RFI-NHLBI-98-02

HEMOCHROMATOSIS STUDY--
FIELD CENTER

RFI-NHLBI-98-02

  1. Project Information

    1. A general description of the required objectives and desired results

      The Genetic Epidemiology Scientific Research Group, Epidemiology and Biometry Program, Division of Epidemiology and Clinical Applications, NHLBI, in conjunction with the Blood Diseases Program, Division of Blood Diseases and Resources, NHLBI, and the Ethical, Legal and Social Implications Research Program, Division of Extramural Research, NHGRI, propose to initiate an epidemiologic study of the prevalence and genetic and environmental determinants and potential clinical, personal, and societal impact of iron overload and hereditary hemochromatosis, in a multi-center, multiethnic, primary care-based sample of 100,000 adults. This information will be used to determine the feasibility and potential individual and public health benefits and risks of primary care-based screening and intervention for iron overload and hereditary hemochromatosis. The specific objectives of the study are to:

      1. Determine the prevalence in a primary care population, by race/ethnicity, of: 1) iron overload, defined as sustained elevation of transferrin saturation, and hereditary hemochromatosis; 2) demonstrable clinical and pathological abnormalities related to iron overload and hereditary hemochromatosis; and 3) genetic variants related to iron overload and hereditary hemochromatosis including the recently identified HFE C282Y and H63D genotypes and other, as yet unidentified, variants.

      2. Identify risk factors influencing the phenotypic expression of iron overload and hereditary hemochromatosis in regard to demonstrable clinical and pathological abnormalities, and examine interactions between risk factors to determine the relationship between genotype and phenotype. Risk factors would include genetic factors, such as specific hemochromatosis genotype or thalassemia trait, as well as environmental or non-genetic factors such as gender, age, alcohol intake or hepatitis C virus.

      3. Examine ethical, legal and social issues related to the possibility of implementation of primary care-based screening for iron overload and hereditary hemochromatosis, including identification of appropriate health care delivery models and potential personal, societal, or family-related impact of and barriers to population-based screening and genetic testing.

      4. Estimate the heritability of iron overload and hemochromatosis, and initiate linkage studies to identify main effect and modifier genetic variants associated with iron overload and hemochromatosis.

      The project duration is planned for five years.

    2. Background information helpful to a clear understanding of the requirements and how they evolved.

      Hereditary iron overload, or hemochromatosis, is a common inherited disorder among Caucasians, though it is mistakenly believed by many to be quite rare. The disease is insidious in onset, and many or even most individuals diagnosed with this disorder are not identified until advanced organ damage is present. However, in the absence of anemia, which can be caused by tissue damage from iron in late stages of the disease, it is relatively easy to treat the disorder by removing the excess iron through repeated phlebotomy. Evidence suggests that early diagnosis and treatment can prevent disease manifestations and enable normal life expectancy. The discovery of the HFE C282Y and H63D variants in the HLA gene region on chromosome 6 provides an opportunity for early and rapid genetic identification of individuals at risk for development of hereditary hemochromatosis. Much remains to be learned about the penetrance and expression of these alleles, including their relevance to the full spectrum of clinical disease. To date, the HFE alleles appear predominantly associated with disease mainly in populations of Caucasian descent. While 80-90% of Caucasian hemochromatosis patients have HFE abnormalities, there are hetero- and homozygotes that do not manifest any evidence of disease, or manifest disease at different ages and with different outcomes, implying the existence of other genetic or environmental factors. Similarly, not all hemochromatosis patients have HFE abnormalities. Other genes yet to be discovered are also likely to be involved in pathogenesis of iron overload and familial hemochromatosis in non-Caucasian populations as well. This project is intended to examine the genetic and environmental determinants and correlates of iron overload and hereditary hemochromatosis in diverse populations.

      Increases in body iron may be due to increased absorption hemochromatosis), increased oral intake of non-therapeutic iron, unneeded iron therapy, or multiple blood transfusions in the absence of bleeding. The excess iron is deposited in body tissues, and can reach toxic levels leading to organ damage. The toxicity can affect most tissues and organs, but particularly the liver, causing cirrhosis; the endocrine system, causing diabetes, hypogonadism, and sometimes hypoparathyroidism; and the heart, causing arrhythmias and cardiomyopathy.

      Iron overload and hereditary hemochromatosis have not been as extensively studied in racial/ethnic groups other than Caucasians. The toxicity of excess iron in non-Caucasians appears to be similar to that in Caucasians, but the prevalence of iron overload is unknown and while a genetic contribution to that overload is suspected it has not been proven in all groups. It has long been assumed that iron storage disease in Bantu peoples of Africa is due to increased iron absorption from beer brewed in iron pots, but more recent information suggests there is also a hereditary component to that accumulation of iron. African iron overload does not appear to be due to HFE abnormalities, nor to other genes in the HLA region at all. Iron overload has been reported in Asian populations, but the frequency and genetic effects (if any) are not known. In some studies where HFE variants have been found in non-Caucasians, additional genetic testing has suggested that Caucasian admixture may have been involved. Hispanic-Americans appear to have a frequency of iron overload similar to non-Hispanic Caucasians, although further study of the genetic and environmental correlates are warranted. There has been almost no study of iron overload and hereditary hemochromatosis in Native American populations.

      Hemochromatosis may be suitable for detection and intervention through primary care or population-based screening strategies because: 1) it is relatively common; 2) it is asymptomatic in its early stages; 3) screening methods are reliable; 4) standard diagnostic methods are widely available in developed countries and relatively inexpensive; 5) it is easily treatable; and 6) if untreated, the subsequent burden of morbidity and mortality is substantial. The feasibility and benefits of such programs remain to be assessed, however, since the prevalence of the disorder and the factors related to its phenotypic expression (such as the optimal age for reliable detection and effective intervention) are unknown. Other questions needing to be addressed include public acceptability of screening and testing; sensitivity and specificity of the screening methods, particularly in non-Caucasians; optimal timing and setting of screening and testing; as well as the benefits and costs and/or other burdens associated with screening and testing.

