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