Grantee Research Project Results
2002 Progress Report: Molecular Epidemiology of Hypospadias
EPA Grant Number: R828599Title: Molecular Epidemiology of Hypospadias
Investigators: Manson, Jeanne M. , Devoto, Marcella , Carr, Michael
Current Investigators: Manson, Jeanne M. , Carr, Michael
Institution: Thomas Jefferson University
Current Institution: The Children's Hospital of Philadelphia
EPA Project Officer: Aja, Hayley
Project Period: July 1, 2002 through October 1, 2007
Project Period Covered by this Report: July 1, 2001 through October 1, 2002
Project Amount: $2,962,288
RFA: Genetic Susceptibility and Variability of Human Malformations (1999) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The objectives of this research are to characterize the genetic and environmental risk factors for hypospadias in the general population. Gene assays have been developed, investigators meetings have been held on a quarterly basis, assays for the SRD5A2 gene have been applied to buccal swab DNA, and quality assurance tasks and protocols have been conducted. There have been some shortfalls in subject recruitment during the second year of the project, which are addressed below. Efforts are being made to reduce the number of families lost to the study through "discards" and "no returns." If these families had been captured, the goal of recruiting 250 families would have been met. With the loss of these families to the study, a total of 207 have actually been enrolled.
Progress Summary:
In the first 22 months of this project a total of 100 case and 100 control families were enrolled. The case and control groups are well balanced in terms of race, ethnicity, and demographics. Potentially eligible families are first identified from a scheduling system (IDX) that gives the primary diagnosis of the infant and the date of the next visit to Pediatric Urology at Children's Hospital of Philadelphia (CHOP). Letters are sent out to potentially eligible families followed by a phone call, and interested families are approached during their clinic visit. Once informed consent is obtained, the parents complete questionnaires and buccal swabs are collected from the mother, father, and infant. If families have time constraints or the father is not available, questionnaires and buccal swabs are sent home with available family members and subsequently mailed. After the infant is examined in the Pediatric Urology clinic, medical records are reviewed to determine the final diagnosis of the infant, including primary and secondary conditions.
To date, a total of 262 families have been recruited into the study. Of these, a total of 7 families have withdrawn from the study after they provided consent. This has occurred when one parent has signed the consent form (usually the mother), while the other parent was not present at the time of enrollment (usually the father). A copy of the signed consent form is then sent home for the missing parent, along with a questionnaire for that parent and buccal swab brushes. Withdrawals typically have occurred because the missing parent did not wish to participate in the study. The concerns expressed have been related to confidentiality and participation in a genetic study. When a consented family withdraws, all data associated with the family are removed from the study and destroyed.
A total of 26 families have been "discarded" from the study because the proband did not meet the case/control definition for the study. Families are first identified from the IDX scheduling system according to age (<12 months), sex (male), and diagnosis. The diagnoses in IDX are provisional, particularly for families coming into Pediatric Urology for the first time. Final diagnosis of the infant's condition is made from a chart review, and if the infant has a condition incompatible for study enrollment from the chart review, the family is discarded from the study. The data from these discarded families are maintained in a separate file and will be used in future studies of urogenital anomalies following Institutional Review Board (IRB) approval.
A total of 22 families did not return questionnaires. These families have signed the consent form, contributed buccal swabs, but left the clinic setting before questionnaires were complete. These families are given a stamped envelope to return the questionnaires, and are followed up with phone calls and remailings of the questionnaires. After a minimum of 3 contacts to have the questionnaires returned, we remove these families from the study. None of these families have indicated any concerns with the study, and the likely cause is inconvenience. All data associated with these families are removed from the study and destroyed. We are offering an incentive of two movie theatre tickets for consented families who want to leave the clinic prior to completing the questionnaires. This was instituted in September 2002, and has been successful in convincing parents to remain in the clinic long enough to complete questionnaires.
There are a total of 104 case families and 103 control families enrolled in the study to date. Maternal questionnaires have been completed for all 207 families, and paternal questionnaires for 181 families. The father was not available for 23 families (11 percent total), and mailed paternal questionnaires are pending for 3 families. Medical record review is completed for 175 families, including 86 cases and 89 controls, and is pending for 32 families. Medical record review tends to lag behind collection of other data as the review is not undertaken until the infant has been examined and diagnosed for urogenital anomalies. Buccal swabs for the mother and infant have been obtained in duplicate for all 207 families, and from 181 fathers to date.
