Juvenile myelomonocytic leukemia due to a germline CBL Y371C mutation: 35-year follow-up of a large family

Abstract

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that arises from malignant transformation of the stem cell compartment and results in increased production of myeloid cells. Somatic and germline variants in CBL (Casitas B-lineage lymphoma proto-oncogene) have been associated with JMML. We report an incompletely penetrant CBL Y371C mutation discovered by whole-exome sequencing in three individuals with JMML in a large pedigree with 35 years of follow-up. The Y371 residue is highly evolutionarily conserved among CBL orthologs and paralogs. In silico bioinformatics prediction programs suggested that the Y371C mutation is highly deleterious. Protein structural modeling revealed that the Y371C mutation abrogated the ability of the CBL protein to adopt a conformation that is required for ubiquitination. Clinically, the three mutation-positive JMML individuals exhibited variable clinical courses; in two out of three, primary hematologic abnormalities persisted into adulthood with minimal clinical symptoms. The penetrance of the CBL Y371C mutation was 30% for JMML and 40% for all leukemia. Of the 8 mutation carriers in the family with available photographs, only one had significant dysmorphic features; we found no evidence of a clinical phenotype consistent with a "CBL syndrome". Although CBL Y371C has been previously reported in familial JMML, we are the first group to follow a complete pedigree harboring this mutation for an extended period, revealing additional information about this variant's penetrance, function and natural history.

Conflict of interest statement

Conflict of interest

The authors declare that they have no conflict of interest.

Figures

Fig. 1

Pedigree of NCI JMML family. III.4 died in childhood of non-leukemic causes; III.9 died in a motor vehicle accident.CBL Y371 genotyping was performed on all individuals with available DNA. Filled symbols JMML cases; symbol with a dot in the center obligate carrier. Hemhematologic abnormalities without meeting JMML diagnostic criteria, M4 acute myelomonocytic leukemia/M4

Fig. 2

Dysmorphic features in the JMML CBL p.Y371C heterozy- gote proband (IV.2). At 18 months of age (a), the proband exhib- its dysmorphic features including down-slanting palpebral fis- sures which are also apparent at 5 years of age (b). In an adult (age 36 years) frontal (c) photograph, the proband continues to have down-slanting palpebral fissures. d Adult profile (side) photograph (Swerdlow et al. 2008). In addition, the patient’s recent complete blood counts (CBCs) (2010–2013) indicate a per- sistently elevated absolute monocyte count, though she is currently clinically asymptomatic off-therapy.

Fig. 3

Sequence electrophero- gram demonstrating a germline CBL mutation in the proband (IV.2 of NCI JMML family). There is a chr11:119,148,892, A>G mutation (highlighted in blue) in exon 8 of theCBL gene. Codon 371 is normally TAC (tyrosine 371). The proband is heterozygous for the TGC (cysteine 371) codon

Fig. 4

a Structural comparison of inactive CBL-UbcH7(E2)-Zap70 with the active pY371-CBL-UbcH5B(E2)-Zap70 complex to high- light the key role of Y371 phosphorylation (pY371). E2 proteins are colored in cyan and shown inribbon form. The substrate peptides (Zap70) are displayed in tube format with N-terminal (blue)-to-C- terminal (red) coloring scheme. The Y371 residue in the inactive and the corresponding pY371 in the active forms are shown in CPK mode. Location of catalytic cysteine in E2 is also shown inball-and- stick and marked in yellow. The inactive conformation of CBL shows the TKBD and the ring finger domain (RING) in a closed conforma- tion and the Y371 is buried inside the protein core. The interaction between TKBD and RING is tighter, not allowing E2 to bind in con- formation required for ubiquitin transfer. In the active conformation (right) there is a significant conformational movement around the linker domain (commonly referred to as the LL1-LL-LL2 segment, see Fig. 4a, right panel). An opening of the TKBD-linker-RING seg- ment creates a favorable CBL conformation that allows E2 to bind favorably to the RING domain while becoming optimally positioned to interact with the substrate.b The possible structural impact of Y371C is shown using the inactive CBL structure (PDB:1FBV). Res- idues surrounding Y371 (left) and Y371C (right) are shown in stick mode. The domain residues are also colored (TKBD: green, LH: blue and LL2: red) to indicate their orientation in the CBL architecture. Residues that can form H-bonds (green dotted lines) or Pi–Pi interac- tion (orange line) are also marked