32 6 Vol. 32 No Journal of Jinan University Medicine Edition Dec TYR PCR TYR DHPLC DNA

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1 32 6 Vol. 32 No Journal of Jinan University Medicine Edition Dec Ⅰ TYR OCA TYR PCR TYR HA DHPLC DNA TYR c. 232 _ 233insGGG /c. 841G > T c. 232_233insGGG c. 841G > T E281X c. 232_233insGGG c. 841G > T HA DHPLC c. 841G > T Ⅰ TYR R A Application of the mutation analysis for oculocutaneous albinism typeⅠ in the prenatal diagnosis and a novel pathogenic mutation of TYR gene MIAO Chun-yue 1 WEN Ren-qing 1 ZHENG Hui 1 LI Hong-yi 2 1. Department of Physiology Medical College Jinan University Guangzhou China 2. Department of Medical Genetics Zhongshan Medical College of Sun Yat-Sen University Guangzhou China Abstract Aim Mutation analysis and prenatal diagnosis for the mutation of tyrosinase TYR gene in a family with oculocutaneous albinism type Ⅰ OCA1. Methods Polymerase chain reaction PCR technique was applied to amplify the regions of exon exon-intron and promoter of TYR gene while heteroduplex analysis HA denaturing high performance liquid chromatography DHPLC technique and DNA sequencing technology were used to analyze gene mutation sites of the proband and her parents. The pathogenic mutations were identified and the fetus genotypes were obtained by detecting TYR gene corresponding sites of the DNA sequence. Results The patient of this family was found to be a compound heterozygote with mutants c. 232_233insGGG and c. 841G > T. The parents of proband showed the same mutants c. 232_233insGGG and c. 841G > T. The nonsense mutation E281X was found to be pathogenic. However the fetus got neither of the two mutations. The results obtained by HA and DHPLC methods were fully consistent with the DNA sequencing results. Conclusion The proband's genotype was deter A Tel tlihzh@ jnu. edu. cn

11 A 1000-9965 2011 06-0624 - 05 Application of the mutation analysis for oculocutaneous albinism typeⅠ in the prenatal diagnosis and a novel pathogenic mutation of TYR gene MIAO Chun-yue 1 WEN

2 6 Ⅰ TYR 625 mined which was found to be a novel pathogenic mutation of TYR gene c. 841G > T. The fetus did not inherit any pathogenic mutations. Key words oculocutaneous albinism type 1 TYR gene gene mutation prenatal diagnosis Albinism DNA 2 DNA Taq oculocutaneous albinism OCA 1 /20 000OCA 100 bp DNA OCA1 ~ OCA OCA1 tyrosinase TYR PCR OCA PerkinElmer WAVE Transgenomic uvp-gds DNA HA DHPLC TYR 1-7 DNA TYR OCA1 OCA1A OCA1B OCA1A OCA1B DNA 2 ml EDTA OCA DNA 2 PCR PCR UCSC Genome Bioinformatics 12 TYR OCA1 1 NM bp DNA TYR 5 - PCR 30 μl Mg OCA mmol /L dntp 2. 0 mmol /L Taq 1. 5 U μmol /L DNA 0. 5 μg 10 3 μl 94 3 min ~ 60 s 49 ~ 65 OCA1 30 ~ 45 s ~ 60 s min 4 TYR 2%

tyrosinase TYR PCR OCA PerkinElmer WAVE 2100 1 5-10 Transgenomic uvp-gds7600 11 1.

3 PCR 8% 0. 5 TBE V 30 min PCR 18 μl 2 μl 200 V 3. 5 h mol /L + 10% 4 PCR 95 WAVE DNA DHPLC WAVEMARKTM DNA ± DNA TYR. DNA 2 2 DHPLC 2 DNA PCR 2 _233insGGG /c. 841G > T TYR 2. 1 PCR c. 232_233insGGG c. 841G > T DNA TYR TYR 2 % c. 232_233insGGG c. 841G > T DNA 2. 3 TYR 1 2 c. 841G > T c. 841G > Tc. 841G / > T E281X c c. 232 _ 233insGGG /c. 841G > T TYR c. 232 _ 233insGGG c. 841G > T OCA1 c. 841G > T c G TYR DHPLC CuA CuB E 2 DHPLC

841G > T TYR 2. 1 PCR c. 232_233insGGG c. 841G > T DNA TYR TYR 2 % c. 232_233insGGG c. 841G > T 3 2. 2 DNA 2. 3 TYR 1 2 c.

