Universal newborn screening: A roadmap for action.

Newborn screening (NBS) prevents morbidity and mortality by screening babies for selected disorders in the first days of life so that early diagnosis and treatment can be initiated. Congenital disorders impact an estimated 8 million or 6% of annual births worldwide, and of the top five that contribute 25% to the global burden of these disorders, three can be identified and managed by NBS. There are determined pockets of activity in Latin America, Sub-Saharan Africa, and the Asia Pacific region, where partnerships among government, non-governmental organizations, academia, the private sector and civil society are developing novel NBS programs that are both saving lives and preventing disability in those who survive.

U.S. newborn screening policy dilemmas for the twenty-first century.

Newborn screening has traditionally referred to biochemical testing for inherited disorders, generally metabolic in origin, that are usually correctable by dietary or drug interventions. As new tests have been developed, state public health newborn screening systems have slowly evolved without the benefit of national policies. Thus, newborn screening program changes, when viewed nationally, have been uncoordinated. The net result has been unequally applied mandated screening and, consequently, unequal availability of related public health disease prevention services. Technological advances in laboratory testing over the past 10 years have resulted in limited program changes in some state newborn screening systems, and even greater program disparities. A recent Newborn Screening Task Force identified numerous issues of concern and proposed elements for a plan of action involving public health programs, healthcare providers, and consumers. This minireview details past policy history in newborn screening and identifies some of the current issues confronting programs as they seek to move ahead with the technologies and medical treatments for the twenty-first century.

Newborn screening for congenital adrenal hyperplasia.

Classic CAH (salt-wasting and simple virilizing) meets all of the recommended criteria for newborn screening. There are reliable and efficient newborn screening tests, the disorder results in high morbidity and mortality if left undetected, there is effective treatment that reduces negative outcomes, and there is a relatively high incidence. When compared with the case findings without the benefit of screening, the data from screening programs show reduced adrenal crises, reduced incorrect sex assignments, and reduced deaths. Racial/ethnic prevalence differences are present in newborn screening program data. The Texas data indicate a lower disease frequency in African-Americans when compared with Caucasians, and international data indicate higher frequencies in native Yupik Eskimos, Brazilians, residents of La Reunion, and Filipinos. When worldwide clinical ascertainment data are compared with newborn screenng data, it is clear that newborns with CAH (especially males) die when screening is not done. To be effective in reducing mortality, newborn screening must be performed soon after birth, and the results must be available quickly so that early salt-wasting crises can be averted. It is preferable that newborn screening laboratiories be operational 7 days a week, and that sample delivery from the collection site to testing laboratory be as efficient as possible, including weekends and holidays, so that undue testing delays are not encountered. These two requirements pose major challenges for most programs, but they are critical to optimal screening outcome. Based on the studies in Texas, with second screening samples collected at approximately 2 weeks of age, some newborns with simple virilizing CAH are missed on initial screening using current testing protocols. There is need to set a screening cut-off such that the false-positive rate does not oversaturate the follow-up system, in part owing to the insensitivity of current kit methodologies and the biochemical manifestations of CAH. With advances in genetic testing procedures and improved automation techniques, it may soon be possible for CAH screening programs to include genotyping as a second-tier confirmation as a part of the newborn screening protocol. Despite the fact that CAH is a continuum of disorders, the correlation between genotype and phenotype is fairly consistent in most cases. For the purpose of screening, genotyping will likely be useful only for differential diagnoses of non-salt wasters, given the necessary time constraints and expense of obtaining genotypes and the need for immediate diagnosis/treatment of salt wasters. It is hoped that newborn screening programs will begin to provide answers to some of these question in addition to their primary function of reducing the morbidity and mortality resulting from CAH.

AluY insertion (IVS4-52ins316alu) in the glycerol kinase gene from an individual with benign glycerol kinase deficiency.

Glycerol kinase deficiency has three distinct forms: an isolated form which may be benign or symptomatic, and a complex form which is symptomatic and part of an Xp21 contiguous gene syndrome. Here we report the case of a male with benign isolated glycerol kinase deficiency who was incidentally identified after observation of pseudohypertriglyceridemia. DNA sequencing of this subject's glycerol kinase gene showed the insertion of an AluY sequence in intron 4 of the glycerol kinase gene. Although Alu insertions have been implicated in other diseases, and a closely related AluY element is found as an insert in the C1 inhibitor gene in patients with hereditary angioedema, this is the first case of glycerol kinase deficiency caused by an Alu insertion.

A comparative cost analysis of newborn screening for classic congenital adrenal hyperplasia in Texas.

