Screening For High-frequency Mutations Of SLC25A13Gene By Means Of Melting Curve Analysis In Guangdong Province

Background and Aims Citrin deficiency （CD） is an autosomal recessive diseasecaused by SLC25A13mutations, and Neonatal Intrahepatic Cholestasis caused byCitrin Deficiency （NICCD, OMIM#605814） is the main clinical phenotype of CD inpediatrics. Our previous study has revealed that the five mutations, i.e.851₈54del,1638₁660dup, IVS16ins3kb, IVS6+5G>A, and g.1399C>T, were on top of the list ofSLC25A13mutations in Chinese CD patients. Melting curve analysis has beendeveloped recently as a convenient, accurate and rapid method for the screening ofSLC25A13mutations and for CD diagnosis. This study aims to screen forhigh-frequency mutations of SLC25A13gene using this method, and then calculatethe carrier rate of SLC25A13mutations and the theoretical frequency of CD patientsin Guangdong province, China.Subjects and Methods A total of2428blood samples for healthy examinationwere collected from the medical centers in different cities in Guangdong province.Most of them were collected from the cities in Pearl River Delta area, including599samples from Guangzhou,306from Shenzhen,223from Foshan,240fromZhongshan, and190from Huizhou, respectively. The remaining were from theperipheral cities outside of Pearl River Delta area, including213from Meizhou,190from Heyuan,260from Zhanjiang, and200from Qingyuan respectively. DNA wasextracted with Blood DNA Mini Kit, and then the5high-frequency SLC25A13mutations screened using melting curve analysis. The carrier rate of SLC25A13mutations was represented as the proportion of the number of confirmed carriers in allthe research subjects, and then the theoretical morbidity of CD calculated according toHardy－Weinberg principle. Finally, the carrier rates of SLC25A13mutations indifferent cities were compared using Chi square tests, with P<0.05as the significantcriterion.Results1. A total of52carriers of SLC25A13mutation were detected in this study,including41carriers of851₈54del,5IVS6+5G>A,21638₁660dup,2IVS16ins3kb,and a novel g.1420G>A （V474M, n=2） mutation. The g.1399C>T mutation was notdetected in this study.2. The carrier rate of high-frequency mutations of SLC25A13gene is around1/47（52/2428）, while CD theoretical morbidity about1/8800in Guangdong province. The carrier rate and theoretical morbidity rate are about1/60（26/1558） and1/14400in Pearl River Delta area, respectively, whilst about1/33（27/870） and1/4600in the peripheral cities, respectively.3. The carrier rates in thecities of Guangzhou, Shenzhen, Foshan, Huizhou, Zhongshan, Meizhou, HeyuanZhanjiang and Qingyuan are about1/54,1/61,1/56,1/95,1/60,1/107,1/20,1/33and1/33, respectively, while CD theoretical morbidity about1/11700,1/14800,1/14900,1/36100,1/14400,1/45800,1/1600,1/4400, and1/4400, respectively.4.The difference of the carried rate between Pearl River Delta area and the peripheralarea is significant statistically （2=4.639，P<0.05）. The carried rate in Heyuan is muchhigher than that in most of the cities in Pearl River Delta area （when compared withGuangzhou,2=6.058, P<0.05; Shenzhen,2=4.908, P<0.05; Huizhou,2=5.211,P<0.05; Zhongshan,2=4.056, P<0.05）. In the peripheral cities, the carried rate inHeyuan is higher than in Meizhou （2=6.166，P<0.05）, but no significant differencewas observed when compared with that in Zhanjiang and Qingyuan （Heyuan andZhanjiang:2=1.184， P>0.05, Heyuan and Qingyuan:2=1.106， P>0.05）. Nosignificant difference of the carrier rate is observed among the cities in Pearl RiverDelta area.Conclusions1. Melting curve analysis was confirmed in this study as a convenient,accurate and rapid method for the screening of SLC25A13mutations with thecapability to detect novel mutation, and the novel mutation g.1420G>A expanded themolecular spectrum of citrin deficiency.2. The carried rate of high-frequencymutations of SLC25A13gene in the peripheral cities is significantly higher than thatin Pearl River Delta area.3. Based on the results in this study and latest populationstatistical findings, it is speculated that at least11800patients with CD exist inGuangdong province currently, and enough emphasis should be place onto theirdiagnosis and treatment.4. The screening data of SLC25A13mutations in this studyconstitute a primary evidence of molecular epidemiology for the effect evaluation ofCD on the population in Guangdong province.