Molecular basis of von Hippel-Lindau syndrome in Chinese patients

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1 Chinese Medical Journal 2011;124(2): Original article Molecular basis of von Hippel-Lindau syndrome in Chinese patients SIU Wai-kwan, MA Ronald Ching-wan, LAM Ching-wan, MAK Chloe Miu, YUEN Yuet-ping, LO Fai-man Ivan, CHAN Kin-wah, LAM Siu-fung, LING Siu-cheung, TONG Sui-fan, SO Wing-yee, CHOW Chun-chung, TANG Mary Hoi-yin, TAM Wing-hung and CHAN Albert Yan-wo Keywords: von Hippel-Lindau syndrome; VHL gene; Chinese; VHL mutations V Background Von Hippel-Lindau (VHL) syndrome is an autosomal dominant familial cancer syndrome predisposing the affected individuals to multiple tumours in various organs. The genetic basis of VHL in Southern Chinese is largely unknown. In this study, we characterized the mutation spectrum of VHL in nine unrelated Southern Chinese families. Methods Nine probands with clinical features of VHL, two symptomatic and eight asymptomatic family members were included in this study. Prenatal diagnosis was performed twice for one proband. Two probands had only isolated bilateral phaeochromocytoma. The VHL gene was screened for mutations by polymerase chain reaction, direct sequencing and multiplex ligation-dependent probe amplification (MLPA). Results The nine probands and the two symptomatic family members carried heterozygous germline mutations. Eight different VHL mutations were identified in the nine probands. One splicing mutation, NM_ : c.463+1g>t, was novel. The other seven VHL mutations, c.233a>g [p.asn78ser], c.239g>t [p.ser80ile], c.319c>g [p.arg107gly], c.481c>t [p.arg161x], c.482g>a [p.arg161gln], c.499c>t [p.arg167trp] and an exon 2 deletion, had been previously reported. Three asymptomatic family members were positive for the mutation and the other five tested negative. In prenatal diagnosis, the fetuses were positive for the mutation. Conclusions Genetic analysis could accurately confirm VHL syndrome in patients with isolated tumours such as sporadic phaeochromocytoma or epididymal papillary cystadenoma. Mutation detection in asymptomatic family members allows regular tumour surveillance and early intervention to improve their prognosis. DNA-based diagnosis can have an important impact on clinical management for VHL families. on Hippel-Lindau syndrome (VHL) is an autosomal dominant familial cancer syndrome. Typical VHL tumours include haemangioblastoma of the central nervous system (CNS) and retina, renal cell carcinomas (RCC), and phaeochromocytomas. It is also associated with renal cysts, pancreatic cysts and tumours, endolymphatic sac tumours as well as epididymal and broad ligament cystadenoma. 1 Clinically, VHL is diagnosed in patients with a positive family history, and a CNS haemangioblastoma, phaeochromocytoma or RCC. For those patients without family history, two haemangioblastomas or one haemangioblastoma plus a visceral tumour are required for diagnosis. Patients without phaeochromocytoma are classified as VHL type 1 and those with phaeochromocytoma as VHL type 2. Type 2 patients are further divided into three groups: type 2A patients with low risk of RCC, type 2B patients with high risk of RCC and type 2C patients with isolated phaeochromocytoma. 2 The responsible gene VHL was first identified in It is a tumour suppressor gene located on the short arm of chromosome 3 (3p25-26). It consists of three exons and encodes a protein with 213 amino acids. 3 The identification of pathogenic germline VHL mutations provides a reliable means for confirmation of diagnosis particularly in patients who do not meet the clinical diagnostic criteria. The mutation detection rate by sequencing and Southern blotting in peripheral blood is up to 100%. 4 DOI: /cma.j.issn Department of Pathology, Princess Margaret Hospital, Hong Kong, China (Siu WK, Mak CM, Yuen YP and Chan YW) Department of Medicine & Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, China (Ma RC, So WY and Chow CC) Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China (Lam CW and Tong SF) Clinical Genetic Service, Department of Health, Hong Kong, China (Lo FM) Department of Medicine and Geriatrics, Princess Margaret Hospital, Hong Kong, China (Chan KW and Lam SF) Department of Paediatrics and Adolescent Medicine, Princess Margaret Hospital, Hong Kong, China (Ling SC) Department of Obstetrics & Gynaecology, Tsan Yuk Hospital, Hong Kong, China (Tang MHY) Department of Obstetrics & Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, China (Tam WH) Correspondence to: Prof. LAM Ching-wan, Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China (Tel: Fax: ching-wanlam@ pathology.hku.hk)