      A major objective of the proposed project is to gather information needed to develop recommendations regarding possible population-based screening for hemochromatosis. Estimating the burden of preventable illness from unrecognized hemochromatosis is the most important of these needs. Comparing the relative value of diagnosis and screening by genotype vs phenotype is also important. In particular, differences by racial/ethnic group, age and other characteristics will need to be examined. Some of these issues, such as appropriate thresholds for transferrin saturation screening, may be resolved during the proposed study's planning phase, while others will constitute key research questions to be addressed by the study itself.

  2. Work Statement

    Introduction

    The Iron Overload and Hereditary Hemochromatosis Study is a multi-center study of the prevalence and genetic and environmental determinants of iron overload and hereditary hemochromatosis in a diverse and representative primary care-based sample of men and women aged 25 and older. One hundred thousand participants undergoing routine screening or testing involving a blood draw will be recruited from five to seven Field Centers and screened for transferrin saturation levels. A repeat fasting transferrin saturation screen will be used to identify potential 'case' participants with sustained elevated transferrin saturation levels and matched random 'control' participants with non-elevated transferrin saturation levels.

    In order to obtain data on the prevalence of genetic factors in a primary care population, a random subgroup of approximately 20-40% of the 100,000 screenees will be genotyped for known variants, such as HFE C282Y and H63D, related to iron metabolism and overload. The panel of genotypes to be assayed will reflect the state of knowledge at the time this phase of the study is conducted. In particular, any newly discovered variants related to iron overload and hemochromatosis in non-Caucasian populations, such as for 'African iron overload', will be included. The results of the genotyping will not directly impact the selection of case and control participants; case/control selection will be based only on the transferrin saturation screen results. It is expected that most HFE genotype positive persons will have sustained elevations of transferrin saturation and be included as case participants. Genotype positive persons with non-elevated transferrin saturation levels who are not randomly selected as controls will constitute a third group and undergo the same intensive studies as cases and controls.

    A random sample of the individuals being recruited to participate in the genotyping subgroup will be surveyed to determine their knowledge and attitudes about, interest in, and support for such screening programs. Both qualitative and quantitative measures may be employed. Efforts will be made to ascertain reasons for refusal and related information from those who decline participation.

    Following these transferrin and random subgroup genotyping screens, a comprehensive clinical examination will be conducted in the sustained elevated transferrin saturation potential case participants, the genotype-positive non-elevated participants, and the non-elevated control participants to assess iron stores, distinguish between primary and secondary causes of iron overload and to examine the associated hepatic, endocrinologic, hematologic and cardiovascular disease correlates and sequelae of hemochromatosis. A detailed family and medical history will be obtained. Examination participants not previously genotyped will undergo genotyping, with a panel of genotypes as described above, for use in association analyses. The participants will receive counseling on their results. The examination will also include an extended ELSI assessment of issues related to genetic screening and testing and diagnosis of disease. Data will be collected on the participants' experience with screening, their understanding and interpretation of their results, and on the impact this information is having on their own lives as well as those of their families. Specific components of the comprehensive clinical examination will be determined during protocol development. Follow-up ELSI assessments will examine issues such as impact of the screening program on relationships with family members, and any experiences with stigmatization and discrimination.

    A family study, using comprehensive clinical examinees as probands, will seek to identify modifier genetic variants related to the expression of iron overload and hereditary hemochromatosis disorders via genome scanning and assessment of linkage. Identification of new genetic variants, particularly in minorities, is also of interest, but it is possible this study will not achieve sufficient power to do so. Proposed efforts to improve the power, such as combining data from other studies, will be considered. The family study ELSI assessment will examine family members' experiences with the screening program, the impact of this information on their lives and relationships, and any experiences with stigmatization and discrimination.

    A repository of blood specimens will be established to permit additional studies of genetic and environmental factors relating to iron overload. This will require careful attention to the details of informed consent. For some later studies, the specimens may be anonymized.

    The study will involve five to seven Field Centers, a Coordinating Center (which will subcontract for any necessary Reading Centers such as an ECG Reading Center), and a Central Laboratory. Principal Investigators from each of these seven to nine Centers plus the NHLBI Project Officer form the Steering Committee. A Data Safety and Monitoring Board (DSMB) will be appointed by NHLBI to oversee the project and make recommendations on various aspects (e.g., protocol approval, participant safety, project activation, and later milestones). The DSMB is expected to meet initially to review the "final" protocol, to meet routinely (annually) during the study, and to meet on an ad hoc basis when needed for major protocol revisions or for the evaluation of unexpected results.

    Time lines for the study are provided in Table 1 and Figure 1, and a list of probable exam components is shown in Table 2. Protocol planning, informed consent form design, OMB clearance, training and pilot testing will occupy the first twelve months of the study. The initial transferrin saturation screen will then be conducted over a two year period. The repeat fasting transferrin saturation screen will follow shortly, preferably within one month of the initial screen of each participant. Appropriate threshold values for each of the screens should be proposed, with discussion of specificity and sensitivity issues and expected numbers of participants meeting these criteria. It should be recognized that the sustained elevated transferrin saturation group will be much smaller in number than the initial group of 100,000 screenees. As these sustained elevated transferrin saturation participants and non-elevated control participants are identified, scheduling for the comprehensive clinical examination will begin. These examinations will also take place over a two year period, overlapping the transferrin saturation screen period. Similarly, as examinees with hereditary hemochromatosis are identified, recruitment of relatives for the family study will begin. During the last year of the study, follow-up of comprehensive clinical examinees will be performed. Depending on when the examination is performed, the follow-up interval will be between one and three years. Concurrently, final data analysis and study close-out will be completed, although initial data analysis is expected to begin much earlier, using data from the initial transferrin saturation screen and random subgroup study.

