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1 HEREDITAS (Beijing) , 30(4): ISSN 研究报告 DOI: /SP.J Waardenburg MITF 陈静 1, 杨淑芝 2, 刘军 1, 韩冰 1, 王国建 1, 张昕 1, 康东洋 1, 戴朴 1, 杨伟炎 1, 袁慧军 1., ; 2., 摘要 : Waardenburg 综合征 (WS) 是临床上常见的常染色体显性遗传性耳聋综合征, MITF 基因突变与部分 Waardenburg 综合征 Ⅱ 型 (WS2) 病例的发病有关 MITF 属于碱性螺旋 - 环 - 螺旋亮氨酸拉链转录因子家族, 能调节酪氨酸酶基因, 参与黑色素细胞的分化文章报道了 1 个携带 MITF 基因点突变的 3 代 Waardenburg 综合征 Ⅱ 型中国家系先证者表现为先天性重度感音神经性聋虹膜异色面部雀斑 ; 其他家系成员除一名仅表现为先天性耳聋外, 均表现为颜面上肢雀斑和 / 或早白发患者可检测到 c.639dela 杂合突变, 该突变在 MITF 基因第 7 外显子上产生了终止密码子 (p.i220x), 突变产生的截短的 MITF 蛋白没有 DNA 结合活性该突变是 WS2 病例中第 3 个位于 MITF 第 7 外显子的突变, 尚未见报道该突变与已报道的位于第 7 外显子其他两个突变仅相差 1 个碱基, 氨基酸改变十分相似, 但在表型上有显著差别, 提示遗传背景对 WS 临床表型有重要影响关键词 : Waardenburg 综合征 Ⅱ 型 ; 遗传性耳聋 ; MITF 基因 ; 基因突变 1 Mutation screening of MITF gene in patients with Waardenburg syndrome type 2 CHEN Jing 1, YANG Shu-Zhi 2, LIU Jun 1, HAN Bing 1, WANG Guo-Jian 1, ZHANG Xin 1, KANG Dong-Yang 1, DAI Pu 1, YOUNG Wie-Yen 1, YUAN Hui-Jun 1 1. Institute of Otolaryngology, Chinese PLA General Hospital, Beijing , China; 2. Department of Otolaryngology, the First Affiliated Hospital of Chinese PLA General Hospital, Beijing , China Abstract: Warrgenburg syndrome type 2 (WS2) is the most common autosomal dominantly-inherited syndrome with hearing loss. MITF (microphthalmia associated transcription factor)is a basic-helix-loop-helix-luecine zipper (bhlhzip) factor which regulates expression of tyrosinase, and is involved in melanocyte differentiation. Mutations in MITF associated with WS2 have been identified in some but not all affected families. Here, we report a three-generation Chinese family with a point mutation in the MITF gene causing WS2. The proband exhibits congenital severe sensorineural hearing loss, heterochromia iridis and facial freckles. One of family members manifests sensorineural deafness, and the other patients show premature greying or/and freckles. This mutation, heterozygous deletion c.639dela, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking normal interaction with its target DNA motif. This mutation is a novel mutation and the third case identified in exon 7 of MITF in WS2. Though there is only one base pair distance between this novel mutation and the other two documented cases and similar amino acids change, significant difference is seen in clinical phenotype, which suggests genetic background may play an important role. 收稿日期 : ; 修回日期 : 基金项目 : ( ) [Supported by National Natural Science Foundation of China (No and )] 作者简介 : (1978 ),,,, Tel: ; jingchen91@yahoo.com.cn 通讯作者 : (1936 ),,,,, Tel: ; YWY@263.com (1966 ),,,,, Tel: ; yuanhj310@yahoo.com.cn

, 100073 摘要 : Waardenburg 综合征 (WS) 是临床上常见的常染色体显性遗传性耳聋综合征, MITF 基因突变与部分 Waardenburg 综合征 Ⅱ 型 (WS2) 病例的发病有关 MITF 属于碱性螺旋 - 环 - 螺旋亮氨酸拉链转录因子家族, 能调节酪

2 434 HEREDITAS (Beijing) Keywords: waardenburg syndrome type 2; hereditary hearing loss; MITF gene; mutation Waardenburg (Waardenburg syndrome, WS),, 2 Waardenburg [1] Waardenburg, ; WS 4 : I (WS1), (WS2), WS1 (WS3) WS1 (WS4) WS2 Waardenburg,, [2],,, WS [3] WS WS1 WS3 PAX3, MITF WS2, WS4 EDNRB EDN3 SOX10 WS2 MITF - - MITF,, [4] WS, WS,, WS, WS Waardenburg 3, WS2, MITF 7 c.639dela,, 212, 1 1.1,, Cyrillic2.1, DNA 50 DNA, 30, 20, 12~60, 1.2, (auditory brainstem response, ABR) 40 Hz (40 Hz auditory evoked relative potential, AERP) CT MRI 1.3 DNA 3~5 ml, DNA ( ) DNA, DNA, DNA 20 ng/μl 1.4 NCBI MITF (NM_00248), bp, ORF bp, 9, PRIMER bp PCR, (PCR) MITF ABI 2700, PCR 30 μl

WS Waardenburg 3, WS2, MITF 7 c.639dela,, 212, 1 1.1,, Cyrillic2.1, DNA 50 DNA, 30, 20, 12~60, 1.

