NAT2*16 is assigned decreased function based on definitive evidence. The NAT gene nomenclature committee has historically assigned acetylation activity to alleles as either "rapid" or "slow", classifying the NAT2*16 allele as "slow" . Under the CPIC allele function terminology, slow acetylation alleles are categorized as "Decreased Function". NAT2*16 is characterized by the core SNVs c.341T>C (p.I114T) and c.803G>A (p.R268K). c.803G>A defines NAT2*4, formerly considered the reference allele, which has repeatedly exhibited increased function in vivo and in vitro. The functional SNV c.341T>C (p.I114T) in NAT2*16 has been studied for more than 25 years and has shown decreased NAT2 N-acetylation in numerous clinical studies using caffeine (7668286, 9333101, 9891107, 10744128, 20801937, 24221535), sulfamethazine (21946899), dapsone (10762005), or isoniazid (9321764) as a substrate. Five out of nine variant effect predictors tested by CPIC staff predict that c.341T>C impairs protein function. An in vitro study using a bacterial expression system measured the N-acetyltransferase capacity of a NAT2 construct containing the SNVs c.341T>C (p.I114T) and c.481C>T (p.L161=) and found this enzyme exhibited decreased activity compared to the former reference NAT2*4, using either 2-aminofluorene or sulfamethazine as substrates (7669073 ). Another in vitro study using a yeast expression system found that NAT2*16 displayed decreased thermostability relative to the former reference NAT2*4 resulting in decreased N-acetylation activity toward sulfamethazine (10574910). NAT2*16 was also shown to have decreased N-acetyltransferase activity in human hepatocytes (genotyped as NAT2*4/*16 or NAT2*6/*16) using isoniazid as a substrate (28752039). In vitro studies also show that NAT2*5 (which contains the core SNV c.341T>C, p.I114T) is subjected to enhanced protein degradation by the proteasome (15564878). Thus, NAT2*16 would presumably be degraded in the same manner.