BACKGROUND: Notch3 mutations are the molecular genetic foundation for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Of all currently available detection methods, direct sequencing or restriction enzymes are frequently used, but the cost is relatively high, because the Notch3 gene is composed of many exons and mutational sites are widely distributed. Denaturing high-performance liquid chromatography (DHPLC) exhibits high efficiency and specificity and has been applied to gene detection. To date, there has no report regarding DHPLC in gene detection of large-scale CADASIL families in China. OBJECTIVE: To explore the application and value of DHPLC in the diagnosis of CADASIL by a mutation screening for Notch3 gene in CADASIL probands and their family members. DESIGN, TIME AND SETTING: A comparative observation was performed at the Genetic Diagnosis Laboratory of Institute of Geriatrics, Xuanwu Hospital of Capital Medical University and the Key Laboratory for Neurodegenerative Disease of the Ministry of Education between August 2003 and May 2004. PARTICIPANTS: Fourteen CADASIL patients and their family members, comprising eight males and six females, aged 38-62 years, were included. Their key features included recurrent sub-cortical ischemic events and vascular dementia. In addition, 100 healthy physical examinees were selected as controls, including 52 males and 48 females, aged 56-72 years, who had no neurodegenerative disease or psychosis, and no history or high risk for cerebrovascular disease. METHODS: DNA was extracted from white blood cells. Ten hotspots of the Notch3 gene for sequence variation were first amplified by PCR, and the products were detected using DHPLC. Exons exhibiting a variant in the DHPLC profile underwent another PCR amplification, followed by DNA sequencing to identify the mutation type. In addition, patients with normal DHPLC peak profiles underwent PCR amplification for the remaining 13 exons. DNA sequencing was performed for the exons, exhibiting a variation in the DHPLC profile to identify the mutation type. At the same time, Notch3 gene detection was undertaken in 100 healthy controls. MAIN OUTCOME MEASURES: Peak profile changes of PCR products under different column temperatures during DHPLC detection; Notch3 pathogenic mutations and polymorphisms. RESULTS: Three heterozygous missense mutations at exon3 and exon4, as well as 15 polymorphisms, were detected in DHPLC patients and their family members. Of the three heterozygous missense mutations, Cys134Tyr, a novel mutation, had not been previously reported in China. None of these mutations was found in 200 chromosomes of the controls. In addition, DHPLC did not exhibit noticeable changes after a 1 ℃ temperature adjustment, while 3 ℃ was too great and might result in DNA melting and indiscernible peak profile, so 2 ℃ was determined to be appropriate. CONCLUSION: DHPLC technique exhibits efficiency, sensitivity, and specificity in screening for Notch3 gene mutations. The optimal column temperature allows for two degrees of fluctuation based on the temperature recommended by applied software and peak alterations during screening.