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Jul 15, 2014 - in Chinese Von Hippel–Lindau Disease Families. Xiang-hui ...... initiation is associated with shorter telomere length in Li-Fraumeni syndrome.
Cancer Research

Prevention and Epidemiology

Telomere Shortening Is Associated with Genetic Anticipation in Chinese Von Hippel–Lindau Disease Families Xiang-hui Ning1,3,4, Ning Zhang5, Teng Li1,3,4, Peng-jie Wu1,3,4, Xi Wang1,3,4, Xue-ying Li2, Shuang-he Peng1,3,4, Jiang-yi Wang1,3,4, Jin-chao Chen1,3,4, and Kan Gong1,3,4

Abstract Von Hippel–Lindau (VHL) disease is a rare autosomal dominant cancer syndrome. A phenomenon known as genetic anticipation has been documented in some hereditary cancer syndromes, where it was proved to relate to telomere shortening. Because studies of this phenomenon in VHL disease have been relatively scarce, we investigated anticipation in 18 Chinese VHL disease families. We recruited 34 parent–child patient pairs (57 patients) from 18 families with VHL disease. Onset age was defined as the age when any symptom or sign of VHL disease first appeared. Anticipation of onset age was analyzed by paired t test and the other two special tests (HV and RY2). Relative telomere length of peripheral leukocytes was measured in 29 patients and 325 healthy controls. Onset age was younger in child than in parent in 31 of the 34 parent–child pairs. Patients in the first generation had older onset age with longer age-adjusted relative telomere length, and those in the next generation had younger onset age with shorter age-adjusted relative telomere length (P < 0.001) in the 10 parent–child pairs from eight families with VHL disease. In addition, relative telomere length was shorter in the 29 patients with VHL disease than in the normal controls (P ¼ 0.003). The anticipation may relate to the shortening of telomere length in patients with VHL in successive generations. These findings indicate that anticipation is present in families with VHL disease and may be helpful for genetic counseling for families with VHL disease families and for further understanding the pathogenesis of VHL disease. Cancer Res; 74(14); 3802–9. 2014 AACR.

Introduction Von Hippel–Lindau (VHL) disease (MIM 193300) is an autosomal dominant hereditary cancer syndrome caused by germline mutations in VHL gene (1, 2). The incidence of this disease is roughly one of 36,000 living births, and its penetrance is estimated to be more than 90% by 65 years of age (3). Clinically it is characterized by a wide spectrum of tumors, including central nervous system (CNS) hemangioblastoma, clear cell renal cell carcinoma (RCC), retinal angioma, pancreatic cyst and tumor, pheochromocytoma, endolymphatic sac tumor, and papillary cystadenoma in epididymis or broad ligment (4–6). The risk of a patients with VHL disease developing CNS hemangioblastoma, retinal angioma, and/or clear cell RCC is up to 70% to 80% (3). The variable phenotype of VHL disease may relate Authors' Affiliations: Departments of 1Urology and 2Medical Statistics, Peking University First Hospital; 3Institute of Urology, Peking University; 4 National Urological Cancer Center; 5Department of Urology, Beijing Chaoyang Hospital, Capital University of Medicine Science, Beijing, P.R. China Corresponding Author: Kan Gong, Institute of Urology, Peking University, National Urological Cancer Center, Department of Urology, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, P.R. China. Phone: 86-10-66551032; Fax: 86-10-66551032; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-14-0024 2014 American Association for Cancer Research.

