Table 1 Overview of variants and rationale for testing in accessory assays

Gene	Protein variant^a	IARC class (InSiGHT)^b	Prior-P (in silico)^c	MMR activity (%)^d	IARC class (two-component)	Rationale for Y2H and localization testing	Binding partner interaction^e	Nuclear localization^f
MLH1	Wild type	NA	NA	100	NA		+	+
	p.R265S	4	0.884	27.0	4	(Likely) pathogenic	+	+
	p.A608D	4	0.9	37.3	4		-	+
	p.R659L	5	0.850	33.8	4		-	-
	p.N64S^g	3	0.718	51.6	3	Uncertain	-	+
	p.E268G	1	0.709	67.0	3		+	+
	p.A619P	4	0.273	37.5	3		-	+
	p.Q701K	2	0.1	72.7	1	Reduced activity in CIMRA assay	+	+
	p.N710D	1	0.1	60.4	2	Reduced activity in CIMRA assay	+	+
	p.D132H	1	0.868	97.0	2	Discordant Prior-P/CIMRA	+	+
	p.P581L	2	0.9	96.9	2		+	+
	p.P603R	2	0.872	97.0	2		+	+
	p.K618T	1	0.9	36.1	4	Discordant InSiGHT/two component	-	+
	p.A681T	5	0.1	73.6	1		+	+
MSH2	Wild type	NA	NA	100	NA		+	+
	p.Q419K	2	0.1	97.2	1	(Likely) not pathogenic	+	+
	p.A834T	2	0.428	90.1	1	(Likely) not pathogenic	+	+
	p.L93F	4	0.691	50.5	3	Uncertain	+	+
	p.Y165D	4	0.182	10.7	3		+	-
	p.N331D	2	0.757	62.4	3		+	+
	p.R524L^g	3	0.852	59.5	3		+	+
	p.I577T	2	0.325	54.8	3		+	+
	p.P652H	4	0.665	72.5	3		+	+
	p.T8M	1	0.1	60.1	2	Reduced activity in CIMRA assay	+	+
	p.R96H	2	0.1	69.5	1		+	+
	p.R106K	1	0.1	80.3	1		+	+

CIMRA cell-free in vitro MMR activity, IARC International Agency for Research on Cancer.
^aAmino acid numbering is based on the cDNA with +1 corresponding to the translation initiation codon in the GenBank reference sequence. MLH1: NP_000240.1; MSH2: NP_000242.1.
^bIARC classification for each variant as classified by the InSiGHT Variant Interpretation Committee.
^cIn silico prior probabilities as previously calculated,¹¹ truncated at a minimum value of 0.1 and a maximum value of 0.9.
^dCIMRA assay values as determined experimentally in this work.
^eBinding partner interaction (either MLH1 binding to PMS2 or MSH2 binding to MSH6) as determined by Y2H assays.+ Proficient for interaction, - deficient for interaction.
^fNuclear localization of MMR proteins as determined by transfections of fluorescently tagged proteins combined with microscopy. +Proficient for nuclear localization, - deficient for nuclear localization.
^gVariant reclassified by InSiGHT to class 3 (Uncertain) after our experiments were completed.

Search