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SPECTRUM OF THE BTK GENE
MUTATIONS IN CZECH XLA
PATIENTS
Tomáš Freiberger
Molecular Genetics Lab
Centre for Cardiovascular Surgery and Transplantation
University Centre for Primary Immunodeficiencies
Brno, Czech Republic
X-LINKED AGAMMAGLOBULINEMIA
 males after 6 M of age
 absence of B lymphocytes in PB, severe
hypogammaglobulinemia
 increased susceptibility to infections
 otitis, sinusitis, bronchitis, pneumonia; skin infections;
meningitis, sepsis, osteomyelitis (pyogenic encapsulated
bacteria)
 diarrhea (Salmonella, Giardia lamblia, Campylobacter)
 meningoencephalitis (enteroviruses)
 prognosis improved after introduction of IVIG ther.
X-LINKED AGAMMAGLOBULINEMIA
 mutations in the BTK gene
 BTK important for B cell development, role in preBCR signaling pathway
 cytoplasmic protein: 659 AA, 77 kDa
PH
TH
SH3
SH2
KINASE
~ 140
~ 75
~ 65
~ 100
~ 280 AA
Block in B cell development
Pro-B
CD22
CyIgα
CD22
CyIgα
TdT
PreB-I
Pre-B-II
CD22
CyIgα
CD19
TdT
ψL
CD22
CyIgα
CD19
TdT
ψL
CD22
CyIgα
CD19
CD10++
CD10+
Immature B Mature B
CD22
CyIgα
CD19
CD22
CyIgα
CD19
CD22
CyIgα
CD19
CD10+
CD10+
CD10+
CD10+
Cy μ
Cy μ
SmIgM
SmIgM
SmIgD
ψL
Rag+ Rag+ RagDHJH VHJH
Rag+
VLJL
adapted from Noordzij et al., 2002
pre-BCR signaling pathway
μ
V-preB
λ5/14.1
Igα
Igβ
Syk
Lyn
PLCγ
NF-κB
BLNK
BTK
Ca++ influx
BTK mutations
 BTK gene: Xq21.3-22
 19 exons, 37.5 kb
 mRNA 2591 bp
 mutations scattered all over the gene
http://protein.uta.fi/BTKbase
XLA: genotype-phenotype correlation
! weak genotype-phenotype correlation !
mutations in the same domain
the same type of mutations
the same mutation
various number of B cells in PB
various immunoglobulin levels
various clinical features
Agammaglobulinemia:
one phenotype
various gene defects
BTK (X)
pre-BCR signaling pathway
μ
V-preB
λ5/14.1
Igα
Igβ
Syk
Lyn
PLCγ
NF-κB
BLNK
BTK
Ca++ influx
Agammaglobulinemia:
one phenotype
various gene defects
BTK (X)
μ chain (AR)
pre-BCR signaling pathway
μ
V-preB
λ5/14.1
Igα
Igβ
Syk
Lyn
PLCγ
NF-κB
BLNK
BTK
Ca++ influx
Agammaglobulinemia:
one phenotype
various gene defects
BTK (X)
μ chain (AR)
λ5/14.1 (AR)
pre-BCR signaling pathway
μ
V-preB
λ5/14.1
Igα
Igβ
Syk
Lyn
PLCγ
NF-κB
BLNK
BTK
Ca++ influx
Agammaglobulinemia:
one phenotype
various gene defects
BTK (X)
μ chain (AR)
λ5/14.1 (AR)
Igα (AR)
pre-BCR signaling pathway
μ
V-preB
λ5/14.1
Igα
Igβ
Syk
Lyn
PLCγ
NF-κB
BLNK
BTK
Ca++ influx
Agammaglobulinemia:
one phenotype
various gene defects
BTK (X)
μ chain (AR)
λ5/14.1 (AR)
Igα (AR)
BLNK (AR)
pre-BCR signaling pathway
μ
V-preB
λ5/14.1
Igα
Igβ
Syk
Lyn
PLCγ
NF-κB
BLNK
BTK
Ca++ influx
Agammagobulinemia: candidate genes for causal mutations
μ
V-preB
λ5/14.1
Igα
Igβ
Syk
