Oral Human Papillomavirus Is Common in Individuals with Fanconi Anemia

Fanconi anemia is an inherited autosomal recessive or rare X-linked disorder characterized by genome instability, progressive bone marrow failure, and predisposition to gynecologic and head and neck squamous cell carcinomas (HNSCC). The risk of HNSCC is 1,000-fold greater compared with the general population, remains high even after bone marrow transplantation (BMT), and occurs at strikingly early ages with poor prognoses (1–4). Human papillomavirus (HPV) infection, particularly HPV16, is known to be associated with HNSCC in the general population; specifically oropharyngeal cancers (5). Interestingly, HPV positivity was significantly higher in oral samples from individuals with Fanconi anemia living in Brazil than in non-Fanconi anemia controls (6) suggesting a role for HPV in Fanconi anemia–associated cancer. This report was consistent with an earlier U.S.-based study that found HPV DNA prevalence to be significantly greater in 25 Fanconi anemia–related HNSCC tumors compared with non-Fanconi anemia controls (7). However, a Dutch study failed to detect HPV DNA in any of their Fanconi anemia–related tumors tested (8) and HPV was not detected in 5 HNSCC or 4 anogenital squamous cell carcinoma samples from subjects with Fanconi anemia in a more recent study (9). Therefore, the extent to which HPV is associated with Fanconi anemia or HNSCC in individuals with Fanconi anemia remains unclear. While HPV is commonly sexually transmitted, it has been detected in healthy children and virgin adults in a number of studies (10–12), likely as a result of horizontal (nonsexual, perinatal, or parenteral) transmission. Better characterization of the natural history of oral HPV in the Fanconi anemia population is therefore needed.

Fanconi anemia is a consequence of mutations in one of 16 genes whose respective protein products assemble in the nucleus to repair DNA damage (13, 14). Our recent studies indicate that under normal circumstances, activation of this important DNA repair pathway limits the HPV life cycle and prevents malignant transformation. Specifically, loss of function of the Fanconi anemia–related DNA repair pathway in HPV-positive keratinocytes stimulates cellular and viral DNA replication (15). Conversely, rescue experiments in cells obtained from individuals with Fanconi anemia reduced HPV-mediated DNA damage and suppressed tumor growth (16) indicating that Fanconi anemia DNA repair pathway–deficient keratinocytes uniquely support HPV infection and/or replication. These findings are reinforced by studies in mice demonstrating that deficiencies in the Fanconi anemia DNA repair pathway are associated with an increased incidence of HNSCCs, and with the ability of the HPV E7 protein to induce DNA damage (17–19). In addition, recent immunophenotyping and immune function studies in 10 children with Fanconi anemia suggest heterogeneous immune defects (20). The extent to which Fanconi anemia DNA repair pathway deficiency in keratinocytes and variable or inadequate immune cell function contributes to early HPV acquisition, maintenance, and/or susceptibility to cancer remains unknown. Given that HPV vaccination has been shown to offer protection against some high-risk HPV types in the general population, now is a critical time to investigate HPV in these vulnerable individuals to determine the need for earlier vaccination and to develop targeted treatments.

In this study, we examined the prevalence of HPV infection in a sample of individuals with Fanconi anemia together with their parents who are obligate heterozygotes for the Fanconi anemia mutation, and siblings who are possible heterozygotes for the Fanconi anemia mutation. Study of family members helped to control for possibly nonsexual routes of exposure to HPV. Importantly, we observed a higher prevalence of HPV in individuals with Fanconi anemia as compared with their family members, particularly those that had been sexually exposed.

This study was approved by Cincinnati Children's Hospital Medical Center (CCHMC) institutional review board. Briefly, following a detailed verbal description of the study, written informed consent/parental permission was obtained by a trained clinical research coordinator from participants' ≥18 years old or participants' parent(s)/legal authorized representatives. Participants that were ≥8 years old were asked to sign the consent document along with their parents/guardians to indicate their assent. Younger children or children unable to provide written assent indicated their assent by willingly providing their sample(s).

