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 = 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.

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 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).

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 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 check-up as controls.

Table 1.

Genotype and phenotype in 18 families with VHL disease

Germline mutationSymptoms diagnosed ageGermline mutationSymptoms diagnosed age
Family (pair)PatientsExonNucleotide and protein changeCNSRARCCPCTPheoELSTECOnset agebFamily (pair)PatientsExonNucleotide and protein changeCNSRARCCPCTPheoELSTECOnset ageb
1 (1) Father 2,3 Deletion 50 — — — — — — 50 9 (2) Mothera  No DNA sample available 33 — — — — — — 33 
  (Proband 1, M) 2,3 Deletion 37 31 — — — — 16 16   (Proband 9, M) c.269A>T p.Asn90Ile 28 — 38 38 — — — 28 
2 (1) (Proband 2, M) Deletion 28 41 41 42 42 — — 28  Aunta  No DNA sample available 32 — — — — — — 32 
  Daughter Deletion 16 — — — — — — 16   Cousina  No DNA sample available 30 — — — — — — 30 
3 (1) (Proband 3, M) c.280G>T p.Glu94Stop 54 — 53 53 — — — 53 10 (1) (Proband 10, M) c.533T>G p.Leu178Arg 39 57 57 57 — — — 39 
  Son c.280G>T p.Glu94Stop — — 29 29 — — 23 23   Son c.533T>G p.Leu178Arg — — 30 — — — — 30 
4 (2) Mother c.269A>T p.Asn90Ile — — 63 — — — — 63 11 (1) Mother c.263G>A p.Trp88Stop 36 — — — — — — 36 
  Brothera  No DNA sample available 19 — — — — — — 19   (Proband 11, M) c.263G>A p.Trp88Stop — — 34 38 — — — 34 
  (Proband 4, M) c.269A>T p.Asn90Ile 22 32 — — — — — 22 12 (1) Mothera  No DNA sample available 30 — — — — — — 30 
5 (3) Father c.499C>T p.Arg167Trp — — 54 — — — — 54   (Proband 12, M) c.280G>T p.Glu94Stop 31 — — — — — — 31 
  (Proband 5, F) c.499C>T p.Arg167Trp 37 — 37 37 39 — 37 37 13 (2) Mothera  No DNA sample available 43 — — — — — — 43 
  Sister 1 c.499C>T p.Arg167Trp 38 — — 36 — — — 36   Sister Deletion 30 — 30 30 — — — 30 
  Sister 2 c.499C>T p.Arg167Trp — 34 — — — — — 34   (Proband 13, M) Deletion 23 24 23 23 — — — 23 
6 (2) Mothera  No DNA sample available 45 — — — — — — 45 14 (3) Mothera  No DNA sample available 50 — — 62 — — — 50 
  (Proband 6, F)  Not assayed — — 42 42 — — — 42   Sistera  No DNA sample available 40 — 49 — — — — 40 
   Daughter  Not assayed — — — — — —   (Proband 14, M) c.292T>A p.Tyr99Asn 40 — 41 41 40 — — 40 
7 (4) (Proband 7, M) c.349T>G p.Trp117Gly 36 44 44 40 — — — 36   Brothera  No DNA sample available 27 20 — — — — — 20 
  Son c.349T>G p.Trp117Gly 13 — — — — — — 13 15 (1) Fathera  No DNA sample available 42 — — — — — — 42 
 Brother 1a  No DNA sample available 47 — 51 — — — — 47   (Proband 15, M) c.481C>T p.Arg161Stop 29 — 29 36 — 33 29 29 
  Nephew 1 c.349T>G p.Trp117Gly — — — — — — 37 37 16 (2) (Proband 16, M) c.500G>A p.Arg167Gln 29 — 46 — 46 — — 29 
 Sister 1a  No DNA sample available — — — 46 — — — 46   Daughter  No DNA sample available 24 14 — — — — — 14 
  Niece c.349T>G p.Trp117Gly — 13 — — — — — 13  Sistera  No DNA sample available 50 — — — — — — 50 
 Brother 2a  No DNA sample available — 43 44 — — — — 43   Nephewa  No DNA sample available 15 — 26 — — — — 15 
  Nephew 2 c.349T>G p.Trp117Gly 12 11 — — — — — 11 17 (1) Mothera  No DNA sample available — — 43 43 — — — 43 
8 (4) Grandfathera  No DNA sample available 40 — 60 — — — — 40   (Proband 17, M) Deletion — 21 — — — — 21 21 
  Mother Deletion 43 35 — — — — — 35 18 (2) Mothera  No DNA sample available — 29 — — — — — 29 
   (Proband 8, M) Deletion 23 17 — 19 — — 19 17   (Proband 18, F) c.288insA Frameshift 29 20 29 — — — 40 20 