    The initial 100,000 screenees will include roughly equal numbers of men and women aged 25 and older. Individuals requiring repeated blood transfusions for treatment of other conditions will be excluded. Since iron accumulation increases with age, this broad age span will lead to inclusion of participants with a wide range of iron overload states and related sequelae to be studied. Screening of children and adolescents, however, is not expected to be productive because iron overload is generally not detectable until at least the third or fourth decades of life. Inclusion of slightly younger adults, that is those 25 and older, will enable analysis of gene and environmental interactions related to the penetrance of HFE and other hemochromatosis-related gene expression during the age span when iron overload is developing but before such interactions may be confounded by hemochromatosis-related organ damage. The initial screenees should represent a range of racial and ethnic groups, with the goal of enrolling approximately 40-50% minority participants. To enable comparisons within as well as across Field Centers, each Center's sample is expected to enroll from two or more racial/ethnic groups, one of which may be non-Hispanic Caucasians. For Field Centers with two racial/ethnic groups in the study, each group must comprise 20% or more of that Center's study population. For Field Centers with three or more racial/ethnic groups, at least two of the groups must each comprise 20% or more of the study population at the Center. Selection of Field Centers will be partly governed by the need to achieve the overall racial/ethnic and gender targets for the study as a whole.

    Clinical sites will be expected to assess the clinical, personal, and societal impact of the screening program. During planning, a common approach to this assessment will be developed and implemented. The development of guidelines for informed consent and the protection of individual rights or prerogatives is an important component of this study. All clinical sites will thus be asked to address issues of consent, protection against discrimination, confidentiality, etc.

    Public policy implications of screening programs that identify risk or disease states, in which much about the natural history of the disease remains unknown, but for which existing medical interventions appear to substantially reduce risk or morbid states, are of interest, but may be beyond the scope of the main study. Applicants are thus encouraged to propose and participate in substudies and ancillary studies, as described in task 3.e. below, to address the broader ethical, economic and health policy issues related to the possible implementation of such screening programs, or other issues related to iron overload and hemochromatosis.

    Ethical considerations mandate that subjects found to have evidence of iron overload or deficiency be treated to prevent or attempt to reverse clinical disease. Accordingly, offerors must be prepared to refer those patients to appropriate sources of standard clinical care for follow-up and/or treatment as indicated, which will not be supported by this research program. Although not a primary goal of the program, differences in the response to treatment by genotypes will be sought through follow-up procedures. Study-wide guidelines for treatment, including assessment of family members as guidelines warrant, will be developed and recommended to care providers of cases detected through the study. The final, detailed protocol will be developed and approved by a Steering Committee-appointed subcommittee, and will be reviewed and approved by the Data and Safety Monitoring Board and NHLBI/NHGRI staff.



Figure 1. Time line for the Iron Overload and Hereditary Hemochromatosis Study

|..........Year 1.........|

..........Year 2.........|

..........Year 3.........|

..........Year 4.........|

..........Year 5.........|

Protocol Development |-----------------------|
OMB Clearance |-----------|
Screening Exam |------------------------ ------------------------|
Random Sample Genotyping* |------------------------ ------------------------|
Case/Control Intensive Exam* |----------- ------------------------- ------------------------|
Family Study* |------------------------ ------------------------|
Case Follow-Up* |---------- ------------------------- ------------------------- ------------------------|
Analyses |--------- ------------------------- ------------------------- ------------------------- ------------------------|
Publications





|------------------------

-------------------------

------------------------|

|..........Year 1.........| ..........Year 2.........| ..........Year 3.........| ..........Year 4.........| ..........Year 5.........|

*includes Ethical, Legal, and Social Implications (ELSI) assessment



Table 2. Examination Components

Data to be collected is approximated by the following list of components, which may be modified during protocol development, for the approximate following number of participants (across all Field Centers):

  1. Initial Transferrin Saturation Screen (N=approximately 100,000):
    1. Serum collection

  2. Random Subgroup:
    1. Whole blood for DNA extraction (N=approximately 20,000-40,000)
    2. Initial Ethical, Legal, and Social Implications (ELSI) (N=approximately 2,000-4,000)

  3. Repeat Transferrin Saturation Screen (N=approximately 2,500 initial transferrin saturation elevated participants, and 1,000 non-elevated transferrin saturation participants)
    1. Serum collection

  4. Comprehensive Clinical Examination (N=approximately 750 sustained elevated transferrin saturation participants and 1,000 sustained non-elevated transferrin saturation participants)
    1. Extended ELSI assessment
    2. Dietary questionnaire
    3. Demographic, medical history, and medication use uestionnaires
    4. Fasting whole blood, serum, plasma, DNA and lymphocyte collection
    5. Anthropometry
    6. Resting blood pressure
    7. Assessment of liver disease
    8. Assessment of blood disorders
    9. Assessment of diabetes
    10. Assessment of arthropathies and arthritis
    11. Assessment of heart failure, and arrhythmias

  5. Family Study (N=approximately 2,000 relatives of comprehensive clinical examinee cases)
    1. Fasting whole blood, serum, plasma, DNA and lymphocyte collection
    2. Components of comprehensive clinical examination as above
    3. ELSI assessment

  6. Follow-up for ELSI, morbidity and mortality:
    1. Follow-up ELSI assessment
    2. Dates of deaths and hospitalizations
    3. Interview data from participants and relatives and other informants
    4. Hospital discharge diagnosis and ICD9 codes



Figure 2 Iron Overload and Hemochromatosis Study Design.


