We designed the NDSP as a national, multisite, population-based, case-control study with headquarters in the Department of Human Genetics at Emory University in Atlanta, Georgia. contains a summary of the study design. We defined cases and controls on two levels: eligibility and recruitability. Eligibility described the ideal set of cases and controls that would fit the study criteria. For example, eligible cases would all be live births with standard trisomy 21 or mosaic trisomy 21. Recruitability then placed practical limits on which eligible cases and controls could be recruited. For example, because of limited resources, we could recruit only mothers who were fluent in either English or Spanish. Further, case families whose live-born child died or was put up for adoption were not recruitable because a biological sample could not be obtained from the case child. Even though our protocol did not require biological samples from controls, for the sake of comparability we extended these latter criteria to controls.
National Down Syndrome Project design
Each site was responsible for ascertaining, contacting, and enrolling its own cases and controls, administering parental questionnaires, and obtaining biological samples. Study personnel made intensive efforts to contact all eligible and recruitable families. They used commercial tracking services and street tracking where resources were available. Emory provided training and guidelines to each site including lay explanations of the components of the study and lists of frequently asked questions that study personnel might encounter when recruiting families. The NDSP produced all written materials in English and Spanish, and bilingual personnel were available to each site to communicate with Spanish-speaking families.
Sites initially contacted families with a letter of introduction explaining the aims of the NDSP. In a follow-up telephone call, study personnel invited the families to participate. At all sites the mother was considered the “gatekeeper.” If she agreed to the study, the recruiter obtained her informed consent and administered the maternal questionnaire by telephone. The interviewer then made arrangements with each mother in the case group to obtain biological samples from her, her child, and the father of the child (if available). Sites reimbursed participating parents a nominal amount for their time and effort. The reimbursement procedure and amount varied based on site-specific Institutional Review Board (IRB) regulations. Reimbursement for completing the questionnaire was usually $10 per parent for both case and control groups. Case families were also reimbursed for providing biological samples. The amounts varied by site and ranged from $20 to $40 for the nuclear family (child, mother, and father). The IRB at each site typically dictated whether reimbursement was to be provided with the introductory letter or after the questionnaires and biological sample collection were completed.
The combined annual birth population of all six sites was approximately 472,500 (). Before beginning active recruitment for the NDSP, each site obtained the necessary IRB approval. Recruitment for the NDSP occurred from 2000 to 2004 and the ascertainment periods varied by site (range: 2.5–3.75 years). Each site ascertained cases and selected controls from a defined geographical area that ranged from three counties to the entire state. All participating sites were part of the National Birth Defects Prevention Network (NBDPN); a state-by-state description of their surveillance systems is available elsewhere.12
The geographical area of California included in the NDSP was not the same as that included in the NBDPN.
National Down Syndrome Project sites
Case identification/control selection
Live born infants with standard trisomy 21 (47,XX, +21 or 47,XY, +21) or mosaic trisomy 21 were eligible. Additional chromosome abnormalities or variations did not disqualify an infant from being identified as a case. Infants with DS due to a translocation involving chromosome 21 were not eligible.
NDSP sites selected infants for the control group randomly from among all infants born in the same study period and geographic area but without DS or other major birth defects (). Sites used either birth certificate data or hospital records for control selection (). We did not match on maternal age because one of the primary aims of the study was to investigate the relationship between meiotic nondisjunction and the age of the mother. Where appropriate, we will control for maternal age in our analyses. Major birth defects that would exclude an infant as a control included those registered by the states as well as those eligible for the National Birth Defects Prevention Study (NBDPS) being conducted separately by CDC at all sites. Details of the state surveillance systems and the NBDPS are available in other publications.12,13
For the purposes of analyzing responses to the maternal questionnaire, our goal was to enroll an equal number of infants as cases and controls. Because our previous experience indicated a somewhat lower participation rate for control families, each site used its number of expected cases as a reference point and adjusted upward the number of controls to be identified.