Patients visiting the Cincinnati Children's Fanconi Anemia Comprehensive Care Center (CCFACCC) were approached to participate. Furthermore, attendees of the Fanconi Anemia Research Fund (FARF) Adult and Family Meetings were invited to participate. Individuals of all ages were eligible to participate if they reported a diagnosis of Fanconi anemia or were a parent or sibling of an individual with Fanconi anemia, and were willing to complete study-related surveys and provide oral rinse samples. A subset (N = 68) also provided a venous blood sample for HPV serology and immune studies. A total of 150 subjects with Fanconi anemia have been enrolled in the study as well as 247 family members (biologic siblings and parents). Of these, 126 individuals with Fanconi anemia, 41 siblings, and 121 parents currently have at least one HPV test result.

Oral exfoliated cells or oral-rinse and gargle samples were collected in 15 mL normal saline and 2.5 mL of 100% ethanol was added immediately after collection. Samples were stored for no more than 4 days at room temperature and then spun at 3,200 rpm for 10 minutes at 4°C, the pellet washed in 10 mL of phosphate-buffered saline (PBS), spun again, and resuspended in 1 mL PBS and stored in 500 μl aliquots at −80°C. In the laboratory of Darron R. Brown at Indiana University (Indianapolis, IN), DNA was isolated using the 5 Prime ArchivePure DNA kit (5 Prime Inc.) according to the manufacturer's protocol. DNA samples were genotyped by The Linear Array HPV Genotyping Test (Roche Molecular Diagnostics; ref. 21). Reactions were amplified in a PerkinElmer TC9600 Thermal Cycler (PerkinElmer), and positive and negative controls (included in the assay) were performed with every PCR run. The GH20/PC04 human β-globin target was coamplified to determine sample adequacy, and detection of specific HPV types was performed as previously described (22). The 37 individual HPV types detected are comprised of high-risk HPV types (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, and subtype 82 W13B) and low-risk HPV types (6, 11, 40, 42, 54, 55, 61, 62, 64, 72, 81, 83, 84, and 89).

Venous blood was collected observing appropriate aseptic techniques and universal precautions by trained personnel. Fifty percent were collected at the CCFACCC and immediately placed into storage at −80°C. Samples collected at FARF Meetings were typically kept at room temperature and centrifuged at 2,400 × g within 8 hours of collection, aliquoted, and then stored on dry ice until they were shipped via Same-Day FedEx (N = 20). Otherwise, they were shipped via Same-Day FedEx to CCHMC, centrifuged within 24 hours, aliquoted, and stored at −80°C (N = 14). An aliquot of each serum (0.5–1 mL typically) was sent via Next-Day FedEx for HPV serology testing in the HPV laboratory at the Centers for Disease Control and Prevention (Atlanta, GA). The HPV4-plex virus-like particle (VLP)-based IgG ELISA for HPV6, 11, 16, and 18 was performed as previously described (23). Cut-off values of median + 2 SDs were calculated using children's sera (gift from Joakim Dillner, Karolinska Institutet, Sweden) for each type. The pseudovirion neutralization assay (PBNA) was performed as described (24).

Humoral immune function was analyzed to assess effective or ineffective immune responses to HPV infection and or vaccination. Patient sera were tested for lymphocyte subsets, B-cell panel, immunoglobulin levels (IgA, IgG, and IgM) along with tetanus and diphtheria titers. Immunoglobulin levels were determined by standard methods in the CCHMC clinical laboratory. Tetanus and diphtheria titers were determined by Quantitative Multiplex Bead Assay in the clinical diagnostic laboratory at CCHMC. Remaining assays were performed at the Diagnostic Immunology Laboratories at CCHMC. Results were interpreted with respect to age-appropriate reference ranges established in the laboratory. Peripheral blood mononuclear cells were isolated from heparinized blood by centrifugation with lymphocyte separation media (MP Biomedicals). Evaluation of patient lymphocyte subsets was performed via routine four-color flow cytometric analysis of EDTA-preserved whole blood using fluorochrome-labeled monoclonal antibodies to lineage-specific cell surface markers for T cells (CD3, CD4, CD8), B cells (CD19), and natural killer (NK) cells (CD16, CD56). All antibodies were obtained from BD Biosciences. Briefly, erythrocytes were lysed by incubation in FACSLyse (BD Biosciences). Samples were then stained with antibody and analyzed on a FACSCalibur flow cytometer (BD Biosciences) using multiset software (BD Biosciences). Similar flow based approach was applied for assessment of B-cell panel (25).

