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