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Autosomal dominant conditions are known to be associated with advanced paternal age, and it has been suggested that retinoblastoma (Rb) also exhibits a paternal age effect due to the paternal origin of most new germline RB1 mutations. To further our understanding of the association of parental age and risk of de novo germline Rb mutations, we evaluated the effect of parental age in a cohort of Rb survivors in the United States. A cohort of 262 retinoblastoma patients was retrospectively identified at one institution, and telephone interviews were conducted with parents of 160 survivors (65.3%). We built two sets of hierarchical stepwise logistic regression models to detect an increased odds of a de novo germline mutation related to older parental age compared to sporadic and familial Rb. The modeling strategy evaluated effects of continuous increasing maternal and paternal age and five-year age increases adjusted for the age of the other parent. Mean maternal ages for patients with de novo germline mutations and sporadic Rb were similar (28.3 and 28.5 respectively) as were mean paternal ages (31.9 and 31.2 respectively), and all were significantly higher than the weighted general U.S. population means. In contrast, maternal and paternal ages for familial Rb did not differ significantly from the weighted U.S. general population means. Although we noted no significant differences between mean maternal and paternal ages between each of the three Rb classification groups, we found increased odds of having a de novo germline mutation for each five-year increase in paternal age, but these findings were not statistically significant (de novo versus sporadic ORs: 30-34 = 1.65 [0.69-4], ≥35 = 1.34 [0.54-3.3]; de novo versus familial ORs: 30-34 = 2.82 [0.95 – 8.4], ≥35 = 1.61 [0.57-4.6]). Our study suggests a weak paternal age effect for Rb resulting from de novo germline mutations consistent with the paternal origin of most of these mutations.
Retinoblastoma (Rb) is a malignant tumor of the retina that occurs in children typically under the age of five. It is estimated that Rb affects 1:15,000 births in the United States (U.S.) (Abramson and Schefler 2004; Lin and O'Brien 2009). RB1 is a tumor suppressor gene involved in regulating the cell cycle, and malignant tumors occur in retinal cells with mutations in both copies of the RB1 gene. Approximately 65-75% of Rb cases are unilateral, occurring in only one eye, and occur sporadically when “two hits” occur in the same retinal cell (Knudson 1971). Approximately 25-35% of Rb cases are bilateral, affecting both eyes. Individuals who inherit an RB1 mutation, either in an autosomal dominant manner from a parent or from a de novo germline mutation, typically develop bilateral Rb. Approximately 10% of all retinoblastoma cases can be attributed to family history with autosomal dominant inheritance. In addition, family history information and genetic testing has revealed that some unilateral cases (10-15%) involve a germline mutation in the RB1 gene (Lohmann et al. 1997; Lohmann and Gallie 2004; Newsham et al. 2009; Richter et al. 2003).
Several autosomal dominant genetic conditions are now known to be associated with advanced paternal age (generally considered to be 40 to 45 years of age or older) (Thacker 2004; Sartorius and Nieschlag 2010). These include Marfan syndrome, achondroplasia, and Apert syndrome, among others. It has also been suggested that there is a paternal age effect with Rb, albeit a weaker effect than the conditions mentioned above (Kühnert and Nieschlag 2004; Risch et al. 1987; Sivakumaran et al., 2000), Further, it has been estimated that 85% of new RB1 germline mutations are paternal in origin, therefore, it would be expected that older paternal age might be related to the appearance of de novo Rb (Dryja et al., 1989).
Studies in Sweden, the Netherlands, Japan, France and more recently the U.S., have examined the association between Rb and advanced paternal and maternal ages (Bunin et al., 1989, DerKinderen et al. 1990; Johnson et al. 2009; Matsunaga et al. 1990; Moll et al. 1996; Pellié et al., 1973, Yip et al. 2006). Some but not all of these studies reported older paternal and maternal age related to occurrence of retinoblastoma. At present, there is no consensus regarding the impact of advanced paternal or maternal age on the incidence of Rb caused by de novo germline mutations. This study aims to further our understanding of the association of parental age and risk of de novo germline Rb mutations by evaluating parental age in a cohort of Rb survivors in the United States. Our hypothesis is that Rb survivors with a de novo germline mutation are more likely to have a father of advanced paternal when compared to survivors of sporadic or familial Rb, and to the general population.
