What is the consequence of a mutation in either of the

recombinase activating genes (RAG-1 or RAG-2)

Antibody Variation + VDJ

Genetic recombinationAntibody diversity depends on:- Independent heavy and light chain combinations- small number of constant genes - Modular coding of variable regions (VDJ)- Imprecision in splicing / nucleotide addition- Somatic mutation- Class switching by DNA deletion

VDJ – Variable, Diverse, Joining Region

RAG + structureRAG = Recombinase Activating Genes.

RAG-1 and RAG-2 are proteins at the end of the VDJ genes; they associate with each other to recognise the recombination signal sequences and induce DNA cleavage between the Antigen receptor coding segment and RSS..

RAG-1 RAG-2

No. of amino acids 1040 537

Location of main enzymatic activity

Residues 384-1008 Residues 1-387

Core region 3 Acidic residues in DDE-motif

Predicted to have a 6-beta bladed propellers

Specificity More specific for target. Less specific for target

V(D)J recombination with RAG

1. Hydrolysis2. Transesterification

forming hairpin3. Signal joint formed4. Coding joint

formed5. DNA ligase forms

coding joint.

• RAG mainly involved in DNA cleavage.

• Assisted by other protein complexes

• RAG essential.

MutatioMutationsnsMutation = structural Mutation = structural

alteration in DNA or RNA. alteration in DNA or RNA. They are usually harmless They are usually harmless

but are occasionally but are occasionally harmful. harmful.

There are several types of mutation; e.g. insertions, deletions and

substitutions, and can be caused by a variety of things that fall into the categories of inherited or somatic

mutations.

For example: a mutation in the DDE region (Asp, Asp and Glu) of RAG1:If a Cys replaces an Asp residue there is partial conservation of catalytic function BUT if a Ala replaces the Asp it looses its cleaving ability.

Mutations are usually harmless and go unnoticed, but can sometimes be

harmful if they change the amino acid sequence. This results in the incorrect protein being translated

and a loss or reduction in its function.

Autosomal Recessive diseases = therefore child inherits both abnormal genes from parent.

Specific RAG mutations have been linked to a certain clinical presentations

SCIDSevere combine Immune

DeficiencyWHAT: Autosomal recessive inheritance

of mutated RAG-1 and RAG-2.

DIAGNOSIS: Recurrent infections + lymphopenia on full bloodcount

Confirmed by absence of T-cells + decreased B-cells.

PHYSIOLOGY: No T/B-lymphocytes means there is no production of antibodies to defend the body from pathogenic attack. Patient = v.vulnerable to opportunistic infections and unless defects are corrected or precautions taken will have died before 1st/2nd birthday.  TREATMENTS: Bone marrow transplant, bubble, gene therapy.

TYPES: B-cell +ve and B-cell -ve

Omenn’s Syndrome -Autosomal recessive form of SCID

-Missense mutations in RAG-1 or RAG-2 genepartial or no V(D)J recombination activitynon-functioning or poorly functional B and T cells

-Symptoms: erythroderma, desquamation, chronic diarrhoea, swollen lymph nodes and enlargement of the liver and spleen

-Treatment: bone marrow transplant, isolation, antibiotics, gene therapy

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The Case

2 month year old female from Saudi Arabia

Presents aged 2 weeks with scaly erythroderma associated with persistent fever and later developed pneumonia, siblings died within first year of life

The InvestigationFlow cytometry showed a critically

low (absent) B-cell count with a predominance of T cells (82%)

Humoral immunity was also abnormal, with elevated serum IgE levels.

RAG mutation analysis revealed a homozygous missense mutation.

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The Risk, Prevention and Treatment

RISK Consanguineous marriage increase

AR disorders.PREVENTION Importance of premarital, pre-birth

screening and genetic counsellingTREATMENT 1st line – gene therapy Specific therapy for dermatitis and

eosinophilia in Omenn syndrome is immunosuppression with cyclosporine

Future – gene transfer

References

E medicine (date unknown) Treatment of Ommens [Online] Available from http://emedicine.medscape.com/article/887687-treatment Accessed 30/10/2010

Case journal (date unknown) [Online]Ommen Case Available from http://casesjournal.com/content/2/1/8391 Accessed 30.10/2010

Janeway. R, 2010, Immunobiology, 7th Edition, Elsevier, London.