Survey completion was done either by paper or by electronic survey [Research electronic data capture (REDCap) software; ref. 26]. Participants ages 15 years and older were asked to complete surveys for themselves unless there was a developmental delay, in which case a parent or guardian was asked to complete the survey. For children ages 12 to 15 years, a parent was asked either to complete the survey or assist the child in completing the survey. However, answers to the sexual questions were ascertained by study staff interview whenever possible. Surveys collected demographic and clinical data including Fanconi anemia complementation group, disease severity, and date/age at bone marrow transplant (if any). In addition, other potential risk factors for HPV-related disease such as vaginal versus Cesarean section mode of delivery at birth, gestational age at birth, tobacco smoke exposure, alcohol consumption, and sexual history (including questions about oral sex and kissing), family history of Fanconi anemia and cancer, history of personal immunization, and/or HPV testing, and presence and frequency of oral lesions were ascertained by survey and/or staff interview. When available, biologic mothers of participants were asked whether they have had genital or anal warts, a dysplastic Pap smear, or had a positive HPV test within a 3-year period before the birth of the individual(s) with Fanconi anemia and siblings.

Statistical analyses were performed using Statistical Analysis Software (SAS), version 9.3 (SAS Institute Inc.). Before analyses, data qualities were examined. Demographic variables and potential risk factors of HPV were first summarized as median and interquartile range (IQR) or frequencies (proportions) and compared in Fanconi anemia, siblings, and parents. These factors were then compared between HPV negative and positive individuals with Fanconi anemia. Using Fisher's exact tests, the impact of Fanconi anemia status on HPV prevalence was stratified by sexual experience or HPV vaccination status. Using a multivariable logistic regression, we further tested the effects of HPV risk factors on the prevalence of HPV, adjusting for sexual experience, HPV vaccination status, and Fanconi anemia status in the model. Finally, the association between BMT and oral HPV was assessed considering HPV infection and sexual experience. Effects were considered significant if P ≤ 0.05.

A total of 288 individuals with oral rinse samples and risk factor questionnaires were included in this study. Of these, 126 individuals had Fanconi anemia, 121 were parents of individuals with Fanconi anemia (74 mothers and 47 fathers), and 41 were siblings of the individuals with Fanconi anemia (Table 1). Participants with Fanconi anemia ranged in age from 6 months to 55 years. Family members' ages ranged from 4 months to 79 years. As expected, ages were significantly different between subjects with Fanconi anemia and family members (P < 0.0001). There were more females in each group than males, and more Caucasians than any other race. Individuals with Fanconi anemia were living in families across all income levels (26% <40 K, 36% 40–90 K, 39% > 90 K) and most had health insurance (32% public, 54% private, and 11% had both public and private insurance; Table 1).

Among those individuals with Fanconi anemia, 14 (11.1%) tested positive for one or more HPV types. Indeed, oral HPV positivity in Fanconi anemia was significantly greater than in non-Fanconi anemia family members (parents and siblings; 2.5%; P = 0.003; Fisher exact test; Fig. 1A), whose prevalences were consistent with published levels in the general population (range from 1.9% to 7%; refs. 10–12). Also consistent with published studies, the most common types of HPV detected among those with Fanconi anemia were HPV6 (50%) and HPV16 (64%), although HPV 18, 51, 66, 84, and CP6 were also detected. Only three of the HPV-positive individuals with Fanconi anemia had previously been vaccinated for HPV (all receiving the GARDASIL Quadrivalent HPV Vaccine, Merck & Co., Inc.) and of these three vaccinated HPV-positive individuals, only a single person with Fanconi anemia tested positive for an HPV type included in the vaccine (HPV16). We also examined HPV positivity in people based on HPV vaccine history. As shown in Fig. 1B, in both vaccinated and unvaccinated groups, individuals with Fanconi anemia tested positive for HPV more often. Importantly, in our study population, we observed significantly higher rates of vaccination in individuals with Fanconi anemia compared with family members (38% vs. 21% in siblings and 1% of parents, P < 0.0001, Fisher exact test; Table 2). It is therefore interesting that HPV prevalence is higher in Fanconi anemia despite greater rates of vaccination.