 Aunt 1a  No DNA sample available 41 — — — — — — 41   Brother c.288insA Frameshift — 34 43 43 — — — 34 
 Aunt 2  No DNA sample available 19 — — — — — — 19             
Germline mutationSymptoms diagnosed ageGermline mutationSymptoms diagnosed age
Family (pair)PatientsExonNucleotide and protein changeCNSRARCCPCTPheoELSTECOnset agebFamily (pair)PatientsExonNucleotide and protein changeCNSRARCCPCTPheoELSTECOnset ageb
1 (1) Father 2,3 Deletion 50 — — — — — — 50 9 (2) Mothera  No DNA sample available 33 — — — — — — 33 
  (Proband 1, M) 2,3 Deletion 37 31 — — — — 16 16   (Proband 9, M) c.269A>T p.Asn90Ile 28 — 38 38 — — — 28 
2 (1) (Proband 2, M) Deletion 28 41 41 42 42 — — 28  Aunta  No DNA sample available 32 — — — — — — 32 
  Daughter Deletion 16 — — — — — — 16   Cousina  No DNA sample available 30 — — — — — — 30 
3 (1) (Proband 3, M) c.280G>T p.Glu94Stop 54 — 53 53 — — — 53 10 (1) (Proband 10, M) c.533T>G p.Leu178Arg 39 57 57 57 — — — 39 
  Son c.280G>T p.Glu94Stop — — 29 29 — — 23 23   Son c.533T>G p.Leu178Arg — — 30 — — — — 30 
4 (2) Mother c.269A>T p.Asn90Ile — — 63 — — — — 63 11 (1) Mother c.263G>A p.Trp88Stop 36 — — — — — — 36 
  Brothera  No DNA sample available 19 — — — — — — 19   (Proband 11, M) c.263G>A p.Trp88Stop — — 34 38 — — — 34 
  (Proband 4, M) c.269A>T p.Asn90Ile 22 32 — — — — — 22 12 (1) Mothera  No DNA sample available 30 — — — — — — 30 
5 (3) Father c.499C>T p.Arg167Trp — — 54 — — — — 54   (Proband 12, M) c.280G>T p.Glu94Stop 31 — — — — — — 31 
  (Proband 5, F) c.499C>T p.Arg167Trp 37 — 37 37 39 — 37 37 13 (2) Mothera  No DNA sample available 43 — — — — — — 43 
  Sister 1 c.499C>T p.Arg167Trp 38 — — 36 — — — 36   Sister Deletion 30 — 30 30 — — — 30 
  Sister 2 c.499C>T p.Arg167Trp — 34 — — — — — 34   (Proband 13, M) Deletion 23 24 23 23 — — — 23 
6 (2) Mothera  No DNA sample available 45 — — — — — — 45 14 (3) Mothera  No DNA sample available 50 — — 62 — — — 50 
  (Proband 6, F)  Not assayed — — 42 42 — — — 42   Sistera  No DNA sample available 40 — 49 — — — — 40 
   Daughter  Not assayed — — — — — —   (Proband 14, M) c.292T>A p.Tyr99Asn 40 — 41 41 40 — — 40 
7 (4) (Proband 7, M) c.349T>G p.Trp117Gly 36 44 44 40 — — — 36   Brothera  No DNA sample available 27 20 — — — — — 20 
  Son c.349T>G p.Trp117Gly 13 — — — — — — 13 15 (1) Fathera  No DNA sample available 42 — — — — — — 42 
 Brother 1a  No DNA sample available 47 — 51 — — — — 47   (Proband 15, M) c.481C>T p.Arg161Stop 29 — 29 36 — 33 29 29 
  Nephew 1 c.349T>G p.Trp117Gly — — — — — — 37 37 16 (2) (Proband 16, M) c.500G>A p.Arg167Gln 29 — 46 — 46 — — 29 
 Sister 1a  No DNA sample available — — — 46 — — — 46   Daughter  No DNA sample available 24 14 — — — — — 14 
  Niece c.349T>G p.Trp117Gly — 13 — — — — — 13  Sistera  No DNA sample available 50 — — — — — — 50 
 Brother 2a  No DNA sample available — 43 44 — — — — 43   Nephewa  No DNA sample available 15 — 26 — — — — 15 
  Nephew 2 c.349T>G p.Trp117Gly 12 11 — — — — — 11 17 (1) Mothera  No DNA sample available — — 43 43 — — — 43 
8 (4) Grandfathera  No DNA sample available 40 — 60 — — — — 40   (Proband 17, M) Deletion — 21 — — — — 21 21 
  Mother Deletion 43 35 — — — — — 35 18 (2) Mothera  No DNA sample available — 29 — — — — — 29 
   (Proband 8, M) Deletion 23 17 — 19 — — 19 17   (Proband 18, F) c.288insA Frameshift 29 20 29 — — — 40 20 
 Aunt 1a  No DNA sample available 41 — — — — — — 41   Brother c.288insA Frameshift — 34 43 43 — — — 34 
 Aunt 2  No DNA sample available 19 — — — — — — 19             