                                              ------------
                                              |          |
                                              | 100,000* |
                                              |          |
                                              ------------
1.  Initial Transferrin                             |
Saturation Screen (TS1)             ---------------(1)--------------
[Random Subgroup**;                 |                              |
Genotyping Initial            ------------                   -------------
ELSI***]                      |          |                   |           |
                              |   2,500  |                   |   97,500  |
2.  Repeat Transferrin        |   TS1+   |                   |    TS1-   |
Saturation Screen (TS2)       ------------                   -------------
                                   |                               |
                            ------(2)------                       (2)
                            |             |                        |
                     -------------- --------------          ---------------
                     |            | |            |          |Random Sample|
                     |   1,750    | |     750    |          |    1,000    |
                     | TS1+  TS2- | | TS1+  TS2+ |          |  TS1-  TS2- |
                     -------------- --------------          ---------------
3.  Clinical                              |                        |
Assessment,                        ------(3)------                (3)
Extended ELSI***,                  |             |                 |
Genotyping                    ------------  --------------  ---------------
                              |   250    |  |   500      |  |    1,000    |
                              | 2o Iron  |  |1oIron Over-|  |  TS1-  TS2- | 
                              | Overload |  | load CASES |  |   CONTROLS  |
                              ------------  --------------  ---------------
4.  Family Study                                 |
(4 or more members                              (4)
per case)                                        |
                                           -------------
                                           |   2000    |
                                           |  Family   |
                                           |  Members  |
                                           -------------

* All numbers after the initial 100,000 are estimates and may change during the protocol development or as cases are identified during the course of the study.

** During step 1, a random subgroup of 20-40% participants will have additional blood drawn for candidate gene testing concurrent with transferrin saturation assays. Any genotype-positive (for example, HFE C282Y/C282Y or HFE C282Y/H63D) individuals not otherwise selected for steps 2-4 will be invited to participate in these steps (although not shown above).

***Ethical, Legal, and Social Implications assessment. An initial ELSI assessment will be conducted in a random subgroup at the beginning of step 1. All step 3, and a subset of step 4 participants will undergo an extended ELSI assessment. A follow-up assessment will be conducted in the hemochromatosis cases (not shown above).


DRAFT

    STATEMENT OF WORK--FIELD CENTER

    [Note: Throughout this statement of work, the terms "Contractor" and "Field Center" are used interchangeably.]

    Independently and not as an agent of the Government, the Contractor shall furnish all the necessary services, qualified personnel, material, equipment, and facilities not otherwise provided by the Government, as needed to perform the Statement of Work below.

    Throughout the period of performance, the Contractor shall provide appropriate senior personnel with expertise in the epidemiology of iron overload and hereditary hemochromatosis; clinical hepatic, endocrinologic, hematologic and cardiovascular disease; genetic epidemiology; genetic counseling; the ethical, legal and social implications (ELSI) of genetic screening and testing; laboratory measurements; and clinical epidemiology study management to:

      1. Take responsibility in particular scientific areas for protocol development, including leadership and participation in subcommittees;

      2. Recruit and examine a representative sample of primary care patients, men and women aged 25 and older, at each stage of the planned program: transferrin saturation screens, ELSI assessments, a comprehensive clinical examination and family study;

      3. Participate in meetings and activities of committees, such as the Steering Committee, Publications Committee, Laboratory Committee, and Ethical, Legal and Social (ELSI) Committee, and other subcommittees, and present status reports to the NHLBI-appointed Data Safety and Monitoring Board (DSMB), as appropriate;

      4. Be involved and closely familiar with data collection and quality assurance activities at own center;

      5. Maintain confidentiality and security of all forms and data files;

      6. Work cooperatively with all study centers, which include the other Field Centers, Coordinating Center, Central Laboratory, and the NHLBI Project Office staff in all relevant aspects of study execution;

      7. Participate actively in data analysis and publication.

    1. SPECIFIC REQUIREMENTS AND TASKS

      The Field Center contract will be awarded for a five year duration with Phases I, II, III, and IV described below:

      1. During Phase I (September 1, 1999 through August 31, 2000), the Contractor shall:

      [Note: See time lines and flowchart in Table 1 and Figures 1 and 2 for overview of phases, tasks and study design. A list of examination components is in Table 2.]

        1. Provide representation for Steering Committee meetings, DSMB meetings, and for subcommittee meetings as appropriate.

      [Note: For planning purposes, assume the Offeror will be represented on the Steering Committee and three subcommittees and that Steering Committee meetings will take place in Bethesda, Maryland. Assume there will be 4 two-day Steering Committee meetings during the first year, 3 during the second year, and 2 each year thereafter. DSMB and Subcommittee meetings will generally take place in conjunction with Steering Committee meetings, but in the first year assume one additional meeting may need to take place separately.]

        1. Actively participate in development of study protocol and forms including the assumption of lead roles for development of particular aspects of the protocol;

      [Note: It is anticipated that a total of 100,000 participants will be recruited among five to seven Field Centers for an initial transferrin saturation screen. For purposes of the proposal, assume that 20,000 participants will be recruited by each of five Field Centers. Concurrent with this screen, a genotyping screen is expected to be performed on a 20-40% random subgroup, and an initial ELSI assessment on a 2-4% random subgroup. Following the initial screen, approximately 3,600 participants are expected to participate in a repeat transferrin saturation screen, with approximately 1,800 participants continuing on to a comprehensive clinical examination, and approximately 560 participants and 2240 of their family members are expected to participate in a family study. A morbidity, mortality and ELSI follow up of the comprehensive clinical examinees, for up to two years, is also planned. An additional 2,000 participants will participate in an ELSI-related substudy comparing acceptability of genotype vs phenotype screening methods (with approximately half of the participants in each of the two groups).]

        1. In conjunction with the other Field Centers and Coordinating Center, develop a core informed consent document to address but not necessarily be limited to specimen collection and disposition, the public release of study data without identifiers, and apply for the Certificate of Confidentiality for DHHS Funded Studies;

      [Note: While a core informed consent form will be developed for use across Field Centers, it is recognized that local IRBs may require modifications for a particular Field Center.]

