Cold Agglutinin Disease (CAD)

Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia (AIHA) that leads to the premature destruction of red blood cells.1 CAD represents about 15% of AIHA cases,2 and primary disease results from a B-cell lymphoproliferative disorder of the bone marrow.3 In this clonal lymphoproliferative disorder, autoantibodies designated by cold agglutinins (CAs) are produced and bind to the red blood cells’ surface blood I antigen at cold temperatures (between 0 and 4 °C). CAs’ attack on the red blood cells causes their agglutination and complement activation by the classical pathway.1 

Molecular Biology of Cold Agglutinins

CAs in CAD are mostly immunoglobulin (Ig) M produced by monoclonal B-cells in the lymphoplasmacytic cell stage.1 Patients with CAD therefore have monoclonal antibodies circulating in their blood that are mainly encoded by the IGHV4-34 gene (initially designated as VH4-21 gene).1 The IGHV4-34 gene is located on the q arm of chromosome 14 and encodes for the CA immunoglobulin heavy-chain.1,4 The sequences Gln6-Trp7 (QW) and Ala23-Val24-Tyr25 (AVY) within the framework region 1 (FR1) of the IGHV4-34 gene are determining for binding to the I antigen.5 Both affinity and specificity to I antigen binding depend on the heavy chain complementarity determining region 3 (CDR3) and light chain variable region.1

It has been reported that inactivating mutations observed in the N-glycosylation sequon located in the gene IGHV4-34, specifically in CDR2, lead to decreased hemoglobin levels. This occurs because the localization of the N-glycosylation site in the antigen-binding pocket can favor the blocking of glycan binding. The study also revealed that a higher number of mutations within the FR3 is located in a particular hotspot germline amino acid sequence Lys90-Leu91-Ser92 (KLS), that may additionally contribute to a decrease in hemoglobin levels.6

Although less frequently, anti-I CAs may be encoded by a different IGHV3 gene family such as IGHV3-23 and IGKV3-20.7 In a recent study, 1 of 27 patients with CAD was described as producing CAs that were encoded by the genes IGHV3-23 and IGKV3-20.6 The CDR3 region of the IGKV3-20 gene was shown to be homogeneous within a subgroup of patients, and the low level of mutations associated with the gene correlated with younger ages at diagnosis.6

Mutations in Cold Agglutinin Disease

CAD is not a hereditary disease, however, recurrent mutations in the genes KMT2D (MLL2) and CARD11 were identified in 69% (11 of 16 patients) and 31% (5 of 16 patients) of a small group of patients with CAD, respectively.2 In this study, antibody-producing B-cells were sourced from the bone marrow of patients aged 56 to 84 years, and whole exome sequencing (WES) was used for studying the landscape of mutations.2 The KMT2D gene encodes for the methyltransferase 2D protein, which is important for development regulation and tumor suppression.8 There were 7 mutations in the KMT2D gene that led to inactive enzymes due to the formation of a truncated protein. KMT function can be also compromised through missense mutations of the C-terminal domain.2 KMT2D mutations have also been identified in different types of lymphoma, congenital heart disease, and Kabuki syndrome.3 Mutations of CARD11 were identified on exon 6, which encodes the BAR domain of CARD11. The CARD11 gene encodes for a protein that regulates immune cell activity.9 Mutations in this domain and in the coiled-coil region where the domain is located are shown to impact NF-kB activation and consequently the proliferation of B-cells and autoantibody production.10

Other studies point to a potential interference of chromosome instability in CAD, as trisomy 3, 12, and 18 were identified in patients with CAD.11,12 In a recent study using cytogenetic microarray and WES, the gain of chromosome 3 was a recurrent finding in CAD-associated lymphoproliferative disease and chromosomes 12 and 18 were suggested as therapeutic predictors.11 

The possibility of exploring the genetic changes observed in CAD for the development of targeted therapies has motivated a wider study including 18 patients with CAD who were participants in a clinical trial. Mutations were detected in genes involved in lymphoma development.3 Between 36 and 163 mutations were identified in the exome region and 4 genes, KMT2D, IGLL5, CARD11, and CXCR4, were reported with nonsynonymous mutations in more than 20% of patients. Other recurrent nonsynonymous mutations were also detected in several other genes (11% to 17%), such as HIST1H1E, CSMD3, and FAT1. Patients presenting with a KMT2D mutation together with a CARD11, CXCR4, or both mutations showed lower hemoglobin levels than patients with KMT2D isolated mutations.3

CAD can be distinguished from lymphoplasmacytic lymphoma through the MYD88 L265P mutation, which appears in 90% of the lymphoma cases but not typically in CAD-associated lymphoproliferative disease.1,2 Mutations in KMT2D, CARD11, and CXCR4 may potentially aid in CAD diagnosis and support a targeted treatment development for patients. 


1. Berentsen S. New insights in the pathogenesis and therapy of cold agglutinin-mediated autoimmune hemolytic anemia. Front Immunol. 2020;11:590. doi:10.3389/fimmu.2020.00590

2. Małecka A, Trøen G, Tierens A, et al. Frequent somatic mutations of KMT2D (MLL2) and CARD11 genes in primary cold agglutinin disease. Br J Haematol. 2018;183(5):838-842. doi:10.1111/bjh.15063

3. Małecka A, Trøen G, Delabie J, et al. The mutational landscape of cold agglutinin disease: CARD11 and CXCR4 mutations are correlated with lower hemoglobin levels. Am J Hematol. 2021;96(8):E279-E283. doi:10.1002/ajh.26205

4. Berentsen S, Malecka A, Randen U, Tjønnfjord GE. Cold agglutinin disease: where do we stand, and where are we going? Clin Adv Hematol Oncol. 2020;18(1):35-44.

5. Potter KN, Hobby P, Klijn S, Stevenson FK, Sutton BJ. Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded Igs in recognition of the red blood cell I antigen. J Immunol. 2002;169(7):3777-3782. doi:10.4049/jimmunol.169.7.3777

6. Małecka A, Trøen G, Tierens A, et al. Immunoglobulin heavy and light chain gene features are correlated with primary cold agglutinin disease onset and activity. Haematologica. 2016;101(9):e361-e364. doi:10.3324/haematol.2016.146126

7. Jefferies LC, Carchidi CM, Silberstein LE. Naturally occurring anti-i/I cold agglutinins may be encoded by different VH3 genes as well as the VH4.21 gene segment. J Clin Invest. 1993;92(6):2821-2833. doi:10.1172/JCI116902

8. Froimchuk E, Jang Y, Ge K. Histone H3 lysine 4 methyltransferase KMT2D. Gene. 2017;627:337-342. doi:10.1016/j.gene.2017.06.056

9. CARD11 gene. MedlinePlus. Updated August 18, 2020. Accessed September 14, 2021.

10. Lenz G, Davis RE, Ngo VN, et al. Oncogenic CARD11 mutations in human diffuse large B cell lymphoma. Science. 2008;319(5870):1676-1679. doi:10.1126/science.1153629

11. Małecka A, Delabie J, Østlie I, et al. Cold agglutinin-associated B-cell lymphoproliferative disease shows highly recurrent gains of chromosome 3 and 12 or 18. Blood Adv. 2020;4(6):993-996. doi:10.1182/bloodadvances.2020001608

12. Michaux L, Dierlamm J, Wlodarska I, et al. Trisomy 3 is a consistent chromosome change in malignant lymphoproliferative disorders preceded by cold agglutinin disease. Br J Haematol. 1995;91(2):421-424. doi:10.1111/j.1365-2141.1995.tb05315.x

Reviewed by Debjyoti Talukdar, MD, on 9/16/2021.