3 4 : Waardenburg MITF 435 表 1 MITF 基因引物序列及扩增条件 Table 1 Primers of MITF gene and PCR conditions No. (5 3 ) Forward primer (5 3 ) Reverse primer ( ) Annealing temperature PCR (bp) PCR products 1 tggtgtctcgggataccttg tggcatcaaataataaacagca tggcatcaaataataaacagca gtggccacaaggacaaacta catcttgttgctgtgccatc aaggtgtgatccaccacaaa gaccattattgctttgggtaaaa tgtgatcctgagataattctccatt tgaggagatcctgtacctctctt aaaagttacgtccatgagttgga gcttttgaaaacatgcaagc gctgtaggaatcaactctcctct cgttgtcatgacctggagaa caaagggagaggggagactt cttatccatgtaaccaagca cacacacacagaatccacaaa ctaatgacgcgcatctacca tcctgggctattgataaagca ( DNA 20 ng, 10 PCR 3 μl, 3 mmol dntp, 5 U Taq DNA, 10 pmol 30 μl) PCR : 94 4 min, s, ( ) 30 s, s, 30, 72 7 min, 4 PCR -, ABI 3100 GeneTool MITF MITF 7 c.639dela ( delt ), Ⅳ-8, 4,,, : ; : ; :, ABR 100 db nhl 40 Hz AERP 120 db nhl CT MRI Ⅲ-11 2 Ⅲ-11 Ⅲ-6 Ⅲ-8,, ; 20,, 30 WS, W [5] 1.95,, WS2-2, -1 Ⅳ-3-6,, 图 1 Waardenburg 综合征 Ⅱ 型患者 MITF 基因第 7 外显子测序分析 1: Ⅳ-8 exon7 c.639dela ( ); 2: Ⅲ-11 exon7 c.639dela delt ( ); 3: Ⅲ-12 exon7 ( ) Fig. 1 Sequence analysis of MITF gene exon 7 in patients with Waardenburg syndrome type 2 1: Forward sequencing of MITF exon7 in Ⅳ-8 reveals heterozygous mutation c.639dela(mutant type); 2: Reverse sequencing of MITF exon7 in Ⅲ-11 reveals heterozygous mutation delt corresponding to the c.639dela (mutant type); 3: Forward sequencing of MITF exon7 in Ⅲ-12 displays normal sequence (wild type).

catcttgttgctgtgccatc aaggtgtgatccaccacaaa 57 253 4 gaccattattgctttgggtaaaa tgtgatcctgagataattctccatt 56 343 5 tgaggagatcctgtacctctctt aaaagttacgtccatgagttgga 56 425 6 gcttttgaaaacatgcaagc

4 436 HEREDITAS (Beijing) 图 2 Waardenburg 综合征 Ⅱ 型中国家系系谱图 Fig. 2 Pedigree of a Chinese family with Waardenburg syndrome type Waardenburg 3 MITF c.639dela, exon7, MITF 212 ERRRRFNI DEEEDLT * ( * ) MITF, MITF DNA (basic region 198~217 ), - - (helix-loop-helix leucine zipper region 218~288 ) 213~220, 3, ( ), - - MITF DNA, -1,,, Lalwani [2] MITF c.760c>t, CpG, c.639dela c.760c>t 1 ( Anil K.Lalwani, c.640c>t), 3 WS2 7 c.760c>t, 1 3 1,,, Liu [6] WS2,, -8-1, 1 c.760c>t, MITF 7,, 图 3 MITF 蛋白结构域模式图, 220 (p. I220X) Fig. 3 Schematic of MITF domain structure Dash line frame displays the amino acid variant covers two domains, and a nonsense codon emerges in advance at position 220th.

-1,,, Lalwani [2] MITF c.760c>t, CpG, c.639dela c.760c>t 1 ( Anil K.Lalwani, c.640c>t), 3 WS2 7 c.760c>t, 1 3 1,,, Liu [6] WS2,, -8-1, 1 c.