        1. In conjunction with the other Field Centers and Coordinating Center, develop and follow standardized methods of participant recruitment and retention and, using computerized tracking system developed by Coordinating Center, monitor number of contacts, demographic features of persons contacted, outcome of contacts, and limited data collection from non-participants;

        2. Actively plan and conduct pilot testing, including laboratory certification and central training of study personnel, of examination components as specified in the protocol; and

      [Note: The Offeror should plan pilot testing to include components of the initial and repeat transferrin saturation screens, the comprehensive clinical examination, and initial and extended ELSI assessments. The protocol for the initial transferrin saturation screen and ELSI assessment will likely require clearance from the Office of Management and Budget (OMB). The Coordinating Center will coordinate the effort to produce the OMB clearance packet, with assistance from the other Centers as needed. After draft protocols have been developed and approved by the Steering Committee, staff are to be centrally trained for each screen or examination component. Pilot testing will be conducted after training is complete and OMB clearance has been obtained, and will consist of performing the study components, according to the draft Manual of Operations, in approximately 10-20 non-study participant volunteers per Field Center. Repeat measurements should be planned for any examination component for which reproducibility is in question. The pilot tests also are to include shipping blood specimens as specified by the protocol. As the initial transferrin saturation assays are likely to be conducted on-site, rather than at the Central Laboratory, each Field Center's laboratory will need to obtain study certification documenting the ability to conduct standardized assays as specified in the protocol.]

        1. Redesign and implement changes in protocol and final Manuals of Operation, as recommended by the Steering Committee based on pilot testing results.

      1. During Phase II (September 1, 2000 through August 31, 2002), the Contractor shall

        1. Conduct initial transferrin saturation screen:

      [Note: It is anticipated that a total of 100,000 participants will be recruited among five to seven Field Centers. For purposes of the proposal, assume that 20,000 participants will be recruited by each Field Center. It is further expected that approximately 50% of the cohort will be women and that approximately 40-50% of the cohort will be minority participants. Each Field Center is expected to enroll from two or more racial/ethnic groups, one of which may be non-Hispanic Caucasians. Field Centers with two racial/ethnic groups must ensure that each group comprise at least 20% of the Center's study population. Field Centers with three or more racial/ethnic groups in the study must ensure that at least two of the groups comprise 20% or greater of the Center's study population.]

          1. Screen, using methods approved by the Steering Committee, a primary care-based sample of approximately 20,000 men and women aged 25 and older already undergoing a routine screen, test, or examination involving a blood draw, assuring representation from minority racial/ethnic groups and using methods to maximize articipation;

      [Note: It is assumed that the primary care patient is already undergoing routine screening, testing or examination involving a blood draw, so only minimal additional effort for recruitment, informed consent, and phlebotomy tasks is required. If this is not the case, the Offeror should describe the additional steps and costs needed to complete these tasks.]

          1. During routine screen, test, or examination involving a blood draw, obtain informed consent from participant, using methods approved by the Steering Committee;

      [Note: Attempts should be made to obtain information on reasons for refusal from those declining to participate in the study.]

          1. Draw additional blood, using appropriate vials and storage methods as approved by the Steering Committee, for transferrin saturation assay; and

      [Note: For planning purposes, assume the initial transferrin saturation assay will be performed on-site, at a laboratory meeting certification specifications as determined by the Steering and Laboratory Committees.]

          1. Identify participants with elevated transferrin saturation levels.

      [Note: Offerors should propose and provide justification for appropriate threshold values for elevated transferrin saturation levels (including, as necessary, age-, gender- and/or ethnic-specific values) and numbers of participants estimated to meet this criterion. While the focus of this study is on hereditary hemochromatosis and thus on elevated transferrin saturation levels and iron overload, it is recognized that this screen may also identify individuals with possible iron deficiency. Offerors should propose threshold values for low levels of transferrin saturation that would trigger referral to primary care provider and procedures for appropriate follow-up outside of the study. Iron-deficient participants are eligible for selection into the control sample to provide as broad a comparison group as possible.]

        1. In a random subgroup of participants, conduct initial ELSI assessment and genotyping screen:

          1. Identify, using methods approved by the Steering Committee, a 20-40% random subgroup (20,000-40,000 participants) of the initial transferrin saturation screenees, assuring oversampling of minorities;

          2. Obtain informed consent for genotyping and potential to be invited to participate in subsequent study components, using forms and procedures approved by the Steering Committee;

      [Note: The Offeror may propose plans for combining this and subsequently described stages of informed consent with that for the initial transferrin saturation screen.]

          1. Administer initial quantitative ELSI assessment in 10% (2,000-4,000 participants) of the random genotyping subgroup to cover the following:

      [Note: Items (f) and (g) are not traditional elements of ELSI assessments, but are included as these responses may impact the ELSI responses for this particular study. The survey instruments utilized by the NHGRI-NCI-NIMH-NINR-sponsored Cancer Genetics Studies Consortium may serve as a guideline in the planning of the quantitative assessment. A qualitative ELSI assessment is outlined in task b.iii.2. below.]

            1. knowledge, attitudes, and beliefs towards genetic screening, testing and counseling,

            2. knowledge, attitudes, and beliefs towards blood diseases such as iron overload and hereditary hemochromatosis,

            3. knowledge, attitudes, and beliefs toward testing and screening for such disorders by traditional laboratory tests (transferrin saturation) or genotype,

            4. sociodemographic characteristics of participant, including employment and insurance status,

            5. family issues (including desire for biologic offspring, feelings towards family members with possible inherited disease, and history of hemochromatosis-related diseases),

            6. history of blood donations and transfusions, and

            7. history of physician- or self-prescribed iron treatment;

          1. During blood draw for initial transferrin saturation screen, draw additional blood, using vials and storage methods as approved by the Steering Committee, and send to the Central Laboratory for DNA extraction and genotyping for known HFE gene variants, and other variants related to iron overload, hemochromatosis and iron metabolism, and for central storage of DNA and possible immortalization of cells for later testing of newly discovered variants; and

      [Note: It is anticipated that the genotyping will be performed centrally. Offerors should propose and justify the variants to be tested, and estimate the number of participants expected to be genotype-positive. These participants will be invited to participate in the comprehensive clinical examination and family study, regardless of their outcome on the initial or repeat transferrin saturation assay. It is anticipated that variants to be tested will reflect the state of knowledge at the time this phase of the study is conducted.]

