Table 6.

Summary of functional studies in CDKN1B variants and variant classification

CDKN1B variant	LOH	IHC	Migration in denaturing protein gel	CHX chase	ICF	POMC expression	RHOA binding	CDK2 binding	ACMG classification after functional studies
c.-29_-26delAGAG, p.?	No.	Moderate to strong nuclear CDKN1B staining in ∼30% of cells.	Not done (variant previously functionally characterized).						Likely pathogenic.
c.320delA, p.Q107Rfs*12	Yes (in colon cancer).	n/a	Faster than wt (truncated protein).	Shortened half-life/increased degradation speed.	Cytoplasmic localization.	Increased.	–70%	+10%	Pathogenic.
c.356T>C, p.I119T	No.	Weak to moderate nuclear staining in ∼20% of cells.	Slower than wt (possible acquired posttranslational modification).	Shortened half-life/increased degradation speed.	Nuclear localization.	Unchanged.	–30%	–40%	VUS.
c.376G>C, p.E126Q	n/a.	n/a	Normal.	Shortened half-life/increased degradation speed.	Nuclear and cytoplasmic localization.	Unchanged.	–70%	–80%	Likely pathogenic.
c.407A>G, p.D136G	No.	Moderate to strong nuclear staining in ∼55% of cells.	Normal.	Shortened half-life/increased degradation speed.	Nuclear localization.	Unchanged.	–60%	–50%	VUS.

ACMG, American College of Medical Genetics and Genomics; CHX, cycloheximide; ICF, immunocytofluorescence; IHC, immunohistochemistry; LOH, loss of heterozygosity; n/a, not available; VUS, variant of uncertain significance; wt, wild type.