Abbreviations: CNS, hemangioblastomas of CNS; RA, retinal angiomas; PCT, multiple pancreatic cysts or tumors; Pheo, pheochromocytoma; ELST, endolymphatic sac tumor; EC, epididymal/ovarin cystadenoma.

aDeath before diagnosis.

bThe onset age referred to the age when any symptoms or signs of VHL disease began.

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-μL PCR mixture contained 2X SYBR master mix (Takara) 5 μL, genomic DNA 30 ng, 300 nmol/L telomere primer Tel1 (5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT) and 900 nmol/L Tel2 (5′-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA), or 200 nmol/L single copy gene primer 36B4u (5′-CAGCAAGTGGGAAGGTGTAATCC), and 500 nmol/L 36B4d (5′-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 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.

formula

The relationship between age and relative telomere length in 325 normal controls can be expressed by the linear regression equation of Y = 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 age-adjusted 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.

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 (Table 2), which indicated that the mean onset age was 16.8 years earlier (paired t test, P < 0.001), 9.7 years earlier (HV test, P = 0.01), and 19 years earlier (RY2 test, P = 0.04) in children. The distribution of onset age in the 34 parent–child pairs also showed the same tendency of onset age in children and parents (Fig. 1). Therefore, significant difference in onset age was present between children and parents in the 34 VHL disease parent–child pairs.

Figure 1.

Onset age in the parent–child pairs. Kaplan–Meier curve indicates the difference of onset age in 34 parent–child pairs (log-rank test, P < 0.001).

Figure 1.

Onset age in the parent–child pairs. Kaplan–Meier curve indicates the difference of onset age in 34 parent–child pairs (log-rank test, P < 0.001).

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Table 2.

Difference in onset age between parents and children with VHL disease

Paired t testHV testRY2 test
nOnset age (y) mean (range)MOAD (y)aPMOAD (y)PMOAD (y)P
Parents 23 42.9 (28–63) 16.8 <0.001 9.8 0.01 19 0.04 
Children 34 26.1 (8–42)       
Total 57 32.3 (8–63)       
Paired t testHV testRY2 test
nOnset age (y) mean (range)MOAD (y)aPMOAD (y)PMOAD (y)P
Parents 23 42.9 (28–63) 16.8 <0.001 9.8 0.01 19 0.04 
Children 34 26.1 (8–42)       
Total 57 32.3 (8–63)       

aMOAD, mean onset age difference between parents and children.

Relative telomere length was shorter in patients in the next generation than in those in the first generation in the 10 parent–child patient pairs

To assess the difference of relative telomere length between patients with VHL and healthy controls, we measured relative telomere length of blood leukocytes in the 29 patients with VHL disease and compared with 325 normal controls (Fig. 2). Relative telomere length was shorter in the 29 patients with VHL disease than in the normal controls (P = 0.003), and 23 of the 29 patients with VHL disease showed the relative telomere lengths shorter than the average value of normal controls.

Figure 2.

Relationship between age and relative telomere length in patients with VHL disease and normal controls. In normal controls (n = 325), the relative telomere length is negatively correlated with age, with the linear regression equation of Y = 1.4503-0.0117*X, R2 = 0.162 (the continuous line). In patients with VHL disease (n = 29), the average relative telomere length (the dotted line) is slightly lower than that in normal controls.

Figure 2.

Relationship between age and relative telomere length in patients with VHL disease and normal controls. In normal controls (n = 325), the relative telomere length is negatively correlated with age, with the linear regression equation of Y = 1.4503-0.0117*X, R2 = 0.162 (the continuous line). In patients with VHL disease (n = 29), the average relative telomere length (the dotted line) is slightly lower than that in normal controls.

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In the 10 parent–child pairs from eight families with VHL disease, patients in the first generation had older onset age with longer relative telomere length, and those in the next generation had younger onset age with shorter relative telomere length (Fig. 3). The difference of age-adjusted telomere length in the 10 parent–child patient pairs (Table 3) also indicated that the telomere length was significantly shorter in children than in their respective parents (P < 0.001). The relative telomere lengths assayed by PCR are compatible with the telomere lengths measured by Southern blot analysis (Fig. 4).