4 6 Ⅰ TYR OCA1 GAG X TAG DNA TYR TYR 1 CuB Cu OCA TYR c. 232_233insGGG c. 841G > T HA DHPLC 1 SCRIVER C BEAUDET A SLY W et al. The metabolic and molecular bases of inherited disease M. Universi- OCA1 ty of Michigan McGraw-Hill J OCA GR NSKOV K E K J SAND A et al. Birth prevalence andmutation spectrumin Danish patients with autosomal recessive albinism J. Investigative Ophthalmology & TYR Visual Science OCA OCA1 4 WEI AIHUA WANG YU LONG YAN et al. A comprehensive analysis reveals mutational spectra and com- OCA1 mon alleles in Chinese patients with oculocutaneous albi-

3 GR NSKOV K E K J SAND A et al. Birth prevalence andmutation spectrumin Danish patients with autosomal recessive albinism J.

5 nism J. J Invest Dermatol PREISING M N FORSTER H GONSER M et al. Screening of TYR OCA2 GPR143 and MC1R in patients with congenital nystagmus macular hypoplasia and fundus hypopigmentation indicating albinism J. Molecular Vision OETTING W S KING R A. Molecular basis of albinism mutations and polymorphisms of pigmentation genes associated with albinism J. Hum Mutat Ⅰ J KING R A PIETSCH J FRYER J P et al. Tyrosinase gene mutations in oculocutaneous albinism 1 OCA1 definition of the phenotype J. Hum Genet CHAKI M SENGUPTA M MONDAL M et al. Molecular and functional studies of tyrosinase variants among indian oculocutaneous albinism type 1 patients J. Journal of Investigative Dermatology HUTTON S M SPRITZ R A. Comprehensive analysis of oculocutaneous albinism among non-hispanic caucasians shows that OCA1 is the most prevalent OCA type J. J Invest Dermatol NAN HOWGMEI KRAFT P HUNTER D J et al. Genetic variants in pigmentation genes pigmentary phenotypes and risk of skin cancer in Caucasians J. Int J Cancer DNA http / /www. genome. UCSC. edu /genomes /TYR 13 Mutations of the tyrosinase gene associated with OCA http / /albinismdb. med. umn. edu / OCA1 mut. html 14 RAY K CHAKI M SENGUPTA M. Tyrosinase and ocular diseases Some novel thoughts on the molecular basis of oculocutaneous albinism type 1 J. Progress in Retinal and Eye Research SENDOVSKI M KANTEEV M ADIR N et al. First structures of an active bacterial tyrosinase reveal copper plasticity J. Journal of Molecular Biology J ROSENMANN E ROSENMANN A NE'EMAN Z et al. Prenatal diagnosis of oculocutaneous albinism type Ⅰ review and personal experience J. Pediatr Dev Pathol FALIK-BORENSTEIN T C HOLMES S A BORO- CHOWITZ Z et al. DNA-based carrier detection and prenatal diagnosis of tyrosinase2negative oculocutaneous albinism OCA1A J. Prenat Diagn OETTING W S FRYER J P SHRIRAM S et al. Oculocutaneous albinism type 1 the last 100 years J. Pigment Cell Res TANITA M MATSUNAGA J MIYAMURA Y et al. Polymorphic sequences of the tyrosinase gene allele analysis on 16 OCA1 patients in Japan indicate that three polymorphic sequences in the tyrosinase gene promoter could be powerful markers for indirect gene diagnosis J. J Hum Genet LEE SEUNGTAEK PARK S K LEE HAEYUL et al. DNA-based prenatal diagnosis of a Korean family with tyrosinase- related oculocutaneous albinism OCA1 J. Jpn J Hum Genet HSIEH JERYUARN WU CHICHEN CHANG C C et al. Prenatal diagnosis of oculocutaneous albinism two mutations located at the same allele J. Prenat Diagn

6 OETTING W S KING R A. Molecular basis of albinism mutations and polymorphisms of pigmentation genes associated with albinism J. Hum Mutat 1999 13 2 99-115. 7. Ⅰ J 2006 23 3 280-282.

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