          1. Provide counseling for genotype-positive participants.

        1. Conduct the following two ELSI-related substudies:

      [Note: It is expected that these substudies will be conducted in a subset of the five to seven Field Centers. For planning purposes, assume that two Centers will be chosen and each will enroll half of the participants described.]

          1. Randomly select 20-40 individuals from task b.ii.3. (above) in each of several race/ethnic groups, stratified by screening method, to undergo in-depth qualitative evaluation of ELSI issues outlined in task b.ii.3.

      [Note: Offeror should propose and justify methods to be used. These methods may include qualitative or semi-structured interviews, focus groups, or other appropriate in-person methods. Qualitative issues include, but are not limited to, perceived susceptibility to a condition; perceived severity of the seriousness of developing a condition; perceived benefits of interventions to reduce morbidity or mortality; and perceived barriers to undertaking some preventative behavior.]

          1. Randomly assign 2,000 additional primary care patients undergoing routine blood screening or testing to be invited to participate in a comparison of screening methods. These 2,000 participants will be in addition to the 100,000 undergoing the main study protocol, and are solely to evaluate the acceptability of phenotype screening vs genotype screening in primary care patients. These 2,000 participants will be randomly assigned to screening by phenotype only (transferrin saturation, with follow up for elevated transferrin saturation and case identification as in task b.iv. and c.i. below) or genotype only (initially without transferrin saturation, but to be followed by transferrin saturation and case identification as in task b.iv. and c.i. if genotype positive for known variants). Due to the differential nature of case ascertainment in this group, any cases identified from these 2,000 subjects may not be poolable with those identified through the main study protocol; appropriateness of pooling will be discussed within the Steering Committee during protocol development.

        1. Conduct repeat transferrin saturation screen in potential case, and/or genotype-positive participants:

          1. Recruit participants with initial elevated transferrin saturation (see Task 2.b.i.3. above) and those genotype-positive (Task 2.b.ii.4. above) as quickly as feasible, within approximately two months following the initial screen;

          2. Obtain informed consent for repeat transferrin saturation screen and the potential to be invited to participate in subsequent study components, using forms and procedures approved by Steering Committee;

          3. Draw blood, using methods approved by the Steering Committee, for repeat transferrin saturation assay, and send to the Central Laboratory according to approved protocols. Blood draw should occur in the morning following an overnight fast and with any other precautions to ensure reliability of the results, as determined by the protocol; and

      [Note: Repeat transferrin saturation assay will be conducted at the Central Laboratory.]

          1. Identify participants with sustained, elevated transferrin saturation levels.

      [Note: Offerors should propose and provide justification for methodology to select appropriate threshold values for sustained elevated transferrin saturation levels (including, if necessary, age-, gender- and/or ethnic-specific values) and numbers of participants estimated to meet this criterion. Standardized criteria for all Field Centers will be developed during protocol planning and approved by the Steering Committee. These participants will be considered cases for the comprehensive examination. Genotype-positive participants will be invited to participate in the comprehensive examination, regardless of repeat transferrin saturation assay results.]

        1. Conduct repeat transferrin saturation in potential control participants:

          1. Identify and recruit random sample of participants with initial non-elevated transferrin saturation levels (item b.i.3. above) and genotype-negative (item b.ii.4. above) as quickly as feasible but no later than two months following the initial screen;

      [Note: It is expected that 1,000 control participants will be recruited across the Field Centers. Controls will be matched to cases by Center, at a minimum. For purposes of the proposal, assume that 200 control participants will be selected at each Field Center. Offerors should propose and justify appropriate sampling scheme to result in randomly-selected control participants, and other criteria beyond Center, if any, for matching controls to cases.]

          1. Obtain informed consent, including the potential to be invited to participate in subsequent study components, using forms and procedures approved by Steering Committee;

          2. Draw blood, as in Task 2.b.iii.3. above, for repeat transferrin saturation assay; and

          3. Identify participants with sustained, non-elevated transferrin saturation levels.

      [Note: Use threshold values for sustained transferrin saturation levels as defined above in Task 2.b.iii.4. These participants will be considered controls during the comprehensive examination.]

      1. During Phase III (as potential cases are identified, approximately December 1, 2000 through August 31, 2003), the Contractor shall:

        1. Conduct comprehensive clinical examination in genotype-positive participants, case participants with sustained elevated transferrin saturation, and genotype-negative non-elevated transferrin saturation control participants, as soon as feasible, within approximately six months following repeat transferrin saturation screen:

          1. Obtain informed consent, including the potential to be invited to participate in the family study, using forms and procedures approved by Steering Committee;

          2. Collect data, using standardized procedures, as approved by the Steering Committee during protocol development, to assess:

      [Note: Offerors should provide justification for proposed questionnaires and measures.]

            1. ELSI issues regarding genetic screening and testing and hemochromatosis. The initial ELSI assessment should be extended to examine participants' experience with screening during this study; acceptability and impact of screening methods; their understanding and interpretations of their results; the impact this information is having on their own lives and their family; their plans and/or intentions to communicate this information with their families; any experiences of stigmatization or discrimination; psychosocial measures; and use of preventive services; health-related behaviors and related items,

            2. dietary information, including consumption of red meat, Vitamin C, iron supplements, tea intake, alcohol intake and other intakes as appropriate, and

            3. information on sociodemographic characteristics, medical history (including iron overload, hemochromatosis, therapeutic phlebotomy, other blood donation, blood transfusions, chelator therapy, liver transplantation, menstrual and pregnancy history, as well as related symptoms and disorders), smoking, medication use including oral contraceptives, and family history, including three to four generation pedigree,

          1. Perform phlebotomy, following the study protocol, to obtain specimens to:

      [Note: Unless otherwise determined during protocol development, all assays will be conducted at the Central Laboratory.]