Paternal cases and controls
Our previous population-based studies had determined that at least 90% of the cases of standard trisomy 21 are due to an error in the egg and less than 10% are the result of a similar meiotic error during spermatogenesis.10
Because the overwhelming majority of cases are maternal in origin, we designed a protocol by which we could efficiently collect questionnaire data from fathers of paternal cases while minimizing the number of fathers interviewed where the case was maternal in origin. Furthermore, the protocol had to ensure that the parental origin of the chromosome error was not revealed to the participating family. Once biological samples were collected from the family unit, molecular studies were conducted to determine parental origin of the chromosome error. If the error was paternal, and the father was available, Emory asked the site to recontact and interview the father. In addition, on a periodic basis, the sites were contacted to interview a father of a maternal case. At no time were the sites informed of the parental origin of any case. This ensured that parental origin was not released to the family.
When determining the number needed in the control group for the upcoming year, each site specified a random subset in which the fathers would also be interviewed during the initial contact period. Because we anticipated so few cases would be of paternal origin, we wanted to achieve a 2:1 ratio of controls to cases for data analysis. Our previous experience had been that, of the control mothers who participated, only about half of those fathers also agreed to the study. Therefore, each site adjusted accordingly the number of control fathers to be interviewed.
We developed parental questionnaires () based on ten years of experience with similar survey instruments in the ADSP. To ensure that personnel at all sites were administering the questionnaire accurately and uniformly, we implemented several quality control measures. First, Emory personnel prepared detailed annotations for each question in both maternal and paternal questionnaires. They required interviewers to review the annotations, become familiar with the questionnaires, and pass a test that consisted of satisfactorily administering a questionnaire to Emory personnel by telephone and submitting their completed test questionnaires for review. We found these tests to be crucial in identifying and correcting interviewer errors. In addition, interviewers from all sites attended annual project meetings to review and resolve discrepancies in recruitment and interviewing procedures.
National Down Syndrome Project questionnaires—topics
Trained interviewers at each site administered the questionnaires by telephone or, in rare instances, in-person if the parents had no phone. The average length of time from birth of the index child until administration of the maternal questionnaire varied by site (). Sites mailed each completed questionnaire (excluding personal identifiers) to Emory soon after completion. Emory reviewed the questionnaire and contacted the site if deficiencies were noted. In order to document responses that mothers offered to specific questions about their reproductive histories, interviewers requested written permission from mothers in both the case and control groups to obtain pertinent medical records. Upon receipt of the signed medical record release form, the recruiter requested the appropriate records and forwarded the medical information to Emory after removing all personal identifiers.
National Down Syndrome Project: Selected characteristics of participating mothers and mothers in the birth population
Case infant medical records
The NDSP study design included a plan to link medical information about case infants to both the questionnaire and the molecular data. Linking with parental questionnaires will enable us to explore a number of important topics such as the occurrence of heart or gastrointestinal defects in relation to factors such as race, maternal age, family history, and environmental exposures. Similarly, a link to the molecular data will facilitate our search for genes important in the DS phenotype.
Trained personnel at each site abstracted records from birth hospitals and tertiary facilities. The protocol required documentation of karyotypes for each case to ensure that the NDSP included only infants with standard trisomy 21 or mosaic trisomy 21 and excluded translocations. In addition, we made every effort to obtain information from the most definitive procedures available such as echocardiograms, cardiac catherizations, or operative reports for heart defects and surgical summaries for GI abnormalities. Sites recorded this information on forms designed for the purpose and sent the forms to Emory for review by a single, clinically-trained investigator prior to data entry.
Each site was responsible for obtaining biological samples on case infants and their parents. When the father was not available or not willing to participate, sites collected samples on the mother and child. The latter situation was not optimal, however, because the molecular analyses were less informative for origin of the extra chromosome 21 and recombination patterns.
The Georgia site obtained blood samples and used aliquots of these samples to extract DNA and establish lymphoblastoid cell lines. Study personnel trained in phlebotomy scheduled home visits to obtain written consent and draw the parental blood samples. They usually obtained the infant sample when blood was being drawn for clinical purposes such as thyroid function or pre-operative tests. Although this approach could mean waiting several weeks or months for the sample, it appealed to parents and was undoubtedly one of the main reasons for the high case participation rate at the Georgia site.