Figure 3.

Relationship between onset age and age-adjusted relative telomere length in the 10 parent–child pairs. Arrow, proband; filled square and circle, affected; slant line on square or circle, died; question mark, suspected VHL disease patient. Onset age and age-adjusted relative telomere length of the parent–child pairs are shown in bar charts under pedigrees.

Figure 3.

Relationship between onset age and age-adjusted relative telomere length in the 10 parent–child pairs. Arrow, proband; filled square and circle, affected; slant line on square or circle, died; question mark, suspected VHL disease patient. Onset age and age-adjusted relative telomere length of the parent–child pairs are shown in bar charts under pedigrees.

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Figure 4.

Telomere length measurement by Southern blot analysis for four pairs of parent–child patients. Telomere length was measured by Southern blot analysis for four pairs of parent–child patients (Family 2, I:1 and II:3; Family 4, I:1 and II:3; Family 6, II:1 and III:1; Family 7, II:6 and III:7; see Fig. 3) and four normal controls. TEL age (years), the age when genomic DNA was obtained; TEL length (bp), mean telomere length by Southern blot analysis; qPCR data, relative telomere length by quantitative PCR method. The measurements were separated in two blots.

Figure 4.

Telomere length measurement by Southern blot analysis for four pairs of parent–child patients. Telomere length was measured by Southern blot analysis for four pairs of parent–child patients (Family 2, I:1 and II:3; Family 4, I:1 and II:3; Family 6, II:1 and III:1; Family 7, II:6 and III:7; see Fig. 3) and four normal controls. TEL age (years), the age when genomic DNA was obtained; TEL length (bp), mean telomere length by Southern blot analysis; qPCR data, relative telomere length by quantitative PCR method. The measurements were separated in two blots.

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Table 3.

Age-adjusted relative telomere length and onset age of the 10 parent–child pairs

ParentChild
Parent–child pairFamily No.TumorOnset ageTEL ageaTEL (age adjusted)bTumorOnset ageTEL ageaTEL (age adjusted)bTELchild − TELparent
CNS 50 69 −0.372 CNS, RA 16 38 −0.688 −0.316 
CNS, RA, RCC 28 42 0.074 CNS 16 15 −0.152 −0.226 
CftNS 53 53 −0.262 RCC 23 25 −0.791 −0.529 
RCC 63 63 0.637 CNS, RA 22 32 −0.042 −0.679 
RCC 54 64 −0.007 CNS, RCC 37 37 −0.386 −0.379 
RCC 54 64 −0.007 CNS 36 36 −0.354 −0.347 
RCC 54 64 −0.007 RA 34 34 −0.273 −0.266 
RCC 42 42 0.008 RA 17 −0.121 −0.129 
CNS, RA, RCC 36 45 −0.069 CNS 13 17 −0.283 −0.214 
10 CNS, RA 35 46 −0.328 CNS, RA 17 26 −0.437 −0.108 
ParentChild
Parent–child pairFamily No.TumorOnset ageTEL ageaTEL (age adjusted)bTumorOnset ageTEL ageaTEL (age adjusted)bTELchild − TELparent
CNS 50 69 −0.372 CNS, RA 16 38 −0.688 −0.316 
CNS, RA, RCC 28 42 0.074 CNS 16 15 −0.152 −0.226 
CftNS 53 53 −0.262 RCC 23 25 −0.791 −0.529 
RCC 63 63 0.637 CNS, RA 22 32 −0.042 −0.679 
RCC 54 64 −0.007 CNS, RCC 37 37 −0.386 −0.379 
RCC 54 64 −0.007 CNS 36 36 −0.354 −0.347 
RCC 54 64 −0.007 RA 34 34 −0.273 −0.266 
RCC 42 42 0.008 RA 17 −0.121 −0.129 
CNS, RA, RCC 36 45 −0.069 CNS 13 17 −0.283 −0.214 
10 CNS, RA 35 46 −0.328 CNS, RA 17 26 −0.437 −0.108 

Abbreviations: CNS, hemangioblastoma of the CNS; RA, retinal angioma; TEL, telomere length.

aTEL age, the age when DNA sample was obtained.

bTEL (age adjusted), age-adjusted relative telomere length.

Anticipation and its molecular mechanism have been proved in many hereditary diseases. In this study, we provide the evidence of earlier onset age and shorter telomere length in the successive generations in families with VHL disease, indicating that anticipation exists in families with VHL disease and that it may be attributed to the shortening of telomere length in successive generations.