Typically transmission of high-risk carcinogenic HPV types in the general population occurs predominantly via sexual contact, including oral sex and open-mouthed kissing outside of vaginal sex (27). When we examined only those individuals who were sexually experienced, we observed significantly more oral HPV infections in those with Fanconi anemia (17.7%; P = 0.003) compared with family members (2.4%; Fig. 1C). In participants that were not yet sexually experienced, individuals with Fanconi anemia also tested positive for oral HPV (8.7%) more often compared with siblings (2.9%), though the difference did not reach statistical significance. We next assessed the effect of sex on the association between oral HPV positivity and Fanconi anemia. While having sex did not seem to increase HPV positivity in family members, individuals with Fanconi anemia exhibited a higher prevalence of HPV when they reported having had sexual experience (17.7% vs. 8.7%), but this association again was not statistically significant at the 5% level. In multivariable logistic regression analysis, including sexual experience, HPV vaccination, and Fanconi anemia status, only having Fanconi anemia was significantly associated with oral HPV positivity (P = 0.002). Indeed, having Fanconi anemia increased the odds of HPV positivity by 4.9-fold (95% CI, 1.6–15.4). Greater HPV prevalence was observed in those with Fanconi anemia compared with family members regardless of age. Importantly, among those individuals with Fanconi anemia that were not sexually active, 7 children less than 13 years of age (10.9%) also tested HPV positive (Fig. 1D).

We next evaluated several other factors associated with HPV positivity or HNSCC in the general population and compared those with Fanconi anemia to their family members (Table 2). We observed that 71% of individuals with Fanconi anemia compared with 54% of siblings and 90% of parents were delivered vaginally (P = 0.0001; Table 2). The biological mothers of those with Fanconi anemia compared with sibling group as a whole were not more likely to have reported having an abnormal Pap smear, positive HPV test, or other sexually transmitted disease within 3 years of their children's birth (P = 0.75). Not surprising given the differences in age as well as strength of the anti-smoking and anti-alcohol guidelines and education provided to those with Fanconi anemia by the Fanconi Anemia Research Fund (28), primary smoking and alcohol use were lower for those with Fanconi anemia and their siblings compared with parents (P = 0.0003 and 0.0001, respectively; Table 2). While the frequency of genital warts and common warts did not significantly differ between groups (P = 0.87 and P = 0.81, respectively; Table 2), significantly more individuals with Fanconi anemia reported oral sores (18%) compared with 8% of siblings and 4% of parents (P = 0.006).

Examining only those individuals with Fanconi anemia, no other factor related to Fanconi anemia (complementation group, having had a transfusion or having a family history of cancer) or those known to contribute to HPV or HNSCCs (i.e., tobacco and alcohol use, oral, genital and common warts, and dental hygiene) were found to be significantly different between oral HPV-positive or oral HPV-negative individuals with Fanconi anemia in our study (Table 3). While some have hypothesized that BMT might be associated with the increased risk of HNSCC in individuals with Fanconi anemia, we found that HPV positivity was only slightly higher (12.7%) post-BMT compared with pre-BMT (9.5%) and this association was not statistically significant (P = 0.78; Fig. 2). When we considered BMT and sexual experience simultaneously, we again observed higher levels of oral HPV positivity in those post-BMT particularly if they had had sexual experience, but again these differences were not statistically significant (P = 1.0; Fig. 2). Secondhand smoke exposure, however, was marginally statistically significantly higher in those that were oral HPV positive (29%) compared with oral HPV negative (9%; P = 0.052).

There was a broad range of ages (from 6 to 51 years of age) among the 14 individuals with Fanconi anemia who tested oral HPV positive (Table 3). Importantly, 7 (50%) were under the age of 13 years and 8 individuals with Fanconi anemia indicated that they were not sexually active. The level of oral HPV positivity was similar for males and females and there were no differences by whether or not they had a BMT (57%). While 9 (64%) had the FANCA complementation group, C, F, I, and D2 complementation groups were also represented in those that tested oral HPV positive (Table 4). Only a single oral HPV-positive participant (HPV 84) reported having had a diagnosis of oropharyngeal cancer before sampling.

Furthermore, among all 8 oral HPV-positive individuals with Fanconi anemia who had corresponding immune data, we observed 6 study participants to have normal IgG levels and 5 participants with normal absolute and/or memory B cells (Table 5). Most tetanus and diphtheria titers were also normal. Only 3 of the 8 subjects had been vaccinated for HPV before blood sampling. All vaccinated individuals had positive titers to all four HPV types included in the vaccine (all Gardasil) as well as normal tetanus and diphtheria levels (Table 5). Two of the oral HPV–positive individuals who indicated that they had not yet been vaccinated for HPV were also serologically positive to types other than identified in their oral sample suggesting prior natural infections with these types. Three of the oral HPV–positive participants had no detectable HPV titers. Two of these individuals tested positive for both oral HPV6 and HPV16. Interestingly, these individuals were either deficient in their absolute B-cell count or memory B cells. Overall, there were no unifying characteristics that were shared by those that were oral HPV or serologically positive with Fanconi anemia. In fact, these data support our previous report that individuals with Fanconi anemia have heterogeneous immune responses (20).

