Immunological and hematological findings as major features in a patient with a new germline pathogenic CBL variant

Abstract

Casitas B-lineage lymphoma (CBL) encodes an adaptor protein with E3-ligase activity negatively controlling intracellular signaling downstream of receptor tyrosine kinases. Somatic CBL mutations play a driver role in a variety of cancers, particularly myeloid malignancies, whereas germline defects in the same gene underlie a RASopathy having clinical overlap with Noonan syndrome (NS) and predisposing to juvenile myelomonocytic leukemia and vasculitis. Other features of the disorder include cardiac defects, postnatal growth delay, cryptorchidism, facial dysmorphisms, and predisposition to develop autoimmune disorders. Here we report a novel CBL variant (c.1202G>T; p.Cys401Phe) occurring de novo in a subject with café-au-lait macules, feeding difficulties, mild dysmorphic features, psychomotor delay, autism spectrum disorder, thrombocytopenia, hepatosplenomegaly, and recurrent hypertransaminasemia. The identified variant affects an evolutionarily conserved residue located in the RING finger domain, a known mutational hot spot of both germline and somatic mutations. Functional studies documented enhanced EGF-induced ERK phosphorylation in transiently transfected COS1 cells. The present findings further support the association of pathogenic CBL variants with immunological and hematological manifestations in the context of a presentation with only minor findings reminiscent of NS or a clinically related RASopathy.

Keywords: CBL; CBL syndrome; Noonan syndrome; RAS signaling; phenotypic spectrum.

Figure 1.. Structural and functional consequences of the Cys401Phe change in CBL.

(A) Location of Cys⁴⁰¹ within the RING domain of CBL (PDB 4A49). The four residues (Cys³⁸¹, Cys³⁸⁴, Cys⁴⁰¹, Cys⁴⁰⁴) coordinating the first zinc atom (green), and those (Cys³⁹⁶, His³⁹⁸Cys⁴¹⁶, Cys⁴¹⁹) coordinating the second zinc atom (red) are shown. The mutated residue, Cys⁴⁰¹, is highlighted in yellow. Its substitution is predicted to affect proper folding of the domain. (B) Functional consequences of p.Cys401Phe on intracellular signaling. AKT and ERK phosphorylation assays in COS-1 cells transiently expressing the CBL p.Cys401Phe and p.Arg420Gln mutants, or the wild-type protein. Protein phosphorylation (pAKT, Cell Signaling, #9271; pERK1/2, Cell Signaling, #9106) was evaluated basally or following EFG stimulation (100 ng/ml; 30 min). Equal amounts of cell lysates were resolved on 10% polyacrylamide gel. β-actin (Sigma-Aldrich, #A5316) and CBL (anti-HA-HRP, Roche, #12013819001) levels are also shown for equal protein expression and loading. Representative blots of four independent experiments are shown. (C) Activation of AKT and ERK1/2 is expressed as a multiple of basal activation in cells transfected with wild-type CBL. Mean densitometry values ± SD of the four independent experiments are shown. Asterisks indicate significant differences compared with wild-type CBL at the corresponding time point, basally or upon EGF stimulation (**p<0.01; ***p<0.001; two-way ANOVA followed by Tukey's multiple comparison test). A single letter code is used to specify amino acid substitutions.