Anticipation has been a controversial issue probably due to the bias in clinical data evaluation and the undetermined mechanism behind anticipation (28). The identification of anticipation in an inheritable disease has to rely on the judicious use of statistics methods. Paired t test was commonly used for anticipation analysis, but this method may introduce a truncation bias, leading to the increase of type I error (29). Statisticians have been trying to design better methods to make anticipation test more accurate (30–32). Currently, there are two recommended methods, HV and RY2 tests, for anticipation analysis as they display a balance between reducing truncation bias and increasing examination efficiency (27). In this study, we used paired t test as well as HV and RY2 tests to treat our data and obtained identical results with statistical significance from the three methods (Table 2). Though the truncation bias can be adjusted by HV and RY2 tests, other factors such as the insidious onset of symptoms and the presence of more sensitive diagnosis technology may also affect the recognition of anticipation in hereditary diseases. Despite the fact that the lower incidence of VHL disease limited us to recruit a large cohort of VHL families, anticipation was found in 31 of the 34 parent–child pairs in our 18 families with VHL disease families.

To define the molecular basis for anticipation in patients with VHL disease, we measured relative telomere length in 10 parent–child pairs (totally 18 patients) from eight families with VHL disease and correlated these values with onset age in these patients. To appropriately compare relative telomere length with onset age, the relative telomere length was adjusted by the age when DNA sample was obtained (19, 25). Our results clearly showed that the age-adjusted telomere length was significantly shorter in child than in his or her parent in all of the 10 parent–child pairs in the eight families with VHL disease (Table 3 and Fig. 3), suggesting the close relationship between shortening of telomere length and anticipation in families with VHL disease. Therefore, the anticipation in VHL disease may correlate to the progressive shortening of telomere in successive generations. However, why telomere shortens in the offspring of patients with VHL disease is yet unknown.

Decrease of telomere has been considered to be the cause of genetic anticipation in three cancer syndromes, including dyskeratosis congenital, hereditary breast cancer syndrome, and Li–Fraumeni syndrome. Dyskeratosis congenita is caused by dominant mutations in TERC encoding RNA component of telomerase (33). Most hereditary breast cancer syndrome patients carry mutations in BRCA1 or BRCA2, which are involved in repair of dsDNA breaks, and BRCA2 has been described to affect telomere replication (34–36). Li–Fraumeni syndrome is associated with the mutations in TP53 tumor suppressor gene responsible for initiating DNA repair mechanisms (37). We found that the relative telomere length was shorter than normal controls in 23 of 29 patients with VHL disease (Fig. 2). Besides, relative telomere length was also shorter in patients with VHL disease than in their respective family members who carried normal VHL gene with normal phenotype (data not shown). Together with the recently finding of VHL gene participates in the repair response to DNA damage, we can suppose that the mutation of VHL gene can partly influence the telomere length although the exact mechanism has not been studied fully.

The screening guideline for VHL disease proposes that the offspring of patients with VHL should have ophthalmoscopy and other screening methods examined beginning from infancy (4–6). In the 34 child–parent pairs of our 18 families with VHL disease, the onset age in children was about 10 years earlier than their respective parents, which suggests the time when enhanced screening methods such as CT scanning for VHL tumors should be performed in family members carrying VHL mutations.

We first investigated the anticipation in VHL disease through rigid statistical analyses of clinical data and search for its possible mechanism. These findings indicate that anticipation was present in families with VHL disease and may be helpful for genetic counseling for families with VHL disease and for further understanding the pathogenesis of VHL disease.

No potential conflicts of interest were disclosed.

Conception and design: X.-H. Ning, K. Gong

Development of methodology: X.-H. Ning, N. Zhang, X.-Y. Li, K. Gong

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X.-H. Ning, N. Zhang, T. Li, P.-J. Wu, X. Wang, S.-H. Peng, J.-Y. Wang, J.-C. Chen, K. Gong

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X.-H. Ning, N. Zhang, T. Li, P.-J. Wu, X.-Y. Li, K. Gong

Writing, review, and/or revision of the manuscript: X.-H. Ning, N. Zhang, T. Li, P.-J. Wu, X. Wang, S.-H. Peng, J.-Y. Wang, J.-C. Chen, K. Gong

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Gong

Study supervision: K. Gong

The authors thank Hong Zhang, Renal Division, Peking University First Hospital, and Dingfang Bu, Medical Experiment Center, Peking University First Hospital, for their technical assistance.

This work was supported by the grants from the Program for New Century Excellent Talents in Universities (grant NCET-10-0190) and the National Natural Science Foundation of China (grants 30872560 and 81172418).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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