OBJECTIVE: Texas mandates a two-test newborn screening program for congenital adrenal hyperplasia (CAH): one test at birth and a second test at approximately one to two weeks after birth. The authors compared the dollar cost of detecting infants with CAH clinically and through the screening program. METHODS: The authors estimated the costs of screening newborns in 1994 for CAH, including resources used by the Texas Department of Health and the broader cost to society. RESULTS: Fifteen infants with classic CAH were diagnosed in Texas in 1994 among 325,521 infants born (1:21,701 cumulative incidence). Seven infants were detected clinically and the others were detected through screening, six on the first screen and two on the second screen. The first screen identified all previously undetected infants with severe salt-wasting CAH. The cumulative cost to diagnose the seven infants detected clinically was $79,187. The incremental costs for the screening program were $115,169 per additional infant diagnosed through the first screen and $242,865 per additional infant diagnosed through the second screen. CONCLUSIONS: If the goal is early diagnosis of infants with the severe salt-wasting form of CAH, a single screen is effective. If the goal is to detect infants with the simple virilizing form of the disorder who may benefit from early treatment, the second screen is necessary, but it is not as cost-effective as the first screen.

Neuroblastoma screening in the United States: results of the Texas Outreach Program for neuroblastoma screening.

BACKGROUND: Mass screening of infants for neuroblastoma began in Japan after studies suggested that survival rates could be improved by early detection. This study was initiated in 1991 to test the methodology and feasibility of screening for neuroblastoma within the U. S. health care system. METHODS: Infants ages 5-10 months (mean age, 9 months, 25 days) who were born in Texas were screened for neuroblastoma. An enzyme-linked immunoadsorbent assay (ELISA) for homovanillic acid (HVA) and vanillylmandelic acid (VMA) used to quantify the HVA and VMA was performed on urine extracted from specimens dried on filter paper. Infants were recruited to participate in the study by several methods, and the effectiveness of each method was determined by calculating compliance rates. RESULTS: Between February 1991 and June 1994 a total of 14,046 infants were recruited to participate in neuroblastoma screening. Neuroblastoma was detected in 2 children for an incidence rate of 1 in 7023. A total of 291,158 screening kits were distributed to the parents of these infants, resulting in an overall compliance rate of only 4.8%. Compliance rates varied by method of distribution of the test kits: Houston Women, Infants, and Children (WIC) clinic (53%), volunteers (31%), Rio Grande Valley WIC clinics (14.5%), the patient's private physician (9.9%), and by mail (4.7%). CONCLUSIONS: Early detection of neuroblastoma in infants ages 5-10 months was achieved using ELISA. Compliance rates were poor, but clinics with a preventive health focus, such as the WIC clinics, achieved higher compliance rates than did private physicians.

Results of screening 1.9 million Texas newborns for 21-hydroxylase-deficient congenital adrenal hyperplasia.

OBJECTIVE: To assess results of newborn screening for 21-hydroxylase-deficient congenital adrenal hyperplasia (CAH) in Texas over 6 years of screening 1.9 million infants. METHODS: In 1989, CAH was incorporated into the ongoing Texas Newborn Screening Program, which requires two screens on each newborn. 17-Hydroxyprogesterone was assayed, without extraction, by radioimmunoassay of blood collected from heel sticks onto filter paper collection cards. Infants with elevated levels of 17-hydroxyprogesterone were referred for evaluation, and those considered to have CAH were studied with respect to disease characteristics. Data were collected by pediatric endocrinologists using standardized forms that included type of CAH, results of laboratory tests, treatment regimen, disease symptoms and signs, and, for girls, degree of genital virilization. RESULTS: The incidence of classic CAH in Texas is 1:16 008, with a ratio of salt-wasting to simple-virilizing of 2.7:1. A majority of infants detected were undiagnosed until screened, despite signs of salt-wasting or ambiguous genitalia. It was difficult to differentiate salt-wasting from simple-virilizing CAH in infants who were identified before the onset of adrenal insufficiency or electrolyte abnormalities. A substantial number of infants with nonclassic (NC) CAH also were detected. Not all infants were detected on the initial screen; 14% of infants with classic CAH and 87% with NC CAH were detected on the second routine screening test. CONCLUSIONS: Our findings confirm the benefits of newborn screening for CAH and the importance of a second screening test, and suggest that programs for newborn CAH screening must consider complex issues in diagnosis and treatment. These results also confirm that CAH is a continuum of disorders, rather than a disorder with discrete subtypes. In addition, the difficulties in differentiating CAH subtypes in newborns, and thus deciding appropriate treatment, and the high incidence of NC CAH suggest that standard diagnostic criteria and treatment regimens for CAH may need modification. Where screening exists, physicians will encounter more cases of CAH than in the past.