2 238 Table. Summary of clinical data and mutation analysis results Patient Family history Mutation Type Age at diagnosis (Years) CNS RCC Phaeo EC Proband 1 Yes c.481c>t [p.arg161x] * Sister c.481c>t [p.arg161x] 1 45 * Daughter 1 Not detected Asymptomatic Daughter 2 Not detected Asymptomatic Proband 2 No c.482g>a [p.arg161gln] 2C 18 * Proband 3 No c.499c>t [p.arg167trp] 2B 24 * Proband 4 No c.463+1g>t 1 26 * 43 Son Not detected Asymptomatic Daughter Not detected Asymptomatic Proband 5 No c.233a>g [p.asn78ser] 1 28 * 28 Proband 6 Yes Exon 2 deletion 1 15 * Sister 1 Exon 2 deletion 1 21 * Sister 2 Not detected Asymptomatic Daughter Exon 2 deletion Asymptomatic Proband 7 Yes c.319c>g [p.arg107gly] 1 55 * Father c.319c>g [p.arg107gly] Asymptomatic 80 Brother c.319c>g [p.arg107gly] Asymptomatic Proband 8 No c.482g>a [p.arg161gln] 2C 10 * Proband 9 Yes c.239g>t [p.ser80ile] 2B Fetus 1 c.239g>t [p.ser80ile] Not applicable Fetus 2 c.239g>t [p.ser80ile] Not applicable * The first presenting tumour of the patient. Proband 9 had family history of RCC and Phaeo. - Absence of tumour. CNS: CNS haemangioblastoma; RCC: Renal cell carcinoma; Phaeo: Phaeochromocytoma; EC: epididymal cystadenoma. The estimated incidence of VHL syndrome is 1 in live births in the Caucasian population. 5 The incidence in the Chinese population is unknown. The molecular basis of VHL in mainland Chinese is under-reported. There are only six papers on germline VHL mutations of Chinese patients in English literature. The first paper by Huang et al in 2004 reported a germline mutation in a Chinese VHL family. 6 Tong et al described VHL mutations in five patients with non-syndromic familial phaeochromocytoma in Zhang et al in 2008, determined the germline mutations in another 27 VHL patients. 8 In the next year, Rao et al reported one VHL germline mutation in RCC patients. 9 Mao et al described the VHL genetic findings in three Chinese VHL families 10 and Tong et al described a germline mutation in a VHL family presenting with bilateral phaeochromocytoma. 11 There are another five articles in Chinese reporting germline mutations in Chinese VHL families In Southern Chinese, the data remains scarce. There is only one report on the genetic findings of two Chinese families with cerebellar haemangioblastoma by Tse et al in 1997 from Hong Kong. 17 Here, we conducted a comprehensive mutational analysis of the VHL gene in nine unrelated Hong Kong Chinese families with clinical characterization of VHL syndrome in Hong Kong. METHODS Patients Nine index subjects were referred from clinical departments for the analysis of VHL genes. Seven patients fulfilled the clinical diagnostic criteria of VHL syndrome. Two of them, proband 1 and proband 8, presented with isolated bilateral phaeochromocytoma. Family history was negative in five probands. Two symptomatic and eight asymptomatic family members were investigated for the presence of the mutation. One proband had prenatal diagnosis performed twice. Clinical data of these patients are summarized in the Table. Approval from the local ethic committee was obtained and informed consent was obtained for every individual. Mutation analysis EDTA blood samples were collected for mutation analysis of the VHL gene. Genomic DNA of the patients was extracted from whole blood samples using the QIAamp blood kit (Qiagen, Hilden, Germany) according the manufacturer s instructions. For prenatal diagnosis, DNA was extracted from the chorionic villus sample. Polymerase chain reaction (PCR) All the exons and flanking regions of the VHL gene were amplified by PCR using the following conditions: 1 cycle of 95 o C for 15 minutes; 40 cycles of denaturation at 94ºC for 30 seconds, annealing at 58ºC for 45 seconds, and an extension at 72ºC for 45 seconds. The 25 μl reaction mixture contained 40 ng DNA template, 1 PCR buffer, 1 Q-solution (Qiagen), 0.2 μmol/l of each dntp, 12.5 pmol of each primer, and units of HotStarTaq DNA Polymerase. Primer sequences are available upon request. Direct sequencing PCR products were purified by Exo-sapit (GE Healthcare, Uppsala, Sweden). The purified PCR products were sequenced using BigDyeDeoxy terminator cycle sequencing reagents according to manufacturer s instructions (Applied Biosystems, Foster City, CA, USA). Products of sequencing reactions were purified by Auto-Seq G-50 columns (GE Healthcare). Purified sequencing fragments were separated by capillary electrophoresis and detected by laser-induced fluorescence on an ABI Prism 3100 Genetic Analyzer