The retrospectively defined cohort used for this study consists of 262 Rb patients diagnosed from January 1, 1985 through December 31, 1996 at a medical center in New York, NY. This cohort is currently part of a larger study of secondary cancer incidence and cause-specific mortality in long-term Rb survivors (Kleinerman et al, 2005; Yu et al., 2009). Hospital records were used to identify study subjects and to collect medical history and treatment data. Demographic data, including parental age at the birth of the Rb patient, was collected via telephone interviews conducted in 1998. Of the original 262 Rb patients, four did not survive one year and thirteen had died by the time of the interview in 1998. Therefore, parents of 245 survivors were eligible to be interviewed for the study and parents of 160 (65.3%) survivors agreed to participate. We did not identify statistically significant differences between the respondents and non-respondents for hereditary status, year of birth or sex of their child; however, respondents were more likely to be white and report a family history of retinoblastoma. Family history was defined as a first or second degree relative with Rb.
We excluded one individual from the analysis who was born in 1950 because all other members of the cohort were born between 1975 and 1996. Maternal age data was not available for two survivors and paternal age for five survivors, but all other available data for these survivors were included. Therefore, we analyzed 159 of the 160 survivors for this study.
The cohort survivors were grouped into one of three Rb classifications:
The sporadic and de novo germline mutation classifications based on laterality of Rb tumors are an approximation, because mutation testing data were not available for the cohort. Published mean maternal age data for the U.S. is available for the years 1970 to 2000 (Mathews and Hamilton 2002), however mean paternal age data is not, so we calculated mean paternal age from public use files available from the U.S. Centers for Disease Control and Prevention, National Center for Health Statistics Reproductive Statistics Branch, Division of Vital Statistics (U.S. CDC data) for births for the years 1975 to 1996. We also calculated mean maternal age for each year from 1975 to 1996 and then compared these data to the data published by Mathews and Hamilton (2002) and found our calculations to be accurate to within 0.1 years (Appendix A). Paternal age data for the U.S. was missing for 11.1% of fathers in 1975 up to a high of 16.9% of fathers in 1991. Thus, the mean paternal age data used for this study is an approximation and may overestimate mean paternal age in the U.S from 1975 to 1996. For comparison to the mean parental ages in our cohort, we calculated a weighted mean for mean maternal and paternal age in the U.S. general population based on the years of birth of our cohort of survivors.
Associations between categorical predictors and the three Rb classification groups were assessed with either Chi-square tests or the Freeman-Halton extension of the Fisher's exact test when small expected cell frequencies were noted. Two sets of hierarchical stepwise logistic regression models were built to look for increased odds of a de novo germline mutation versus sporadic and familial Rb associated with increased maternal and paternal age. The first modeling strategy looked for effects of continuous increasing maternal and paternal age while controlling for differences in race (White, African-American, Hispanic, other/unknown) and age of the other parent. To assess nonlinear age effects, a second set of models using parental age groupings of <30, 30–34, and ≥35 years were subsequently analyzed while controlling for differences in race and age of the other parent. We used parental age <30 years as our reference group because we had a limited number of fathers and mothers less than age 25 years. Parental age groupings of <25, 25–29, 30–34, and ≥35 years were also analyzed and are included in Appendix B. All differences between means were compared using either one sample t-tests or one-way ANOVAs. For all tests, statistical significance was declared for two tailed p-values p < .05. All calculations were performed using SAS Enterprise Guide version 4.22, SAS version 9.21 (Cary, NC).
We classified 75 (47.2%) survivors as sporadic, 46 (28.9%) as de novo germline mutation and 38 (23.9%) as familial (Table 1). We found no noteworthy differences between sex, race, year of birth, age of mother at birth, and age of father at birth for each of the three groups. Laterality and family history differed for each of the Rb classification groups by definition. As expected, de novo germline mutation and the familial cases of Rb were more likely to be diagnosed less than 1 year of age as compared to sporadic cases (p <0.001).
Mean maternal and paternal ages for the de novo germline mutation classification were significantly higher than the weighted mean maternal (28.3 vs. 26.2 years, p = 0.003) and paternal (31.9 vs. 29.3 years p = 0.007, ) ages of the general U.S. population, (Table 2). Similarly, maternal and paternal ages for the sporadic Rb classification group were significantly higher than the mean maternal and paternal ages of the general U.S. population (p < 0.001 and p = 0.01, respectively). Conversely, the parental ages for the familial classification group did not differ significantly from the general U.S. population. Although the three Rb classification groups did not differ significantly from each other (Table 3), the mean maternal and paternal ages for both de novo and sporadic Rb were similar (28.3 and 28.5, and 31.9 and 31.2 respectively) whereas the mean ages for the familial parents were approximately 1.7 years younger for mothers of sporadic and de novo survivors and approximately 1.8 years younger than the average age for fathers of sporadic and de novo survivors.
In our analysis of continuous parental age, there was no statistically significant effect of maternal or paternal age when the odds of having a de novo germline mutation were compared to the odds of having sporadic Rb or familial Rb. The odds ratios for almost all scenarios were close to 1.0 (Table 4). In addition, there continued to be no statistically significant effect when the analysis was adjusted for race and the age of the other parent (Table 4).