Preliminary investigation of the use of dried-blood spots for the assessment of in utero exposure to environmental pollutants.

We determined the concentration of dichlorodiphenyldichloroethylene (p,p'-DDE) in dried-blood spot specimens from 2-day-old infants from rural Texas who had never been breast fed. Anonymous, residual whole blood spots on filter paper, previously used for routine newborn screening procedures, were soaked in a phosphate buffer, extracted with an organic solvent, and eluted through silica gel. The concentrated eluates were analyzed by capillary gas chromatography with electron capture detection (ECD). The blood collected from 10 newborns was analyzed and found to contain DDE concentrations ranging from 0.13 to 1.87 pg/microliter with a mean of 0.72 pg/microliter. One of the 10 newborns had a whole blood DDE concentration of 1.87 pg/microliter, which was greater than the concentration of 1.34 pg/microliter in a freshly drawn sample from an adult donor whose blood serum was shown to contain DDE. With improvement in detection limits, this approach has the potential to displace the analyses of mothers' blood (as a surrogate indicator of infants' exposures) and cord blood as standard procedures for determining the newborns' body burden of environmental pollutants.

Multivariant confirmation of sickle cell disease using a non-radioactive minisequencing reaction.

A non-radioactive solid-phase minisequencing method for confirmation of abnormal hemoglobin variants causing sickle cell disease has been developed. In this method amplified 5'-biotinylated target sequences containing normal and mutation sites are immobilized onto streptavidin-coated microplates. Detection primers corresponding to target sequences are annealed immediately adjacent to the mutation site and single-step, hapten-labeled nucleotide primer extension reactions are performed. The incorporation of the labeled nucleotide is detected through immunological reaction with an enzyme-labeled anti-hapten conjugate and a substrate. The method enables confirmation of mutations of the beta-globin gene variants (Hbs S, C, E D-Punjab, O-Arab) and the alpha-globin gene variant (Hb G-Philadelphia). The test was evaluated using characterized dried blood spot specimens (n = 100) The advantages of the procedure are easy performance and objectiveness. The non-radioactive minisequencing assay will prove helpful for genotyping in neonatal screening for hemoglobinopathies and in prenatal and pre-implantational diagnostics.

Demographic risk factors associated with elevated lead levels in Texas children covered by Medicaid.

This is the first large population-based study of demographic risk factors for elevated lead in Texas children. It summarizes data on 92,900 children covered by Medicaid screened for blood lead during the first 6 months of 1993 in Texas. The highest percentage of elevated lead levels (14.3%) was in children 25-36 months of age, with slightly lower percentages in those younger (13% of 19-24 months) and older (12% of 37-48 months) with blood lead levels greater than 10 micrograms/dl. The group with the highest percentage of elevated blood lead levels was 2-4-year-old African American males (17.3%) making this subgroup 3.5 times higher than the group with the lowest percentage-white girls over age 4 (4.8%). Males had higher blood lead levels for all ages and ethnic groups. Three principal risk factors were found for excessive blood lead in children: ethnicity, gender, and age; this is consistent with the second National Health and Nutrition Examination Survey (NHANES II) and Phase I of the NHANES III results demonstrating ethnicity and income association with lead in children in the United States.

Guidelines for the retention, storage, and use of residual dried blood spot samples after newborn screening analysis: statement of the Council of Regional Networks for Genetic Services.

These guidelines provide scientific information for policy development by state health departments considering appropriate use of newborn screening specimens after screening tests are finished. Information was collected, debated, and formulated into a policy statement by the Newborn Screening Committee of the Council of Regional Networks for Genetic Services (CORN), a federally funded national consortium of representatives from 10 regional genetics networks. Newborn screening programs vary widely in approaches and policies concerning residual dried blood spot samples (DBS) collected for newborn screening. Recognition of the epidemiological utility of DBS samples for HIV seroprevalence surveys and a growing interest in DBSs for DNA analysis has intensified consideration of issues regarding retention, storage, and use of residual DBS samples. Potentially these samples provide a genetic material "bank" for all newborns nationwide. Their values as a resource for other uses has already been recognized by scientists, administrators, and judicial officials. Programs should promulgate rules for retention and use of residual newborn screening DBS samples based on scientifically valid information. Banking of newborn samples as sources of genetic material should be considered in light of potential benefit or harm to society.

Application of molecular genetics in public health: improved follow-up in a neonatal hemoglobinopathy screening program.