3 Chinese Medical Journal 2011;124(2): (Applied Biosystems). Sequencing results were compared with NCBI Reference Sequence: NG_ In this study, we annotated the nucleotide position of the mutations by defining the first base of the initiation codon of the VHL gene as nucleotide position 1 (NCBI Reference Sequence: NM_ ). Multiplex ligation-dependent probe amplification (MLPA) A SALSA MLPA P016B VHL kits (MRC-Holland, Amsterdam, the Netherlands) was used to detect deletions and duplications. MLPA of the VHL gene was performed for the index patients with a negative sequencing result according to the manufacturer s instructions. 18 We analyzed negative controls with each sample. The commercial VHL-MLPA assay included eight pairs of probes designed to amplify the three VHL exons, five probe pairs for three neighbouring genes (FANCD2, IRAK2, and GHRL), and 16 probe pairs for other chromosomal locations. The amplification products were separated by capillary electrophoresis on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). RESULTS Each of the nine probands carried one heterozygous germline mutation with eight different mutations identified (Table). Two unrelated probands had the same mutation. Seven mutations were detected by direct sequencing. Theses mutations consisted of five missense (c.233a>g [p.asn78ser], c.239g>t [p.ser80ile], c.319c>g [p.arg107gly], c.482g>a [p.arg161gln], c.499c>t [p.arg167trp]), one nonsense (c.481c>t [p.arg161x]) and one novel splicing mutation (c.463+1g>t) (Figure 1). Of the seven mutations, two were in exon 1, one in exon 2, three in exon 3 and one in intron 2. In addition, MLPA showed an exon 2 deletion in proband 6 (Figure 2). Figure 1. DNA sequencing electrophoretogram shows the novel mutation in VHL NM_ : c.463+1g>t (arrow) in the sense direction. The two symptomatic family members also carried the germline mutations. Three of the eight asymptomatic family members had mutations and five tested negative. Figure 2. Top: MLPA electrophoretogram of exon 2 deletion. The peaks corresponding to the deleted exon 2 are marked by arrows. Lower panel: Normal control. The father of proband 7 was asymptomatic upon genetic testing, but screening of VHL tumours after genetic testing revealed a left RCC with lung metastasis. The other two asymptomatic mutation carriers were screened and found to be negative for VHL tumours. In prenatal diagnosis, the fetuses were positive for the mutation. In our series, five families had VHL type 1 while four families had VHL type 2. All four VHL type 2 families had missense mutations. The deletion, nonsense mutation and splicing mutation were associated with VHL type 1. DISCUSSION In this study, we determined the molecular basis of VHL syndrome in nine unrelated Southern Chinese families by analyzing the VHL gene. Altogether eight different mutations were detected with five missense mutations, one gross deletion, one nonsense mutation and one splicing mutation. The splicing mutation c.463+1g>t was novel. It was analyzed by in silico analysis and it was predicted to abolish the normal donor splice site and conceivably lead to mis-splicing. The other mutations c.233a>g [p.asn78ser], c.239g>t [p.ser80ile], c.319c>g [p.arg107gly], c.481c>t [p.arg161x], c.482g>a [p.arg161gln], c.499c>t [p.arg167trp] and the exon 2 deletion have all been previously reported in different ethic groups such as North Americans, Europeans and Japaneses. 4,19-21 The missense mutations c.233a>g [p.asn78ser] and c.499c>t [p.arg167trp] were determined to have a deleterious effect on the native protein of the VHL gene. 22 MLPA of the VHL gene is a fast and easy to perform method for screening gross deletions for a DNA-based diagnosis of VHL syndrome. Gross deletions account for 20% 30% of the VHL germline mutations. 4,20,23 These mutations are likely to be missed by direct sequencing. Therefore, another analytical method that is able to detect gross deletions should be used for patients with negative