The primary and secondary conditions found in cases and controls from the medical record review revealed that of the 86 cases, 65 have uncomplicated hypospadias, and 21 have additional, secondary conditions of cryptorchidism or chordee. The 89 controls have a spectrum of primary conditions including hydronephrosis, vesicoureteral reflux (VUR), hydrocele, inguinal hernia, obstruction of ureteropelvic junction (OUPJ), absent kidney, and urinary tract infection (UTI).
The case and control groups are well balanced in terms of racial/ethnic groups, marital status, demographics (education, income level), and age at intake. There were differences in maternal reproductive history, with the control group having significantly more miscarriages and preexisting gynecologic conditions (primarily endometriosis and excessive menstrual bleeding) than the case group. There is a trend toward a higher prevalence of male relatives with urogenital anomalies and hypospadias in the case group than the control group but differences were not significant. Maternal obstetrical history during the index pregnancy also is similar between case and control groups with the exception of maternal complications during pregnancy. The case group had a higher prevalence of kidney/bladder infections during pregnancy than the control group.
To date, no environmental risk factors for hypospadias have been identified, although the number of subjects is too small for meaningful evaluation. There have been trends towards increased occupational and home exposures to pesticides, insecticides, and herbicides in case pregnancies, but differences have not always been significant and results have fluctuated as more families are recruited into the study.
Assays for the SRD5A2 gene are fully validated. An ongoing problem has been the quantity and quality of buccal swab DNA collected from parents and infants in the study. The mean concentration of buccal swab DNA collected is 28.5 µg/ml, for a total amount of 1.5 µg in 50 µl. Much of this DNA is bacterial, and does not amplify in polymerase chain reactions (PCR). We have developed assays with degenerate oligonucleotide primed PCR (DOP-PCR) to amplify this small quantity of human genomic DNA. This procedure has markedly expanded the amount of human template DNA available for examining single nucleotide polymorphisms in candidate genes. To date, the only polymorphism identified has been a V89L mutation in exon 1 of the SRD5A2 gene, which is known to reduce enzyme activity by approximately 60 percent. Evaluation of the entire expressed portion of the SRD5A2 gene (all exons and exon-intron boundaries) on the first 100 families (approximately 50 cases and 50 controls) is nearly complete, and it is anticipated these data will be available by the end of the year.
A certificate of confidentiality has been obtained from the National Institute of Health (NIH) for the project (National Institute of Environment and Health Sciences [NIEHS] 02-09). This certificate will greatly facilitate the protection of identity of research subjects from involuntary disclosure that could expose subjects or their children to adverse economic, legal, psychological, and social consequences.
The quantitative measurements being made in this study are gene assays. There are no chemical measurements in environmental media or body fluids. For the gene assays, buccal swabs are collected in duplicate from both parents and the infant at the time of the scheduled visit. Buccal swabs are precoded with unique, four digit, randomized-number codes, allowing DNA analysis to be conducted without knowledge of the name or case status of the infant. The interviewer at CHOP collects these samples, stores them in a refrigerator at 4°C, and transfers them on a weekly basis to Dr. Manson's laboratory. Upon receipt in the laboratory, buccal swabs are stored at 4°C and DNA extracted within a week of receipt. Once extracted, the DNA sample is stored at -80°C until gene analysis is conducted. DNA samples will be analyzed in batches of 96 specimens to minimize interassay variability. As an internal control and consistency check, 5 percent of samples from each batch will be rerun and genotypes will be validated on these samples.
Dr. Manson is responsible for oversight of design and maintenance of the database. Personal identifiers for subjects are kept in a computer file accessible to Dr. Manson and two designated non-laboratory staff. These random numbered identifiers are keyed to a study number (Family Identification Number). Data are entered into the database by study numbers only. Access to the electronic files is password protected and not accessible by the CHOP Information System staff. All files are maintained and safeguarded through daily back ups in peripheral drives not accessible to other staff. Paper copies of personal identifiers used to back up the computer system are maintained in a locked cabinet in Dr. Manson's office area. A management information system (MIS) has been developed to track subjects throughout their participation in the study. The study data management system (SDMS) has been developed to collect and prepare data for statistical analyses. Six data dictionaries are automatically generated by the software to provide primary documentation for all data elements associated with the study. The MIS and SDMS are implemented on a Pentium computer in SPSS software.