Because geographic areas were larger at the other five NDSP sites, the expense of blood collection was outside NIH budgetary constraints, and we were limited to collecting buccal samples for DNA. Each site mailed bar-coded buccal cell collection kits, consent forms, and instructions to participating families. Parents collected the cheek cell samples on themselves and their child using four buccal brushes per individual and returned the kits by regular mail. Sites stored the returned kits in freezers, batched them, and mailed them in cold-packs to Emory for DNA extraction and analysis.
Emory laboratory staff extracted DNA from blood or buccal samples for genotyping chromosome 21-specific polymorphic markers. We chose markers based on their high degree of heterozygosity and placement on chromosome 21 (). Later in the study when whole genome amplification became a potential, we added the step of storing an aliquot of the DNA sample prior to analyses.
Chromosome 21 genetic markers and their physical location along 21q. Marker positions were defined based on information from public databases.
We examined the genotyping data to detect Mendelian inconsistencies, non-paternity, and genotyping errors. Once these were resolved, we used an algorithm to identify origin of the nondisjunction error.
Algorithm for determining origin of the extra chromosome 21
In families for which we had samples from the child and both parents, we required that at least two chromosome 21 markers be informative to assign parent of origin of the extra chromosome. Once parent of origin was established, we used pericentromeric markers to determine the type of nondisjunction error (i.e., MI, MII, PI, PII, or mitotic). To do this, we used the closest informative marker within the predefined pericentromeric marker set (). If parental heterozygosity was retained in the centromeric region in the trisomic offspring (“non-reduction”) (-i), we concluded that the error occurred during MI. If parental heterozygosity of the centromeric marker was reduced to homozygosity (“reduction”) while heterozygosity of other, non-centromeric markers was retained, we declared the error to be meiosis II-type (-ii). We considered the error mitotic if the informative markers were reduced to homozygosity along the entire length of the chromosome (-iii).
Figure 3 Examples of scenarios that define the origin of the nondisjunction error. Hypothetical genotypes of three ordered markers are shown. The most centromeric marker is noted by an arrow. Reduction (R) or no reduction (N) to homozygosity for each marker in (more ...)
If we received DNA from only the mother and child, we considered the error maternal in origin if there were more than eight markers consistent with a maternal error (-i). We considered the error paternal if at least two markers were inconsistent with a maternal error (-ii).
Figure 4 Examples of scenarios that define the origin of the nondisjunction error. Hypothetical genotypes of three ordered markers are shown. The most centromeric marker is noted by an arrow. Reduction (R) or no reduction (N) to homozygosity for each marker in (more ...)
In order to successfully “pass” the algorithm, the family could not have any discrepancies among markers that defined parental origin. A subset of samples failed to “pass” the algorithm due to a lack of DNA or genotype inconsistencies. In these situations, we requested a second sample. When non-paternity was suspected, we excluded the paternal sample from the analysis and repeated the algorithm using only data from the mother and child (-i, -ii).
In addition to establishing the origin of the extra chromosome 21, we determined the recombination profile along the long arm of chromosome 21 based on the non-reduced/reduced status of each informative marker. A change of status from reduction to non-reduction (or vice versa) between adjacent informative markers indicated a recombination event (e.g., -i, -ii).
Data management and security
For correspondence and data transfer between sites including questionnaires, samples, and clinical information, we used only ID numbers to identify individual participants. For Emory to monitor participation rates on an ongoing basis, the remote sites sent bimonthly electronic progress reports to Emory. NDSP personnel at Emory collected the hardcopy questionnaires and clinical data forms from all sites, sent these to a third party company for double entry and cross-checking, then uploaded the data into our primary data repository along with genotyping results from the Emory laboratory. The Emory site applied security at both the database and application levels to properly protect HIPAA-regulated data.