4 240 sequencing findings. Genotype-phenotype correlations in VHL syndrome have been established in previous studies which are useful for genetic counselling and determination of prognosis. 19,20,24 In general, our results correlated with the previous studies. All of our VHL type 2 patients carried missense mutations. Prognostic counselling regarding phaeochromocytoma is possible based on molecular results even if there is no family history. 25 Patient 5 and patient 7 had clinical VHL type 1. However, the missense mutation c.233a>g [p.asn78ser] and c.319c>g [p.arg107gly] were reported to be associate with phaeochromocytoma. 8,21 The genetic analysis findings were important to determine the risk of phaeochromocytoma in these two patients. Deletions, frameshift mutations, nonsense mutations and splice mutations are associated with VHL type 1. 4,20 In our study, all families with deletions, nonsense mutations and splice site mutations were classified as having clinical VHL type 1. The genetic diagnosis is the only method to accurately confirm VHL syndrome when patients have not met the clinical diagnostic criteria at initial presentation. It had been reported that mutations in either the RET, VHL, SDHD or SDHB genes were detected in 24% of patients with apparent sporadic phaeochromocytoma. 21 Patient 2 and patient 8 presented with isolated bilateral phaeochromocytoma at the ages of 18 and 10 years old, respectively. However, mutation analyses of the SDHD and SDHB genes were performed for these two patients according to a method previously described 26 and no mutations were identified. Further analysis found the missense mutation, c.482g>a [p.arg161gln], in the VHL gene. The clinical features of the SDHD and SDHB mutations and VHL syndrome overlap but their prognoses are significantly different. The mutation c.482g>a [p.arg161gln] is known to be associated with retinal and CNS haemangioblastoma and RCC. 20,27 Early diagnosis of VHL for these two young patients would be beneficial as regular surveillance of tumours and early intervention may produce a better prognosis. Identification of the underlying mutation not only confirms the diagnosis, it also provides useful information for counselling and subsequent management decision-making. Genetic analysis is also critical to determine the presence of mutations in asymptomatic family members. In our study, mutations were detected in three asymptomatic family members. Genotype-driven tumour screening detected left RCC with lung metastasis in the father of proband 7. The other two asymptomatic mutation carriers were negative for VHL tumour screening. Genetic analysis could accurately identify mutation carriers who could truly benefit from regular tumour surveillance to facilitate early intervention. On the other hand, asymptomatic family members who did not inherit the mutation could be spared from anxiety and from unnecessary monitoring. Bilateral epididymal papillary cystadenoma is characteristic of VHL syndrome. Papillary cystadenoma of the epididymis is a rare benign tumour, accounting for only 4% of all epididymal tumours. 28 Two-thirds of patients with epididymal papillary cystadenoma have VHL syndrome. 29 Among VHL patients with epididymal papillary cystadenoma, up to 40% of them have bilateral disease. 29 Thus, the presence of papillary cystadenoma in both epididymides is highly suggestive of VHL. Two patients in our cohort have bilateral epididymal cystadenoma and it was the first presentation for one of them. Leung et al 30 also reported a Chinese VHL patient who first presented with epididymal cystadenoma. Therefore, a high level of suspicion for VHL is required for patients with epididymal papillary cystadenoma and they should have genetic analysis of the VHL genes to achieve early diagnosis. In conclusion, our study demonstrates the mutation spectrum in Chinese VHL patients. Genetic analysis can accurately confirm VHL syndrome in patients with isolated tumours, such as isolated phaeochromocytoma or epididymal papillary cystadenoma. Detection of mutations in asymptomatic family members will identify patients who will benefit from regular tumour surveillance and early intervention to improve the prognosis. DNA-based diagnosis has important impacts on clinical management for VHL families. REFERENCES 1. Maher ER, Yates JR, Harries R, Benjamin C, Harris R, Moore AT, et al. Clinical features and natural history of von Hippel-Lindau disease. Q J Med 1990; 77: Lonser RR, Glenn GM, Walther M, Chew EY, Libutti SK, Linehan WM, et al. von Hippel-Lindau disease. 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