Lyn
PLCγ
NF-κB
BLNK
BTK
Ca++ influx
Agammaglobulinemia:
one phenotype
various gene defects
BTK (X)
μ chain (AR)
λ5/14.1 (AR)
Igα (AR)
BLNK (AR)
Igβ (AR)
pre-BCR signaling pathway
μ
V-preB
λ5/14.1
Igα
Igβ
Syk
Lyn
PLCγ
NF-κB
BLNK
BTK
Ca++ influx
Agammagobulinemia: candidate genes for causal mutations
μ
V-preB
λ5/14.1
Igα
Igβ
Syk
Lyn
PLCγ
NF-κB
BLNK
BTK
Ca++ influx
MUTATION DETECTION
 DNA from 27 unrelated Czech XLA patients
 mutation screening (19 exons)
 PCR + DGGE and/or SSCP
 determination of mutation
 direct sequencing
 verification of mutation
 restriction analysis
 second strand sequencing
BTK – 11. EXON (p.E301del)
DGGE
m
wt
wt
SSCP
wt
wt
wt
wt
m
wt
SEQUENCING
ATG
ATT
p.M450I
RESULTS I
 23 unique mutations detected in 24 unrelated patients
• 5 affected male relatives
• 26 female carriers
10 mutations not described previously
 No mutation detected in 3 unrelated patients (all regions
sequenced)
• 1x homozygous deletion of the µH gene (JJ van Dongen)
• 2x analysis in progress
RELATIVE FREQUENCY OF MUTATIONS
Types of unique mutations (total number: 620)
Types of unique mutations (total number: 23)
1%
9%
19%
4%
32%
4%
13%
28%
12%
61%
9%
4%
4%
Missense
Deletions/insertions inframe
Deletions/insertions undef.
Others
Nonsense
Deletions/insertions frameshift
Splice site
BTKbase (http://bioinf.uta.fi)
Czech patients
LOCATION OF MUTATIONS
Total number of unique mutations - domains affected
Total number of unique mutations - domains affected
14
300
12
250
200
10
150
8
100
6
50
4
0
2
PH
TH
SH3
SH2
TK
Other
0
PH
TH
SH3
SH2
TK
Czech patients
BTKbase (http://bioinf.uta.fi)
PH
TH
SH3
SH2
KINASE
~ 140
~ 75
~ 65
~ 100
~ 280 AA
Other
RESULTS II
 6x polymorphism
• 4x previously described: c.908+70t>c; c.980+78g>a;
c.1482-29a>g; c.2031c>t
• 2x novel: c.103-27g>c; c.1763+71c>t
SSCP vs. DGGE
 both methods used in 13 cases with proven
mutation
 DGGE positive in 12/13 patients (sensitivity 92%)
 SSCP positive in 10/13 patients (sensitivity 77%)
 DGGE or SSCP positive in 13/13 patients (= 100%)
 DGGE is efficient method for mutation
screening of the BTK gene
CONCLUSION
 Detection of mutations in the BTK gene
enables:
 confirmation of diagnosis
 identification of mutation carriers
 prenatal diagnosis in affected families
ACKNOWLEDGEMENTS
Barbora Ravčuková
Lucie Grodecká
Jan Nejedlík
Jiřina Bartůňková, Anna Šedivá, Radana
Zachová, Václava Gutová, Eva Pařízková,
Helena Schneiderová, Olga Škopková, Olga
Kryštůfková, Vítězslav Novák
Jiří Litzman
SSCP
wt DNA
mutant DNA
denaturation
different migration pattern of
denatured DNA single strands
(non-denaturing PAGE)
wt
m
DGGE
wt DNA
mutant DNA
denaturation and
slow renaturation
primers - GC clamp
wt
m
low denaturant
high denaturant