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to the various mutation types and other gene modifier effects (7–9). VHL gene is located in chromosome 3p25-26 (2). It displays tumor suppressor effect through the gene product VHL protein to degrade the hypoxia-inducible factor and inhibit the expression of hypoxia response genes such as VEGF, erythropoietin, platelet-derived growth factor, and carbonic anhydrase (10). Recently, the VHL gene product has been proved to participate in DNA damage repair response (11, 12). Anticipation is a phenomenon that the successive generations progressively manifest earlier onset age and more serious presentations for an inherited disease. To date, anticipation has been found in two types of hereditary diseases, neurologic diseases such as fragile X syndrome, X-linked spinal and bulbar muscular atrophy, myotonic dystrophy and Huntington disease, and hereditary cancer syndromes such as dyskeratosis congenita, hereditary breast cancer, Li–Fraumeni syndrome, and hereditary nonpolyposis colorectal cancer syndrome (Lynch syndrome). Two molecular changes may be involved in the anticipation, expanding of trinucleotide repeats found in the anticipation in neurologic diseases, and shortening of telomere detected in the anticipation of hereditary cancer syndromes (13–21). The gradual decrease of telomere length with aging is attributed to the decrease of telomerase function or mutations accumulated in the DNA repair system besides aging (22).

Genetic Anticipation in VHL Disease

Because the study about the anticipation in VHL disease was scarce, we used the clinical data and DNA samples of the families with VHL disease recruited in our research group to evaluate the anticipation and its relevance to telomere length.

Patients and Methods Patients and samples This project was approved by the Medical Ethics Committee of Peking University First Hospital (Beijing, China) and informed consent was obtained from the patients. During the period from 2009 to 2012, 39 families with VHL disease were diagnosed with hereditary VHL disease at the Department of Urology, Peking University First Hospital based on the clinical criteria and mutation detection in VHL as previous described (23). In these families, 19 families had two or more than two patients in two or more than two generations, including one family with obscure onset age. Therefore, a total of 57 patients with VHL disease from 18 families were enrolled in this study. In the 18 families, 23 patients were diagnosed in the first generation and 34 patients in the second or third generation to form 34 parent–child patient pairs (one pair in 8 families, two pairs in 6 families, three pairs in 2 families, and four pairs in 2 families; Table 1). In the 18 families, DNA sample was available for assay in 29 patients, so that the relationship between anticipation and relative telomere length could be evaluated in 10 of the 34 parent–child pairs. Relative telomere length was also measured in 325 healthy individuals (15–90 years of age, mean age 48.7 years) from those for health checkup as controls. Relative telomere length assessment Genomic DNA was isolated from peripheral blood by using a blood DNA extraction kit (Tiangene). We followed the method described by Cawthon to quantify relative telomere length by measuring copy number ratio of telomere repeats (T) to the single copy gene 36B4 (S) using qRT-PCR (24). The 10-mL PCR mixture contained 2X SYBR master mix (Takara) 5 mL, genomic DNA 30 ng, 300 nmol/L telomere primer Tel1 (50 -GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT) and 900 nmol/L Tel2 (50 -TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA), or 200 nmol/L single copy gene primer 36B4u (50 -CAGCAAGTGGGAAGGTGTAATCC), and 500 nmol/L 36B4d (50 -CCCATTCTATCATCAACGGGTACAA; ref. 19). qRT-PCR was run in an ABI 7500 PCR instrument using the profile of 95 C for 30 seconds and 40 cycles of 95 C for 15 seconds, 54 C for 2 minutes, and 72 C for 15 seconds. A standard curve from a control DNA sample (male, 45 years old) by serial 1/4 dilutions from 50 ng to 0.19 ng was constructed to evaluate the amplification efficiency (E), and this sample was measured in every batch of PCRs as the inter-run calibration. Threshold cycle (Ct) values were automatically determined by the 7500 software v2.0.5. The measurements of telomere length and single copy gene 36B4 were triplicate in one batch for each sample, and the mean of the three Ct values (Cm) was used for the calculation. PCR efficiency and the calibration of copy

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numbers were also enrolled in the telomere length calculation (19, 25). The formula to calculate the copy number ratio of telomere repeats (T) to single copy gene 36B4 (S) is as follows, and T/S represents the value of relative telomere length. T=S ¼