Newborn screening for the hemoglobinopathies has been shown to reduce morbidity and mortality, particularly for sickle cell anemia, by facilitating initiation of penicillin prophylaxis by 4 months of age. The purpose of the current investigation was to determine whether molecular genetic follow-up testing could be introduced into a neonatal hemoglobinopathy screening program and, if successfully introduced, whether it would reduce time to diagnostic confirmation. Between July 1, 1991, and October 7, 1992, 518 original dried blood specimens were referred from the Texas Department of Health Neonatal Hemoglobinopathy Screening Program for molecular genetic follow-up testing. Allele-specific cleavage (ASC) after amplification with matched and mismatched polymerase chain reaction primers was compared to allele-specific oligonucleotide (ASO) hybridization. By November 2, 1992, molecular genetic analyses were definitive in 506, and agreement was observed between ASC and ASO hybridization in all specimens analyzed. Approximately 13% of those initially screened FS were considered probable S/beta-thal by DNA and RNA testing. Rapid molecular genetic analysis contributed to a substantial reduction of the mean age at confirmation by approximately 50%, to about 2 months of age. ASC is a reliable method for molecular genetic analysis of dried blood specimens, providing methodology which can be readily automated. An automated method is demonstrated that is based on microtiter plate technology and will significantly reduce labor intensity and costs, while increasing sample throughput. Even with current manual testing methods, DNA and RNA analysis of initial newborn screening specimens will reduce the age at confirmation well under 4 months, the age cut-off for effective initiation of penicillin prophylaxis.

Unbiased analysis of the frequency of beta-thalassemia point mutations in a population of African-American newborns.

OBJECTIVE: To determine frequency of specific beta-thalassemia alleles in the African-American population prospectively, using newborn screening specimens, and to evaluate the need for including these alleles in screening follow-up programs. DESIGN: Allele-specific oligonucleotide tests were developed and used to analyze African-American newborn screening specimens for beta-thalassemia point mutations to determine their frequency. Direct sequencing of amplified DNA from the dried blood specimens was used to confirm the presence of point mutations. POPULATION: African-American newborns in Texas. RESULTS: Allele-specific oligonucleotides identified five newborn specimens carrying beta-thalassemia point mutations among 471 specimens from African-American neonates. Direct sequencing of DNA from the dried blood specimens confirmed that these individuals had a normal and a mutant allele. Five newborn screening specimens in which the results of screening and DNA tests were in disagreement (four with FS by screening and AS by DNA, and one with FC by screening and AC by DNA) were analyzed for these beta-thalassemia point mutations and in each case were found to be S/beta-thalassemia or C/beta-thalassemia compound heterozygotes, respectively. CONCLUSIONS: Allele-specific oligonucleotides accurately identified newborn specimens carrying beta-thalassemia point mutations. Direct sequencing from dried blood specimens confirmed these results. The A(-29)G allele frequency was 0.003, and the C(-88)T frequency was 0.002. These alleles also were observed among positive samples in a neonatal hemoglobinopathy screening program. Therefore, any newborn screening program with a molecular genetic follow-up component must include testing for these beta-thalassemia alleles to assure timely and appropriate management for affected infants.

Genotypic confirmation from the original dried blood specimens in a neonatal hemoglobinopathy screening program.

Dried blood spots are used for newborn screening because of ease of sample collection, handling, and shipment. DNA is stable and accessible in the filter paper matrix. Genotypic confirmation using initial specimens is demonstrated for a regional screening program. Seventy-five blinded samples underwent DNA analysis after Hb electrophoresis. DNA was microextracted from a 1/2-inch semicircle (25 microL whole blood equivalent), amplified, and analyzed by four different methods. Direct amplification without microextraction and automated sequencing from microextracted DNA also was performed. All four analyses agreed for the A and S alleles in 70 of 75 specimens. Three disagreements were clarified by the other semicircle from the original sample: two were due to polymerase chain reaction contamination and one to contamination of one of four analytical tests. Two would have required analysis of a second specimen, one because of polymerase chain reaction failure and the second because the patient had S/beta-thalassemia. Direct amplification without microextraction was successful in an additional 77 of 78 specimens for analysis of the A, S, C, and E alleles. Automated direct sequencing from microextracted DNA was demonstrated for the A, S, and C alleles. Analysis of microextracted DNA from dried blood specimens for A and S alleles reduced the need for and costs of obtaining a second specimen for confirmation by 97%. Direct amplification without microextraction for analysis of A, S, and C alleles permits additional reduction in personnel time and costs. We have demonstrated that microextracted DNA is amenable to automated sequencing after asymmetric polymerase chain reaction. Direct genotypic confirmation can facilitate diagnosis and initiation of medical intervention.