Dr. Manson has worked with programmer Kathleen Gibbs to define a set of standards for supporting the database in an Information Systems Quality Assurance Plan (ISQAP). The ISQAP activities have thus far focused on procedures for preventing data loss prior to and after data entry, ensuring the integrity of all database files, maintaining data security, and continuous quality improvement of all software written for data management. Quality assurance procedures address several key time points during data collection: subject selection, review and correction of data prior to data entry, and master file data integration. Periodic reports and error logs have been routinely generated for comparison with manually maintained records to ensure the MIS is functioning properly. All questionnaires and subject identifiers attached to biosamples are inspected for accuracy. Data are entered into a temporary data file (SPSS Version 3.0 Work Station data file) and keyed for double entry for quality assurance prior to integration into the main SDMS. Frequency, range, and logic checks are preprogrammed in the SPSS Data Entry subfiles so that errors are captured and identified at the time of electronic entry and subsequent double-key entry. Data are then imported into the SDMS and rechecked to ensure corruption did not occur during the integration process.
Programming was developed in SPSS for offsite uses so that replies to questions can be electronically logged into the temporary data files via a laptop computer at the time of interview or shortly thereafter. All (100 percent) questionnaire data are examined weekly to verify consistency with the hard copy data collection form.
Future Activities:
In the next year, it is anticipated that approximately 200 families will be recruited into the study. This estimate is based on recruiting approximately 5 families per week. Questionnaire data and medical record reviews will be collected and entered into the database. Data are evaluated for trends on a monthly basis. By the end of 2003, there should be approximately 4,000 families enrolled in the study out of the total of 800 families, and hopefully there should be sufficient data to begin identifying genetic and environmental risk factors.
Evaluation of study samples for polymorphisms in the SRD5A2 gene will continue next year. DNA from approximately 50 case and 50 control infants is being assayed for any polymorphism in the entire expressed sequence of the gene (exons 1-5). This evaluation will be completed by the end of 2002, and will allow evaluation of 100 alleles from each group. The allelic frequency of polymorphisms will be calculated. Decisions will then be made on the most productive analytic strategy to pursue once results from these 100 infants are available. One scenario is that a broad spectrum of variants will be found in a large proportion of case infants in each exon of the SRD5A2 gene. Under these circumstances, any PCR product with a homozygous mutant or heterozygous migration pattern on partially denaturing high performance liquid chromatography (HPLC) will be sequenced from remaining cases (350) and all control infants (350). Another scenario is that a narrow spectrum of variants will be found in a large proportion of the 50 cases restricted to single sites in the gene. If only a few polymorphisms are found in this initial sample, a PCR-based assay can be developed to amplify only those specific polymorphisms.
The PCR conditions for amplifying the 11 exons of the HSD17B3 gene have been established. It is anticipated that by the end of 2003, study samples will be evaluated for polymorphisms in this gene following procedures described above for the SRD5A2 gene.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 39 publications | 7 publications in selected types | All 7 journal articles |
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Jordan B, Charest A, Dowd JF, Blumenstiel JP, Yeh R-F, Osman A, Housman DE, Landers JE. Genome complexity reduction for SNP genotyping analysis. Proceedings of the National Academy of Sciences of the United States of America 2002;99(5):2942-2947. |
R828599 (2002) |
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Kittler R, Stoneking M, Kayser M. A whole genome amplificaton method to generate long fragments from low quantities of genomic DNA. Analytical Biochemistry 2002;300(2):237-244. |
R828599 (2002) |
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Manson JM, Carr MC. Molecular epidemiology of hypospadias: review of genetic and environmental risk factors. Birth Defects Research Part A:Clinical and Molecular Teratology 2003;67(10):825-836. |
R828599 (2002) R828599 (2003) R828599 (2004) R828599 (2005) R828599 (2006) R828599 (Final) |
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Supplemental Keywords:
infant, epidemiology, genetic, urogenital, hypospadias., RFA, Scientific Discipline, Health, Environmental Chemistry, Health Risk Assessment, Susceptibility/Sensitive Population/Genetic Susceptibility, Biochemistry, Children's Health, genetic susceptability, Biology, male infants, prenatal exposure, infants, endocrine disruptors, Human Health Risk Assessment, hyposadias, children, assessment of exposure, children's vulnerablity, environmental toxicant, epidemeology, human susceptibility, pregnancy, developmental disorders, maternal exposure, toxicsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
- Final Report
- 2007
- 2006 Progress Report
- 2005 Progress Report
- 2004 Progress Report
- 2003 Progress Report
- Original Abstract
7 journal articles for this project