Our cross-sectional study demonstrates a higher frequency of oral HPV positivity in individuals with Fanconi anemia compared with family members. Furthermore, while HPV prevalence was significantly higher for those with Fanconi anemia who reported sexual exposure compared with family members, HPV prevalence tended to also be higher in those who were not yet exposed to sex.

A possible role for HPV in Fanconi anemia (29) has been previously suggested following the report of higher prevalence of HPV in Fanconi anemia–related head and neck tumors (7). In contrast, another equally small study reported the absence of HPV in Fanconi anemia–related HNSCC tumors and cell lines derived from them (8). These opposing results may be due to experimental variability such as types of primers used, quality and content of DNA tested, analysis of tumors or tumor-derived cell lines, or geographic differences. More recently, Alter and colleagues were unable to detect HPV in HNSCCs from individuals with Fanconi anemia (9). However, de Araujo and colleagues have shown an increased prevalence of oral HPV isolated from 42 oral cytobrush samples from individuals with Fanconi anemia without oral malignant lesions (6). One possibility is that the Brazilian population spanned a younger age range comparable with our study population. A second explanation for the discrepancy could be that some/many individuals with Fanconi anemia successfully clear the virus or suppress its replication before tumors develop, thus limiting detection in the tumors themselves. These studies, together with our data presented here, suggest that studies of the association between HPV and Fanconi anemia might have to be conducted on younger individuals, and call for continued longitudinal studies across the lifespan to determine whether early life exposure to HPV affects the development of HNSCCs in individuals with Fanconi anemia later in life. Furthermore, longitudinal studies would better address potential routes of transmission outside of sexual exposure. We have currently not detected significant differences in breastfeeding, birthing method (Caesarean section vs. vaginal), BMT/blood transfusions, and alcohol and tobacco exposure, although in some cases, the data collected were incomplete and likely underpowered to ascertain a true association.

While studies in human subjects remain controversial, there is growing evidence from cell lines, animal studies, expression analysis, and computational biology approaches that support a strong association between the consequences of HPV infection in the context of deficiency in the Fanconi anemia proteins and development of cancer. Organotypic raft cultures of HPV oncogene immortalized, FANCA-deficient patient-derived keratinocytes demonstrate increased DNA damage, and excessive hyperplasia compared with FANCA complemented cell populations (16). In addition, the Fanconi anemia pathway seems to limit the ability of HPV to replicate in cultured keratinocytes (15). Indeed, FANCD2-deficient mice overexpressing the HPV E7 oncoprotein have increased DNA damage and are more likely to develop HNSCCs (17) and cervical and vaginal cancer (18). These effects of E7 are due to inactivation of the Rb family of tumor suppressors that limit DNA damage (19). Microarray analysis of vulvar tissue infected with both low-risk and/or high-risk HPV types indicated that FANCA, FANCD2, and other DNA damage markers were significantly induced following high-risk HPV infection, along with increased DNA damage in the tissues (30). And finally, using a computational biology approach, a link between HPV16 and the Fanconi anemia DNA repair pathway was identified and then validated experimentally by evaluating the impact of overexpressing E6 or E7 proteins in primary fibroblasts and keratinocytes using global gene expression analysis (31).

Together, these diverse approaches point to a common theme. It appears that HPV infection results in elevated DNA damage that then triggers the Fanconi anemia pathway to repair this DNA damage. In individuals where this pathway is defective, it is likely that the DNA damage will not be repaired, and as a consequence increases the likelihood of tumor development in the long term. It becomes important to consider these studies again in the context of the conflicting human data. Even if HPV is suppressed or cleared, and is undetectable by PCR assays, one might speculate that the resulting DNA damage is the trigger for increased HNSCCs and anogenital carcinomas many years later. Additional studies will be required to support this hypothesis.