ðETel;sample ÞCmðTEL;sampleÞ ðE36B4;sample Þ

Cmð36B4;sampleÞ



ðETel;calibrator ÞCmðTEL;calibratorÞ ðE36B4;calibrator ÞCmð36B4;calibratorÞ

The relationship between age and relative telomere length in 325 normal controls can be expressed by the linear regression equation of Y ¼ 1.4503-0.0117 X (see Fig. 2). Normal relative telomere length at the DNA sample-obtained age can then be predicted by this equation. The difference between predicted normal relative telomere length at the DNA-obtained age and the relative telomere length actually measured was the ageadjusted relative telomere length (19), which was used for the comparison among patients with VHL in generations. Telomere length measurement by Southern blot analysis The oligonucleotide (TTAGGG)4 was tailed with DIG-dUTP as the probe by using terminal transferase. Two micrograms of genomic DNA were digested with HinfI/RsaI and separated in 0.8% agarose gel. The DNA fragments in the gel were then transferred onto a Hybond-N membrane by Southern blotting. After UV cross-link and prehybridization, the membrane was hybridized in a solution containing 2 pmol/mL probe, 50% formamide, 2 SSC, 0.1% lauroyl sarcosine, 0.02% SDS, and 1% milk powder at 42 C for 6 hours, washed at room temperature in 2 SSC, 0.1% SDS for two times, and in 0.1 SSC, 0.1% SDS, 15 minutes for two times. Hybridized probe on the membrane was recognized by alkaline phosphatase conjugated anti-DIG antibody and chemiluminescent method. The luminescent image was developed on a phosphoimager. The telomere length was calculated by a telomeric software (version 1.2; ref. 26). Statistical analysis Paired t test was used to examine the difference of onset age between generations. HV (parametric conditional maximum likelihood approach of Huang and Vieland) and RY2 (special nonparametric method of Rabinowitz and Yang) tests were used to lower the truncation bias when paired t test is conducted (27). t test was used to evaluate the difference of relative telomere length between patients with VHL disease and healthy controls, and paired t test to analyze the differences of age-adjusted relative telomere length in parent–child pairs. Statistical analyses were performed using R software. P < 0.05 was considered to be statistically significant.

Results Onset age was earlier in patients in the next generation than in those in the first generation in the 18 families with VHL disease In the 34 child–parent pairs in the 18 families with VHL disease (Table 1), onset age was younger in child than in parent in 31 pairs and was older in child than in parent in three pairs. We compared the onset age between children and parents

Cancer Res; 74(14) July 15, 2014

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Exon

Father 2,3 (Proband 1, M) 2,3 (Proband 2, M) 1 Daughter 1 (Proband 3, M) 1 Son 1 Mother 1 Brothera (Proband 4, M) 1 Father 3 (Proband 5, F) 3 Sister 1 3 Sister 2 3 Mothera (Proband 6, F) Daughter (Proband 7, M) 2 Son 2 Brother 1a Nephew 1 2 Sister 1a Niece 2 Brother 2a Nephew 2 2 Grandfathera Mother 1 (Proband 8, M) 1 Aunt 1a Aunt 2

Patients Deletion Deletion Deletion Deletion c.280G>T p.Glu94Stop c.280G>T p.Glu94Stop c.269A>T p.Asn90Ile No DNA sample available c.269A>T p.Asn90Ile c.499C>T p.Arg167Trp c.499C>T p.Arg167Trp c.499C>T p.Arg167Trp c.499C>T p.Arg167Trp No DNA sample available Not assayed Not assayed c.349T>G p.Trp117Gly c.349T>G p.Trp117Gly No DNA sample available c.349T>G p.Trp117Gly No DNA sample available c.349T>G p.Trp117Gly No DNA sample available c.349T>G p.Trp117Gly No DNA sample available Deletion Deletion No DNA sample available No DNA sample available

Nucleotide and protein change 50 37 28 16 54 — — 19 22 — 37 38 — 45 — — 36 13 47 — — — — 12 40 43 23 41 19

