SPECTRUM OF THE BTK GENE MUTATIONS IN CZECH XLA
PATIENTS
Tomáš Freiberger
Molecular Genetics LabCentre for Cardiovascular Surgery and Transplantation
University Centre for Primary ImmunodeficienciesBrno, 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 pre-
BCR 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 PreB-I Pre-B-II Immature B Mature B
CD22CyIgα
TdT
CD22CyIgα
CD22CyIgαCD19TdTψLCD10++
CD22CyIgαCD19TdTψLCD10+
CD22CyIgαCD19
ψLCD10+
Cy μ
CD22CyIgαCD19
CD10+
Cy μ
CD22CyIgαCD19
CD10+
SmIgM
CD22CyIgαCD19
CD10+
SmIgMSmIgD
Rag+DHJH
Rag+VLJL
Rag+VHJH
Rag-
adapted from Noordzij et al., 2002
pre-BCR signaling pathway
V-preB
λ5/14.1
Syk
Igα
μ
NF-κB
Igβ
Ca++ influx
Lyn
BLNK BTK
PLCγ
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
V-preB
λ5/14.1
Syk
Igα
μ
NF-κB
Igβ
Ca++ influx
Lyn
BLNK BTK
PLCγ
Agammaglobulinemia:
one phenotype
various gene defects
BTK (X)
μ chain (AR)
pre-BCR signaling pathway
V-preB
λ5/14.1
Syk
Igα
μ
NF-κB
Igβ
Ca++ influx
Lyn
BLNK BTK
PLCγ
Agammaglobulinemia:
one phenotype
various gene defects
BTK (X)
μ chain (AR)
λ5/14.1 (AR)
pre-BCR signaling pathway
V-preB
λ5/14.1
Syk
Igα
μ
NF-κB
Igβ
Ca++ influx
Lyn
BLNK BTK
PLCγ
Agammaglobulinemia:
one phenotype
various gene defects
BTK (X)
μ chain (AR)
λ5/14.1 (AR)
Igα (AR)
pre-BCR signaling pathway
V-preB
λ5/14.1
Syk
Igα
μ
NF-κB
Igβ
Ca++ influx
Lyn
BLNK BTK
PLCγ
Agammaglobulinemia:
one phenotype
various gene defects
BTK (X)
μ chain (AR)
λ5/14.1 (AR)
Igα (AR)
BLNK (AR)
pre-BCR signaling pathway
V-preB
λ5/14.1
Syk
Igα
μ
NF-κB
Igβ
Ca++ influx
Lyn
BLNK BTK
PLCγ
Agammagobulinemia: candidate genes for causal mutations
V-preB
λ5/14.1
Syk
Igα
μ
NF-κB
Igβ
Ca++ influx
Lyn
BLNK BTK
PLCγ
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
V-preB
λ5/14.1
Syk
Igα
μ
NF-κB
Igβ
Ca++ influx
Lyn
BLNK BTK
PLCγ
Agammagobulinemia: candidate genes for causal mutations
V-preB
λ5/14.1
Syk
Igα
μ
NF-κB
Igβ
Ca++ influx
Lyn
BLNK BTK
PLCγ
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 SSCP
m wt wt 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
Types of unique mutations (total number: 23)
61%
4%
9%
13%
4%
9%
BTKbase (http://bioinf.uta.fi) Czech patients
Types of unique mutations (total number: 620)
32%
12%
4%
28%
4%
19%
1%
Missense Nonsense
Deletions/insertions inframe Deletions/insertions frameshift
Deletions/insertions undef. Splice siteOthers
RELATIVE FREQUENCY OF MUTATIONS
0
2
4
6
8
10
12
14
PH TH SH3 SH2 TK Other
Total number of unique mutations - domains affected
BTKbase (http://bioinf.uta.fi) Czech patients
0
50
100
150
200
250
300
PH TH SH3 SH2 TK Other
Total number of unique mutations - domains affected
LOCATION OF MUTATIONS
PH TH SH3 SH2 KINASE
~ 140 ~ 75 ~ 65 ~ 100 ~ 280 AA
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
wt mlow denaturant
high denaturant
primers - GC clamp