiskott aldrich syndrome3
Wiskott-Aldrich syndrome
  • eczema
    • mild, transient … severe, difficult to control
  • thrombocytopenia
  • immunodeficiency
wiskott aldrich syndrome4
Wiskott-Aldrich syndrome
  • eczema
    • mild, transient … severe, difficult to control
  • thrombocytopenia
    • small size platelets
  • immunodeficiency
wiskott aldrich syndrome5
Wiskott-Aldrich syndrome
  • eczema
    • mild, transient … severe, difficult to control
  • thrombocytopenia
    • small size platelets
  • immunodeficiency
    • clinical: recurrent infections
    • laboratory: ↓ IgM, ↑ IgA, IgE; ↓ isohemagglutinin

↓ ag induced lymphocyte proliferation

wiskott aldrich syndrome6
Wiskott-Aldrich syndrome
  • eczema
    • mild, transient … severe, difficult to control
  • thrombocytopenia
    • small size platelets
  • immunodeficiency
    • clinical: recurrent infections
    • laboratory: ↓ IgM, ↑ IgA, IgE; ↓ isohemaglutinin

↓ ag induced lymphocyte proliferation

  • - autoimmunity and/or malignancy
scoring system to define phenotype of was
Scoring system to define phenotype of WAS
  • Disease XLT Classic WAS
  • Score 1 2 3 4 5
  • thrombocytopenia + + + + +
  • small platelets + + + + +
  • eczema - (+) + ++ +/++
  • immunodeficiency -/(+) (+) + + +
  • infections - (+) + +/++ +/++
  • autoimmunity - - - - +
  • and/or malignancy
genetic background
Genetic background
  • gene WASP
    • WASP - key regulator of lymphocyte and platelet function
      • critical role in signal transduction
      • regulation of the cytoskeleton reorganization

Derry JM et al, Cell, 1994

slide9

GENE

9 kbp;

cDNA

1821 bp

Jin Y et al, Blood, 2004

slide10

PROTEIN

AA

Jin Y et al, Blood, 2004

slide11

50.7 %

35.3 %

15.4 %

Jin Y et al, Blood, 2004

slide12

7.9 %

17.6 %

19.4 %

4.4 %

Jin Y et al, Blood, 2004

molecular genetic diagnostics of was in centre for pid brno
Molecular genetic diagnostics of WASin Centre for PID, Brno
  • PCR of all coding regions followed by direct sequencing
    • exons 1, 2, 3+4, 5+6, 7, 8+9, 10, 11, 12

(Jones LN et al., Blood Cells, Molecules and Diseases, 2002)

  • confirmation by independent PCR (PCR-SSP or PCR+restriction or PCR+sequencing)
case report
Case report
  • boy
  • petechial exanthema, thrombocytopenia early after birth
  • eczema
  • recurrent purulent otitis, pneumonia (Str. pneumoniae, Staph. aureus, Moraxella catarrhalis)
  • splenomegaly
  • Leu 5600/ul, Ly 2100/ul, T ly 1290/ul (61 %), B ly 11 %;
    • CD4+ 30 %, CD8+ 31 %
  • IgG 10,5 g/l N, IgA 3,96 g/l ↑, IgM 0,17 g/l ↓, IgE 1331 IU/ml ↑
case report15
Case report

Leukemia

transient XLT

score 0.5

WAS

score 3-4

case report16
Case report
  • IVIG, antibiotics
  • HSCT from HLA identical brother at 11 years of age
  • in a good shape 2 years after HSCT
wasp gene exon 10 direct sequencing
WASP gene, exon 10, direct sequencing

10. exon

wild type

10. exon

c.1071delC

p.P346fsX444

wasp gene exon 6 5 direct sequencing19
WASP gene, exon 6+5, direct sequencing

artefact?

somatic mosaicism?

slide20

18/20 clones

2/20 clones

5. exon

wild type

5. exon

c.535_536insT

p.E168fsX168

slide21

STOP

at 444

100 % PBMC

slide22

STOP

at 444

STOP

at 168

100 % PBMC

~ 10 % PBMC

summary
Summary
  • In WAS patient detected:
  • new 1 bp deletion resulting in stop codon at 444 in all PBMC
  • new 1 bp insertion resulting in stop codon at 168 in about 10 % of PBMC
summary24
Summary
  • In WAS patient detected:
  • new 1 bp deletion resulting in stop codon at 444 in all PBMC … primary (?)
  • new 1 bp insertion resulting in stop codon at 168 in about 10 % of PBMC … (secondary (?), selective advantage (?))
significance of the second site mutation26
Significance of the second site mutation
  • - ?? spontaneous reversion of mutation ??
    • nature of mutations … rather against
    • mutations far from each other in different functional domains … rather against
    • tertiary structure?
insufficient information at present time
… insufficient information at present time …
  • separated cell populations before HSCT … not available for examination
  • examination of patient’s dry blood spot
    • mutation status at the time of the birth
  • examination of brother suffering from transient XLT (if available)
  • examination of parents (particularly mother) (if available)
  • … will give us additional pieces of information …