— 31 41 — — — — — 32 — — — 34 — — 8 44 — — — — 13 43 11 — 35 17 — —

— — 41 — 53 29 63 — — 54 37 — — — 42 — 44 — 51 — — — 44 — 60 — — — —

— — 42 — 53 29 — — — — 37 36 — — 42 — 40 — — — 46 — — — — — 19 — —

— — 42 — — — — — — — 39 — — — — — — — — — — — — — — — — — —

— — — — — — — — — — — — — — — — — — — — — — — — — — — — —

— 16 — — — 23 — — — — 37 — — — — — — — — 37 — — — — — — 19 — —

CNS RA RCC PCT Pheo ELST EC

Symptoms diagnosed age

50 16 28 16 53 23 63 19 22 54 37 36 34 45 42 8 36 13 47 37 46 13 43 11 40 35 17 41 19 18 (2)

17 (1)

16 (2)

15 (1)

14 (3)

13 (2)

12 (1)

11 (1)

10 (1)

9 (2)

Mothera (Proband 9, M) Aunta Cousina (Proband 10, M) Son Mother (Proband 11, M) Mothera (Proband 12, M) Mothera Sister (Proband 13, M) Mothera Sistera (Proband 14, M) Brothera Fathera (Proband 15, M) (Proband 16, M) Daughter Sistera Nephewa Mothera (Proband 17, M) Mothera (Proband 18, F) Brother

Onset Family (pair) Patients ageb

1 1

1

3 3

1

3 3

1

3 3 1 1

1

Exon No DNA sample available c.269A>T p.Asn90Ile No DNA sample available No DNA sample available c.533T>G p.Leu178Arg c.533T>G p.Leu178Arg c.263G>A p.Trp88Stop c.263G>A p.Trp88Stop No DNA sample available c.280G>T p.Glu94Stop No DNA sample available Deletion Deletion No DNA sample available No DNA sample available c.292T>A p.Tyr99Asn No DNA sample available No DNA sample available c.481C>T p.Arg161Stop c.500G>A p.Arg167Gln No DNA sample available No DNA sample available No DNA sample available No DNA sample available Deletion No DNA sample available c.288insA Frameshift c.288insA Frameshift

Nucleotide and protein change

Germline mutation

33 28 32 30 39 — 36 — 30 31 43 30 23 50 40 40 27 42 29 29 24 50 15 — — — 29 —

— — — — 57 — — — — — — — 24 — — — 20 — — — 14 — — — 21 29 20 34

— 38 — — 57 30 — 34 — — — 30 23 — 49 41 — — 29 46 — — 26 43 — — 29 43

— 38 — — 57 — — 38 — — — 30 23 62 — 41 — — 36 — — — — 43 — — — 43

b

a

— — — — — — — — — — — — — — — 40 — — — 46 — — — — — — — —

— — — — — — — — — — — — — — — — — — 33 — — — — — — — — —

— — — — — — — — — — — — — — — — — — 29 — — — — — 21 — 40 —

CNS RA RCC PCT Pheo ELST EC

Symptoms diagnosed age

Abbreviations: CNS, hemangioblastomas of CNS; RA, retinal angiomas; PCT, multiple pancreatic cysts or tumors; Pheo, pheochromocytoma; ELST, endolymphatic sac tumor; EC, epididymal/ovarin cystadenoma. Death before diagnosis. The onset age referred to the age when any symptoms or signs of VHL disease began.

8 (4)

7 (4)

6 (2)

5 (3)

4 (2)

3 (1)

2 (1)

1 (1)

Family (pair)

Germline mutation

Table 1. Genotype and phenotype in 18 families with VHL disease

33 28 32 30 39 30 36 34 30 31 43 30 23 50 40 40 20 42 29 29 14 50 15 43 21 29 20 34

Onset ageb

Ning et al.

Cancer Research

Genetic Anticipation in VHL Disease

Table 2. Difference in onset age between parents and children with VHL disease Paired t test

Parents Children Total a

n

Onset age (y) mean (range)

23 34 57

42.9 (28–63) 26.1 (8–42) 32.3 (8–63)

HV test

RY2 test

MOAD (y)a

P

MOAD (y)

P

MOAD (y)

P

16.8