Similar to the Brazilian study (6), we found that oral HPV16 was the most common type detected. We also detected less common HPV types in those with Fanconi anemia (36% of oral samples). It is plausible, although speculative, that less common high-risk types, or even low-risk HPV types, might unusually initiate a cancer cascade in those with Fanconi anemia who have both a DNA repair defect and heterogeneous immune deficiency, suggesting a need to evaluate the broad spectrum of HPV types rather than HPV16 and 18 alone in oral rinse and possibly other sample types. Indeed, among 67 throat swabs from individuals with Fanconi anemia collected at the same time as our oral rinse samples in this report, we identified 5 individuals positive for HPV33, HPV84, HPV66, 51, 52, 62, 82, and HPV51; a 60% agreement between sample types (32).

An important variable to consider is the immune response to HPV infection in individuals with Fanconi anemia. We have previously demonstrated that individuals with Fanconi anemia have fewer B cells and NK cells, as well as decreased NK cell function and cytotoxic T-cell activity (20). It is conceivable that in the absence of a fully functional Fanconi anemia pathway, the ability to suppress or clear HPV is also delayed. On the basis of the data from Table 5, there seems to be variability in the immune responses in HPV-positive individuals with Fanconi anemia. While some subjects with Fanconi anemia had normal immune responses, others seem to be unable to mount a strong response (based on B-cell and memory B-cell numbers, IgG levels, and response to tetanus and diphtheria vaccines). These data indicate that while not all individuals with Fanconi anemia have measurably impaired immune responses, those that do could conceivably be at higher risk. It is reasonable to speculate that viral persistence due to the inability of the immune system to clear the infection or due to fluctuations in host immunity as a result of progressive bone marrow failure or Fanconi anemia–related illnesses, further increases the duration of HPV infection and extent of DNA damage, thereby resulting in an increased risk for carcinomas. Our data showed no differences in the type and distribution of factors known to cause cancers in individuals with Fanconi anemia regardless of their HPV infection status. This would further suggest that it might be the ability of the individual to suppress or clear the infection that most likely determines HPV prevalence, incidence, and susceptibility to HNSCCs.

In conclusion, continued longitudinal collection of oral samples and broad testing for HPV types along with immunologic studies to better characterize response remains critical to address these questions. In addition, studies are needed to determine prospectively whether HPV infection is associated with increased HNSCCs in individuals with Fanconi anemia, and if vaccination at a younger age could be effective in preventing early infection.

D.R. Brown reports receiving a commercial research grant from Merck, speakers bureau honoraria from Merck, and has ownership interest (including patents) from Merck. No potential conflicts of interest were disclosed by the other authors.

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC.

Conception and design: S.I. Wells, S.M. Davies, K.C. Myers, M. Butsch Kovacic

Development of methodology: S.I. Wells, E.E. Hoskins, D.R. Brown, M. Butsch Kovacic

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S.L. Sauter, E.E. Hoskins, S.M. Davies, K.C. Myers, R. Mueller, G. Panicker, E.R. Unger, D.R. Brown, P.A. Mehta, M. Butsch Kovacic

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): X. Zhang, E.E. Hoskins, K.C. Myers, U. Sivaprasad, D.R. Brown, P.A. Mehta, M. Butsch Kovacic

Writing, review, and/or revision of the manuscript: S.L. Sauter, S.I. Wells, X. Zhang, S.M. Davies, K.C. Myers, E.R. Unger, U. Sivaprasad, D.R. Brown, P.A. Mehta, M. Butsch Kovacic

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.L. Sauter, E.R. Unger, M. Butsch Kovacic

Study supervision: M. Butsch Kovacic

Other (discussed the study with patients and scheduled the patients for follow-up and specimen acquisition and informed design of recruitment and data collection plans): R. Mueller

The authors thank the CCHMC Fanconi Anemia Comprehensive Care Center staff, FARF staff, and Camp Sunshine staff for their support of this study. Most importantly, the authors thank all the patients, campers, and their families who participated and continue to participate in the study.

This study was funded through the Fanconi Anemia Research Fund (Principal Investigator, S.I. Wells), a CCHMC-sponsored Translational Research Initiative Grant (Principal Investigator, S.I. Wells), and by an NIH R01 HL108102 (Principal Investigator, M. Butsch Kovacic). REDCap was hosted at CCHMC and supported by the Center for Clinical and Translational Science and Training grant UL1-RR026314-01 NCRR/NIH.

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.