This article illustrates the mutational landscape of cold agglutinin disease and correlation of CARD11 and CXCR4 mutations with lower Hb levels.Anal. of the bone marrow from 18 patients with CAD, included in a recent clin. trial by WES.The total number of nonsynonymous mutations detected in each CAD sample ranged from 13 to 62, while the number of all mutations in the sample cohort in the exome regions ranged from 36 to 163.Four genes showed nonsynonymous mutations in more than 20% of patients: KMT2D (12/18; 67%; including splice region mutation), IGLL5 (8/18, 44% and 11/18, 61% including non-coding region mutations), CARD11 (6/18, 33%), and CXCR4 (4/18, 22% and 5/18, 28% including non-coding region mutations).Addnl., several genes showed recurrent nonsynonymous mutations in two or three cases each (11%-17%): HIST1H1E, TMPRSS7, CSMD3, FAT1, FAT4, PHLDB1, CFTR, EP300, GRIK2, LPIN3, LTB, MACF1, NBEA, NEFH, PCNX2, PKHD1L1, RRAGC, SLC30A9, TMEM132E and ZNF618.All patients with either CARD11 or CXCR4 mutations, or both, had concurrent KMT2D mutations.Almost all patients with KMT2D mutations (10/12, 83%) had at least four other recurrent mutations.Patients with CARD11 (six cases) or CXCR4 (five cases) mutations had at least four other recurrent mutations.The CXCR4 functional mutations were detected in four patients.These mutations are frameshift mutations located very close to the C-terminal end of the protein at positions 316-330, and are projected to prevent receptor internalization creating a state of prolonged activation.The IGLL5 gene mutations were located only in the five prime UTR comprising exon 1 and intron 1, with the region affected being less than 600 bp long.A difference in Hb levels prior to treatment was found between patients with, and those without mutations in either CARD11 or CXCR4, or both.The median difference between the lower limit of normal and actual Hb level (adjusted for sex) for patients with CARD11, CXCR4, or both, mutations was 4.5 g/dL (mean difference 4.7 g/dL) and for patients without CARD11 or CXCR4 mutations 1.6 g/dL (mean difference 2.2 g/dL).The difference between the groups was statistically significant (p = 0.026; Mann-Whitney 2-tailed U test).Pathway anal. showed that chromatin modification and chromatin organization were the most affected pathways in CAD patients.Most of the patients (15/18; 83%) had mutations of at least one gene involved in chromatin modification or organization.In addition to KMT2D there were multiple other genes mutated in different samples: ASH1L, ARID1B, BRD1, CACNA1D, CREBBP, DNMT3A, EP300, HDAC1, HIST1H2BO, HIST1H3G, JADE3, KAT8, KDM6A, NFKB1, PPARD, PRDM16, SAP18, SETD1B, SMARCC1 and TBL1XR1.Addnl., 14/18 (78%) samples showed mutations in genes involved in regulation of the nuclear factor kappa B (NF-κB) pathway.In addition to CARD11 and CXCR4, other genes included were CASP3, COL1A1, CREBBP, DLL1, EP300, ERBB3, FBXW7, FGFR1, HDAC1, IRAK2, LRRC7, LTB, MTOR, NFKB1, RBCK1, TBL1XR1 and TLR3.Only 2/18 patients did not show mutations affecting either chromatin modification or organization, or affecting the NF-κB pathway (CAD-1.34 and CAD-7).Of interest, these two patients had close to normal Hb levels.Somatic KMT2D mutations are found in many lymphoma types, while constitutional KMT2D mutations give rise to Kabuki syndrome.It is suggested that KMT2D is a tumor suppressor gene, and KMT2D mutations might act as driver mutations in lymphoma.NF-κB signaling is important for lymphoma development, due to its role in lymphocyte survival and proliferation.In conclusion, CAD showed a relatively low mutational burden, but with recurrent gene mutations.The most common recurrent mutations were in genes known to be involved in lymphoma development.The CAD patients with a KMT2D mutation associated with CARD11 or CXCR4 mutations, or both, had lower Hb levels at diagnosis compared to patients with absence of KMT2D mutation or patients with KMT2D mutation without CARD11 or CXCR4 mutations.