5 4 : Waardenburg MITF 437, WS,,,, MITF MITF,, 6 WS : MITF PAX3 SOX10 ENDRB SLUG EDN3 [3] WS : PAX MITF, PAX3 WS1 - [7] SOX10 PAX3 MITF [8] SOX10 Sp1 EDNRB, MITF MITF MITF WS, (TYR) WS, WS, [1] WS MITF 4 : 1A 1H 1B 1M( 4) [9] 2~9 1M 1A 1H 1B MITF, 1B 3 1b 1A 1H 2, MITF-M,, PAX3, SOX10, CREB c-kit signaling MITF-M [10] MITF-M MITF, MITF-M MITF, MITF WS2, MITF-M WS2 MITF exon1a 1H 1B MITF WS2 图 4 [9] MITF 基因 4 种不同的转录剪接模式 Fig. 4 Four kinds of splicing patterns of MITF gene transcripts [9] WS, MITF PAX3 WS 20% WS2 MITF [11], WS2 MITF 40 ( 15 ) [12] MITF WS2,,,,, 参考文献 (References): [1] Hodgkinson CA, Nakayama A, Li H, Swenson LB, Opdecamp K, Asher JH Jr, Arnheiter H, Glaser T.Mutation at the anophthalmic white locus in Syrian hamsters: haploinsufficiency in the Mitf gene mimics human Waardenburg syndrome type 2. Hum Mol Genet, 1998, 7(4): [DOI] [2] Lalwani AK, Attaie A, Randolph FT, Deshmukh D, Wang C, Mhatre A, Wilcox E. Point mutation in the MITF gene causing Waardenburg syndrome type II in a three-generation Indian family. Am J Med Genet, 1998, 80(4): [DOI] [3] Yokoyama S, Takeda K, Shibahara S. Functional difference of the SOX10 mutant proteins responsible for the phenotypic variability in auditory-pigmentary disorders. J Biochem (Tokyo), 2006, 140(4): [DOI] [4] Watanabe A, Takeda K, Ploplis B, Tachibana M. Epistatic relationship between Waardenburg syndrome genes MITF and PAX3. Nat Genet, 1998, 18(3): [DOI] [5] Nobukuni Y, Watanabe A, Takeda K, Skarka H, Tachibana

6 438 HEREDITAS (Beijing) M.Analyses of loss-of-function mutations of the MITF gene suggest that haploinsufficiency is a cause of Waardenburg syndrome type 2A. Am J Hum Genet, 1996, 59(1): [6] Liu XZ, Newton VE, Read AP. Waardenburg syndrome type II: phenotypic findings and diagnostic criteria. Am J Med Genet, 1995, 55(1): [DOI] [7] Tachibana M. A cascade of genes related to Waardenburg syndrome. J Investig Dermatol Symp Proc, 1999, 4(2): [DOI] [8] Bondurand N, Pingault V, Goerich DE, Lemort N, Sock E, Le Caignec C, Wegner M, Goossens M. Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome. Hum Mol Genet, 2000, 9(13): [DOI] [9] Udono T, Yasumoto K, Takeda K, Amae S, Watanabe K, Saito H, Fuse N, Tachibana M, Takahashi K, Tamai M, Shibahara S. Structural organization of the human microphthalmia-associated transcription factor gene containing four alternative promoters. Biochim Biophys Acta, 2000, 1491(1 3): [10] Shibahara S, Takeda K, Yasumoto K, Udono T, Watanabe K, Saito H, Takahashi K. Microphthalmia-associated transcription factor (MITF): multiplicity in structure, function, and regulation. J Investig Dermatol Symp Proc, 2001, 6(1): [DOI] [11] Tassabehji M, Newton VE, Liu XZ, Brady A, Donnai D, Krajewska-Walasek M, Murday V, Norman A, Obersztyn E, Reardon W, Rice JC, Trembath R, Wieacker P, Whiteford M, Winter R, Read AP. The mutational spectrum in Waardenburg syndrome. Hum Mol Genet, 1995, 4(11): [DOI] [12] YANG Shu-Zhi. The clinical features and mutation analysis of the responsible gene for the syndrome hearing impairment. CHKD Thesis Database of Chinese Master and Doctor Qualification, CHKD, ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇ ( ), ,,, ,,,,,,,,,,,, ,,,, 50 ( ) : : : 50 : : : : kwyuan@genetics.ac.cn :

[DOI] [7] Tachibana M. A cascade of genes related to Waardenburg syndrome. J Investig Dermatol Symp Proc, 1999, 4(2): 126 129.

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Brochure of Waardenburg syndrome 2 8 13 16 18 22 24 30 34 35 35 38 45 49 51 54 Q&A Q&A 60 Q&A 62 79 3 4 17 5 6 第一章給罕病家庭的話 8 9 10 11 12 13 14 15 --- 16 / / 17 18 1 2 0.25 19 20 第二章疾病面面觀 Waardenburg Syndrome