When we examined the odds of having a de novo germline mutation versus the odds of having familial Rb by five-year age groups for paternal age, we found increased odds of having a de novo germline mutation versus the odds of having either sporadic Rb or familial Rb (Table 5). The effect was highest for the 30-34 paternal age group but no effects were significant, even when adjusted for race and age of the other parent (Table 5). We found a non-significant increase in the odds for maternal ages 30-34 (OR=2.80 [0.9-8.8]). However, the odds of having a de novo germline mutation versus the odds of having familial Rb, for maternal age greater than 35, was reduced (0.80, 95% CI = 0.19-3.3).
In this study, we have assessed the influence of older parental age on Rb caused by de novo autosomal dominant mutations using a cohort of Rb survivors diagnosed and treated at one institution. To our knowledge, this is the first study to evaluate parental age effects for three different categories of Rb (hereditary Rb resulting from a de novo germline mutation, sporadic Rb, and familial Rb inherited from an affected parent), and to investigate parental age differences between these three groups.
Although we found no significant differences between maternal and paternal ages when comparing between the three Rb groups, there was some evidence of a signal for older paternal age and the odds of a de novo germline mutation based on the modeling. We also noted that mean parental ages related to de novo germline mutations and sporadic Rb, but not familial Rb, were higher than the general population This finding supports the notion that, in general, those with familial Rb reproduce at ages similar to the general population, but have a 50/50 chance of passing on the mutated RB1 allele with each pregnancy. It was not surprising to find younger parental ages for familial Rb cases when compared with sporadic Rb because this was reported in a previous study (Yip et al. 2006). However, the similarity of mean maternal and paternal ages for de novo germline mutations and sporadic Rb was unexpected, because it has not been previously reported.
When comparing our study to previous studies regarding parental age data for Rb (see Table 6), it is important to note that we compared our hospital-based cohort to the general population in our analysis of mean maternal and paternal age. We did not use matched general population controls for our logistic regression analysis nor did we use incidence data for Rb in the United States. Unlike previous studies, we compared the odds of being in the de novo germline mutation classification group versus the sporadic and familial Rb groups given increasing parental age in our cohort of individuals. Our comparisons of parental age to general population means were similar to findings reported by DerKinderen et al. (1990) and Moll et al. (1996) for de novo mutations, but in contrast to these studies, we also saw significant differences for sporadic non-hereditary Rb.
In our cohort we had relatively few parents over age 35 years making it difficult to estimate the odds of having a child with a Rb de novo mutation for fathers of advanced paternal age (greater than 40-45 years of age) (Table 1). The theory behind de novo mutations and advanced paternal age is that errors occur in mitotic divisions during male spermatogenesis (Thacker 2004; Sartorius and Nieschlag 2010). Although our results suggested a pattern of increased odds of a de novo mutation with paternal age, we believe this is a preliminary finding that should be followed up with additional analysis with a greater number of Rb survivors with increased enrichment for advanced parental age. Only 65% of the cohort agreed to participate, and having more subjects would allow for statistical power to determine if the patterns we observed in this analysis are valid. In addition, more complete information on subject characteristics, such as race, would allow for a more detailed analysis of this potential confounder. Future research could consider the influence of other covariates such as environmental exposures of one or both parents that could increase the risk of de novo retinoblastoma mutations (Bunin, et al. 2011).
Future work regarding Rb and parental age should include mutation status gathered from genetic testing rather than by proxy with laterality and family history since some unilateral survivors in our cohort may have a de novo mutation that predisposed them to Rb. A dataset including mutation status and mutation type (missense versus nonsense, etc.) would allow for a more precise analysis of the differences in parental ages of the three Rb classification groups, given an adequate number of patients to achieve statistical power. Although prior studies have benefited from larger sample sizes, we believe this is the first study to include familial inherited Rb in an analysis of parental age.
Overall, our findings show that, as previously reported for other countries, the mean parental age of Rb survivors with a de novo mutation is statistically significantly higher than the mean age of the general U.S. population. The similarity of mean maternal and paternal ages for de novo germline mutations and sporadic Rb was unexpected and deserves further attention. Our study suggests a greater paternal rather than maternal contribution to RB1 mutations, perhaps during gametogenesis, for the de novo germline mutations, however we have insufficient data for investigating paternal age over age 40 years to test this hypothesis further. Our findings do not indicate statistically significant effects for advanced paternal age and thus would not be appropriate for use in genetic counseling at this time.
We are grateful for the support of the Stanford University School of Medicine Master's Program in Human Genetics and Genetic Counseling. This research was supported in part by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute, Division of Cancer Epidemiology and Genetics.