Schuetz C, Huck K, Gudowius S, Megahed M, Feyen O, Hubner B, Schnieder D, Manfras B, Pannicke U, Willemze R, Knuchel R, Gobel U, Schuetz C, Huck K, Gudowius S, Megahed M, Feyen O, Hubner B, Schnieder D, Manfras B, Pannicke U, Willemze R, Knuchel R, Gobel U, Schulz A, Borkhardt A, Friedrich W, Schwarz K, Niehues T. 2008. An Immunodeficiency Disease with RAG Mutations and Granulomas. Schulz A, Borkhardt A, Friedrich W, Schwarz K, Niehues T. 2008. An Immunodeficiency Disease with RAG Mutations and Granulomas. N Eng J N Eng J Med Med 358 (19) pp. 2030-8358 (19) pp. 2030-8

Villa A, Sobacchi C, Notatangelo L, Bozzi F, Abinun M, Abrahamsen T, Arkwright P, Baniyash M, Brooks E, Conley M, Cortes P, Duse M, Fasth Villa A, Sobacchi C, Notatangelo L, Bozzi F, Abinun M, Abrahamsen T, Arkwright P, Baniyash M, Brooks E, Conley M, Cortes P, Duse M, Fasth A, Filipovich A, Infante A, Jones A, Mazzolari E, Muller S, Pasic S, Rechavi G, Sacco M, Santagata S, Schroeder M, Seger R, Strina D, Ugazio A, A, Filipovich A, Infante A, Jones A, Mazzolari E, Muller S, Pasic S, Rechavi G, Sacco M, Santagata S, Schroeder M, Seger R, Strina D, Ugazio A, Valiaho J, Vihinen M, Vogler L, Ochs H, Vezzoni P, Friedrich W, Schwarz K. 2001. V(D)J recombination defects in lymphocytes due to RAG Valiaho J, Vihinen M, Vogler L, Ochs H, Vezzoni P, Friedrich W, Schwarz K. 2001. V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations. mutations: severe immunodeficiency with a spectrum of clinical presentations. Blood Blood 97 pp.81-8897 pp.81-88

Wada T, Toma T, Okamoto H, Kasahara Y, Koizumi S, Agematsu K, Kimura H, Shimada A, Hayashi Y, Kato M, Yachie A. 2005. Oligoclonal Wada T, Toma T, Okamoto H, Kasahara Y, Koizumi S, Agematsu K, Kimura H, Shimada A, Hayashi Y, Kato M, Yachie A. 2005. Oligoclonal expansion of T lymphocytes with multiple second-site mutations leads to Omenn syndrome in a patient with expansion of T lymphocytes with multiple second-site mutations leads to Omenn syndrome in a patient with RAG1-RAG1-deficient severe deficient severe combined immunodeficiency combined immunodeficiency Blood Blood 106 (6) pp. 2099-2101106 (6) pp. 2099-2101

Kim D, Dai Y, Mundy C, Yang W, Oettinger M. 1999. Mutations of acidic residues in RAG1 define the active site of V(D)J recombinase. Kim D, Dai Y, Mundy C, Yang W, Oettinger M. 1999. Mutations of acidic residues in RAG1 define the active site of V(D)J recombinase. Genes Genes Dev. Dev. 13(23) pp. 3070-8013(23) pp. 3070-80

• Santagata, S. Et al., 2000. N-terminal RAG1 frameshift mutations in Omenn’s syndrome: internal methionine usage leads to partial V(D)J recombination activity and reveals a fundamental role in vivo for the N-terminus domains. Proceedings of the National Academy of Sciences USA (26):14572-7.

• Couedel, C et al., 2010. Analysis of mutations from SCID and Omenn syndrome patients reveals the central role of the RAG2 PHD domain in regulating V9D0J recombination. Journal of Clinical Investigations.(4):1337-44.

• Schwartz, R., 2010. Omenn Syndrome. [Online] Emedicine. Available at: http://emedicine.medscape.com/article/887687-overview. [Accessed: 31 October 2010]