BackgroundPhenylketonuria (PKU) is an autosomal recessive congenital metabolic disorder caused byPAHvariants. Previously, approximately 5% of PKU patients remained undiagnosed after Sanger sequencing and multiplex ligation-dependent probe amplification. To date, increasing numbers of pathogenic deep intronic variants have been reported in more than 100 disease-associated genes.MethodsIn this study, we performed full-length sequencing ofPAHto investigate the deep intronic variants inPAHof PKU patients without definite genetic diagnosis.ResultsWe identified five deep intronic variants (c.1199+502A>T, c.1065+241C>A, c.706+368T>C, c.706+531>C, and c.706+608A>C). Of these, the c.1199+502A>T variant was found at high frequency and may be a hotspotPAHvariant in Chinese PKU. c.706+531T>C and c.706+608A>C are two novel variants that extend the deep intronic variant spectrum ofPAH.ConclusionDeep intronic variant pathogenicity analysis can further improve the genetic diagnosis of PKU patients. In silico prediction and minigene analysis are powerful approaches for studying the functions and effects of deep intronic variants. Targeted sequencing after full-length gene amplification is an economical and effective tool for the detection of deep intron variation in genes with small fragments.