molecular analysis of b cell subsets in common variable immunodeficiency (cvid)
DESCRIPTION
Molecular Analysis of B cell Subsets in Common Variable Immunodeficiency (CVID). A Ridley , S Harris, J Burden, B Ferry, A Janda, Z Davis, D Oscier, AP Williams, JL Smith, E Hodges. CVID. 1 in 50,000 Most common primary antibody deficiency Diagnosed between 20 & 40 years - PowerPoint PPT PresentationTRANSCRIPT
Molecular Analysis of B cell Subsets in Common Variable
Immunodeficiency (CVID)
A Ridley, S Harris, J Burden, B Ferry,
A Janda, Z Davis, D Oscier, AP Williams,
JL Smith, E Hodges
CVID• 1 in 50,000
• Most common primary antibody deficiency
• Diagnosed between 20 & 40 years
• Heterogeneous syndrome
• Decreased Ig levels
• Recurrent infections
• Other causes of immunodeficiency excluded
CD27
IgM/D
naive memory
IgM Memory ~20%
Class Switched Memory ~20%
naive memory
CD27
IgM/D
IgM Memory ~4%
Class Switched Memory ~1%
naive memory
CD27
IgM/D
IgM Memory ~20%
Class Switched Memory ~4%
MB0 Reduced memory B cells
MB2 Class switched memory B cells
MB1Reduced classswitchedmemory B cells
Classification of CVID
LPD
Granulomatous
Splenomegaly
Splenomegaly
Clinical complications less severe
Somatic hypermutation (SHM): nucleotide substitution in V region to increase affinity for antigen
Class switch recombination (CSR): B-cells substitutes expression of IgM and IgD for IgG, IgA or IgE by deleting DNA between switch regions
IgD+ and CD27-
•IgD+/CD27+
•IgD-/CD27+
Aim• Molecular methodology to develop CVID classification to a
molecular level
• Investigation of the pattern and frequency of somatic
hypermutation in B cell subgroups as defined by CD27 expression
• Restriction enzyme-based hot-spot mutation assay (REHMA):
screening test to detect the presence of SHM in B cells of CVID
patients
• To allow improved characterisation, prognosis and management
of CVID patients
Flow cytometric analysis of B cells using CD19/IgM/IgD/CD27 antibodies
Separation of CD27+ peripheral blood B cells
Extract RNA & set up RT-PCR reactions to obtain cDNA
Amplification with: IgGVH3-23 and C FR1 and FR3 primers
Cloning & sequencing of
PCR products to identify the
pattern of somatic hypermutation
RE digestion & fragment length
analysis by capillary
electrophoresis
Correlate levels of somatic hypermutation to previous phenotypic classification
• 3 CLL patients
• 9 healthy controls
• 10 CVID patients
Ig Heavy Chain SHM Analysis
Specific product
2nd round PCR product
Ava II
Mutated
Alu I RE digest
202 bp
202 bp
Alu I RE digest
119 bp164 bp
175 bpUnmutated
202 bp
IgVH3-23 Cγ
FR1 FR3
175 bp
164 bp
158 bp
119 bp Alu I
Alu I
Alu I
202 bp
Ser31 Ser35 Ala50
RE digestion of CLL patients
158 bp
164 bp
Ava II
Alu I
Ava II
Ava II
Alu I
Alu I
158 bp
158 bp
119bp
164 bp
1
2
3
Ala50 Ser35 Ser31 Hot spots
RE digestion of a healthy control
202 bp
158 bp
119 bp 164 bp202 bp
175 bp
Specific product
Ava II
Alu I
Ala50 Ser35 Ser31 Hot spots
Alu1 digestion of MB0 CVID patients
CVID 1
Control202 bp175bp164 bp119bp
CVID 2
CVID 3
Ala50 Ser35 Ser31 Hot spots
Alu1 digestion of MB1 CVID patients
CVID 5
CVID 4
CVID 6
CVID 7
CVID 8
Control202 bp119 bp 175 bp164 bp
Ala50 Ser35 Ser31 Hot spots
Alu1 digestion of MB2 CVID patients
Control
CVID 9
CVID 10
202 bp119 bp 175 bp164 bp
Ala50 Ser35 Ser31 Hot spots
Mutational status by sequence analysis
Percentages of sequences with 0-1, 2-10, 11-20 and 21-30 mutations from a healthy control, an MB2 patient & an MB1
patient
Summary
• Rapid, non-radioactive screening method to look at SHM in CVID
• REHMA confirmed by sequence analysis
• REHMA patterns heterogeneous in the CVID subgroups defined by phenotype
• Correlation to clinical disease
• Investigation of IgM IgVH3-23 transcripts
• Use of PBMC
Acknowledgments
• SGH, Immunology & Molecular Pathology– Dr E Hodges
– Dr J Smith
– S Harris
– A Williams
– Z Shah
• Churchill Hospital, Oxford– J Burden
– Dr B Ferry
– Dr A Janda
• Royal Bournemouth Hospital– Dr D Oscier
– Z Davis