            1. measure serum ferritin, fasting serum glucose and insulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), whole blood associated acetaldehyde (WBAA), carbohydrate-deficient transferrin (CDT) and other liver function tests, and complete blood count (CBC) including MCV and platelet count;

      [Note: The CBC will be conducted locally. The Offeror should propose, with justification, appropriate components of CBC to be included.]

            1. extract DNA for genotyping HFE variants and other related genetic markers of iron overload, hereditary hemochromatosis and iron metabolism (genotyping subgroup will have had DNA extracted during task 2.b.ii.4. above);

            2. immortalize or cryopreserve ymphocytes;

            3. measure serology of infectious agents, immune or inflammatory markers (such as hepatitis C or other relevant microorganisms, C-reactive protein and erythrocyte sedimentation rate); and

            4. measure other relevant assays as determined by the Steering Committee.

      [Note: Blood shall be drawn for low temperature (-70C) long-term storage at the Central Laboratory. For planning purposes, plan that approximately 90 ml of blood will be drawn into 10 tubes and aliquotted into 30 microvials for shipping to the Central Laboratory and long-term storage.]

          1. Perform some or all of the following measurements, based on the comprehensive clinical examination protocol approved by the Steering Committee, as appropriate to distinguish between primary and secondary causes of iron overload, and diagnose and characterize hereditary hemochromatosis and its correlates including related hepatic, endocrinologic, hematologic or cardiovascular manifestations:

      [Note: Offerors should propose and justify non-invasive and any invasive procedures proposed for inclusion in the comprehensive clinical examination, including, as appropriate, any decision-tree algorithms to be used. Participants' primary care provider may wish to conduct additional procedures outside the study protocol; Offerors should discuss options to obtain these data when possible. Protocol-performed assessments must be standardizable among multiple examinees and across Field Centers; hence Offerors should carefully describe proposed measures and methods for assuring standardization and quality control. During protocol development, a common standardized protocol will be agreed upon, and approved by the Steering Committee, DSMB and NHLBI/NHGRI staff.]

            1. anthropometry and blood pressure,

            2. assessment of liver disease,

            3. assessment of blood disorders,

            4. assessment for diabetes,

            5. assessment of arthropathies and arthritis,

            6. assessment of heart failure, and arrhythmias, and

            7. other measurements of disease related to iron overload, as determined by the Steering Committee and Project Office.

          1. Include approximately 5% duplicate measurements for quality control assessments of hematology, clinical chemistry and genetic assays and disease measurements, as specified in the protocol; and

          2. Identify participants with iron overload and/or hereditary hemochromatosis, and provide post-test counseling as appropriate.

      [Note: Offerors should propose definitions of primary and secondary iron overload and hereditary hemochromatosis based on results of above screens and comprehensive clinical examination.]

          1. Conduct qualitative ELSI substudy on 20-40 randomly selected individuals with hemochromatosis, as defined in task c.i.7. (immediately above), from each of several race/ethnic groups to undergo in-depth qualitative evaluation of ELSI issues outlined in task c.i.2.a.

      [Note: It is expected that this substudy will be conducted in a subset of the five to seven Field Centers. For planning purposes, assume that two Centers will be chosen and each will enroll half of the participants described. Offeror should propose and justify methods to be used. These methods may include qualitative or semi-structured interviews, focus groups, or other appropriate in-person methods. Qualitative issues include, but are not limited to, perceived susceptibility to a condition; perceived severity of the seriousness of developing a condition; perceived benefits of interventions to reduce morbidity or mortality; and perceived barriers to undertaking some preventative behavior.]

        1. Conduct family study in each genotype-positive participant and/or participant with hereditary hemochromatosis:

          1. Recruit each genotype-positive participant and/or participant with hereditary hemochromatosis to be probands for family study;

          2. Obtain informed consent, including permission to contact family members, using forms and procedures approved by Steering Committee;

          3. Recruit and obtain informed consent from four or more family members, aged 25 and older, of each proband to participate in family study;

      [Note: Offerors should discuss logistical issues related to geographical location of family members and examination of family members not part of the primary care organization used to ascertain the probands.]

          1. Perform appropriate components of above transferrin saturation screens, genotyping, comprehensive examinations, as determined by Steering Committee, in family members;

          2. Conduct quantitative ELSI assessment in two members of each family, including issues as covered in comprehensive clinical examination ELSI assessment (task 2.c.i.4. above) as well as individual and family impact of screening; family communication issues related to genetic screening and testing; and any experiences with stigmatization and discrimination.

          3. Conduct qualitative ELSI substudy on 20-40 randomly selected individuals in the family study, from each of several race/ethnic groups, to undergo in-depth qualitative evaluation of ELSI issues outlined in task c.ii.5. (immediately above).

      [Note: It is expected that this substudy will be conducted in a subset of the five to seven Field Centers. For planning purposes, assume that two Centers will be chosen and each will enroll half of the participants described. Offeror should propose and justify methods to be used. These methods may include qualitative or semi-structured interviews, focus groups, or other appropriate in-person methods. Qualitative issues include, but are not limited to, perceived susceptibility to a condition; perceived severity of the seriousness of developing a condition; perceived benefits of interventions to reduce morbidity or mortality; and perceived barriers to undertaking some preventative behavior.]

          1. Provide post-test counseling to family members as appropriate; and

          2. Work cooperatively with Coordinating Center and Central Laboratory and provide data as needed to assist those Centers in the family study genome-wide genotyping and linkage analyses.

      [Note: If there are insufficient numbers of families/family members in a particular race/ethnic group to achieve adequate statistical power for linkage analyses, strategies for supplementing families/family members, for instance remote blood collections or including families from other studies, will be considered.

      1. During Phase IV (as hemochromatosis cases are identified, approximately February 1, 2001 through August 31, 2004, the Contractor shall:

        1. Conduct morbidity and mortality follow-up in all main or family study participants with hemochromatosis:

      [Note: The follow-up interval will vary depending on when the comprehensive clinical examination is performed, ranging from approximately one to three years.]

