ESPE Abstracts

Background: Pathogenic IGFI gene mutations causing childhood growth failure are rare. Only 5 autosomal recessive mutations, one IGFI copy number variant and 2 heterozygous frameshift mutations are reported. Heterozygous missense IGFI mutations have not previously been described.

Objectives: To identify and functionally characterise a novel missense IGFI variant in a patient with postnatal growth failure and delayed bone age. The patient had a normal birth weight (-0.2 SDS) but poor growth was observed from a few months of age. Height was -3.4 SDS and head circumference -1.6 SDS at 10 years of age. She had no dysmorphic features and normal development. Bone age was delayed by 2.5 years. A high peak GH was observed (17.1 mg/l). Baseline IGF-I levels were low/normal (144micrograms/L; -1.3 SDS) and responded poorly (increase <15micrograms/L) following IGF-I generation testing.

Methods: The patient was assessed on our unique targeted short stature gene panel. Functional analysis was performed using immunocytochemistry and furin cleavage assays. For the immunocytochemistry, HEK293T cells were transfected with wild-type and mutant IGFI constructs containing FLAG and HA tags. For the furin cleavage assays, whole cell lysates from HEK293T cells transfected with streptomycin tagged wild-type and mutant IGF-I constructs were incubated with furin to assess cleavage at the target site. IGF-I constructs designed for the furin assay lacked the signal peptide, meaning they could be harvested from the cell lysate and would not be secreted from the cell. Detection of constructs was performed by Western blot with 16% tricine gels to optimise separation of small proteins. Relative quantities were assessed using Image J analysis.

Results: We identified a novel heterozygous IGFI variant (102813333C>T, c.356G>A, p.R119H) which was exceedingly rare (gnoMAD frequency 0.004%) and predicted damaging by SIFT (CADD score 32). The p.R119H variant changed the first amino acid of the IGF-I E domain, the most critical residue for furin binding and highly conserved across species. Furin cleaves pro-IGF-I to mature IGF-I (E domain removal). Functional analysis showed that both wild-type and mutant IGF-I are able to translocate to the endoplasmic reticulum. Furin cleavage assay showed reduced cleavage of mutant p.R119H IGF-I compared to wild-type.

Conclusions: We report the first missense variant identified at the critical furin cleavage site, impairing the cleavage of pro-IGF-I to mature IGF-I. Our findings suggest the novel variant impairs pro-IGF-I cleavage, reducing mature circulating IGF-I levels. Pro-IGF-I is likely to be less biologically active than mature IGF-I, resulting in functional IGF-I deficiency and postnatal growth failure.