          1. Using available electronic records (every 6 months) or by performing telephone or mail contact (yearly), ascertain occurrence of deaths; and hepatic, endocrinologic, hematologic and cardiovascular events, according to a standard protocol approved by the Steering Committee. Information to be collected on potential events includes:

            1. Circumstances and life situations immediately prior to an event or death;

            2. Use of medical services, including diagnostic tests and therapeutic interventions, in conjunction with the event;

            3. Diagnostic information, medical care and clinical characteristics from medical records; and

            4. Dates and recorded causes of all deaths using local vital statistics sources, obituaries, the National Death Index, and other sources;

          2. Participate in classification of type and severity of events, using standard protocol approved by the Steering Committee; and

          3. Conduct follow-up quantitative ELSI assessment of impact of screening and testing, relationship with family members and any experiences of stigmatization or discrimination.

          4. Conduct qualitative ELSI substudy on 20-40 randomly selected individuals from each of several race/ethnic groups to undergo in-depth qualitative evaluation of ELSI issues outlined in task d.i.3. (immediately above).

      [Note: It is expected that this substudy will be conducted in a subset of the five to seven Field Centers. For planning purposes, assume that two Centers will be chosen and each will enroll half of the participants described. Offeror should propose and justify methods to be used. These methods may include qualitative or semi-structured interviews, focus groups, or other appropriate in-person methods. Qualitative issues include, but are not limited to, perceived susceptibility to a condition; perceived severity of the seriousness of developing a condition; perceived benefits of interventions to reduce morbidity or mortality; and perceived barriers to undertaking some preventative behavior.]

      1. Throughout the period of performance, in conjunction with the Coordinating Center and Central Laboratory, the Contractor shall analyze collected data and prepare abstracts, presentations, and manuscripts for publication of results.

      [Note: The proposal should include brief descriptions of at least five potential manuscript topics that could be first-authored by the Offeror. It is anticipated that abstracts and manuscripts proposed for presentation or publication will be submitted to the Publications Committee and the NHLBI Project Office in advance for review and approval, as outlined in the section on Deliverables. Due to overlapping interest and expertise, manuscript writing groups will likely include members from several, most or all Centers.]

    2. SUBORDINATE TASKS

      Throughout the period of performance the Contractor shall participate in other activities related to the successful completion of the project, as specified by the Steering Committee and/or NHLBI Project Office. These include, but are not necessarily limited to the following:

      1. Provide for temporary storage at low temperature, and shipping with dry ice, of all blood specimens to be processed or stored at Central Laboratory;

      2. Enter and edit the results from study interviews, examinations and quality assurance activities, including blind duplicates, into a computer, using software designed in cooperation with the Coordinating Center, and transmit the data to the Coordinating Center on a regular basis, approximately weekly, as specified in the study protocol. Transmit quality control data to the Coordinating Center according to specifications of the study protocol. Store copies of all original informed consent and data forms at the Field Center;

      3. In collaboration with the Coordinating Center, provide results of each transferrin saturation assay and comprehensive examination and an indication of any abnormalities of clinical significance to the participant with counseling as appropriate, and, with consent, to his/her physician. Specific abnormalities to be reported will be determined by the Steering Committee during protocol development, and may be modified by individual Field Centers to conform with local community practices. Provide recommendations for follow-up of abnormalities and contact of siblings and other family members of participants with hereditary hemochromatosis as appropriate;

      4. Participate in site visits to Field Centers, as needed;

      5. Participate in ancillary studies and substudies, as desired, feasible, and recommended by the Steering Committee. Such studies may include, but are not limited to, examinations of ethical, economic and health policy issues related to implementation of screening programs, or other questions related to iron overload and hemochromatosis;

      [Note: Investigators may propose ancillary studies and substudies to be conducted in one or more study Centers. A substudy is an investigation which, although not part of the core exam protocol, is funded by Contract funds, and will yield additional information related to study objectives. An ancillary study is a study not funded by contract funds. Substudies and ancillary studies may include all or a subgroup of the cohort at a given center, and may involve additional interviews or examinations of study participants as well as analysis of blood or tissue specimens, tapes, or images collected previously.

      Ancillary studies and substudies are subject to the same policies, reviews and approvals as the core protocol. Substudies involving additional participant burden will likely require OMB clearance. Investigators proposing substudies will also prepare a request for OMB clearance for the substudy; or, for case-control studies, a request for exemption from OMB review. Examples of such submissions will be provided on request.

      Ancillary study data, collected under a grant mechanism, are not required to become part of the public use data set (see Coordinating Center RFP, task2.b.xi); however, these data will be incorporated into the study data set after an appropriate period of time (generally 12 months after completion of data collection). Investigators conducting ancillary studies are to be viewed as collaborating investigators of the primary study, with appropriate access to the full data set. However, use of analytic resources of the study will require additional support from the ancillary study investigators.

      Ancillary studies and substudies will be evaluated by the Steering Committee. Highest priority will be given to studies which: 1) have the highest scientific merit, 2) do not interfere with the main study objectives, 3) produce the least burden on participants, 4) have objectives directly related to the study, and 5) require the unique characteristics of the study participants.

      For all substudies and ancillary studies, the contractor shall define the hypotheses to be investigated and the methodology to be used, and should estimate the cost and burden on participants. Study data collection must not interfere with the conduct of the core examination. All substudies in the proposal should be distinctly identified, with separate descriptions and estimates of costs. Ancillary studies and substudies may be proposed before and/or after contract award as scientific opportunities arise. All studies must be approved by the Steering Committee, the Data and Safety Monitoring Board, and the NHLBI Project Office before initiation.]

      1. Cooperate in assisting Coordinating Center with OMB clearance package, as needed;

      2. Cooperate in providing up-to-date lists of publications and presentations coming from the Field Center for a data base to be managed by the Coordinating Center;

      3. Cooperate in providing updated information as needed by Coordinating Center in producing public use data sets.

      4. Provide raw or summary data as required by the Project Office.


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