Newborn screening for autism: in search of candidate biomarkers.

BACKGROUND: Autism spectrum disorder (ASD) represents a wide range of neurodevelopmental disorders characterized by impairments in social interaction, language, communication and range of interests. Autism is usually diagnosed in children 3-5 years of age using behavioral characteristics; thus, diagnosis shortly after birth would be beneficial for early initiation of treatment. AIM: This retrospective study sought to identify newborns at risk for ASD utilizing bloodspot specimens in an immunoassay. MATERIALS & METHODS: The present study utilized stored frozen specimens from ASD children already diagnosed at 15-36 months of age. The newborn specimens and controls were analyzed by immunoassay in a multiplex system that included 90 serum biomarkers and subjected to statisical analysis. RESULTS: Three sets of five biomarkers associated with ASD were found that differed from control groups. The 15 candidate biomarkers were then discussed regarding their association with ASD. CONCLUSION: This study determined that a statistically selected panel of 15 biomarkers successfully discriminated presumptive newborns at risk for ASD from those of nonaffected controls.

A microsphere-based assay for mutation analysis of the biotinidase gene using dried blood spots.

Biotinidase deficiency is an autosomal recessive syndrome caused by defects in the biotinidase gene, the product of which affects biotin metabolism. Newborn screening (NBS) for biotinidase deficiency can identify affected infants prior to onset of symptoms; biotin supplementation can resolve or prevent the clinical features. In NBS, dry blood spots (DBS) are usually tested for biotinidase enzyme activity by colorimetric analysis. By taking advantage of the multiplexing capabilities of the Luminex platform, we have developed a microsphere-based array genotyping method for the simultaneous detection of six disease causing mutations in the biotinidase gene, thereby permitting a second tier of molecular analysis. Genomic DNA was extracted from 3.2 mm DBS. Biotinidase gene sequences, containing the mutations of interest, were amplified by multiplexed polymerase chain reaction, followed by multiplexed allele-specific primer extension using universally tagged genotyping primers. The products were then hybridized to anti-tag carrying xTAG microspheres and detected on the Luminex platform. Genotypes were verified by sequencing. Genotyping results of 22 known biotinidase deficient samples by our xTAG biotinidase assay was in concordance with the results obtained from DNA sequencing, for all 6 mutations used in our panel. These results indicate that genotyping by an xTAG microsphere-based array is accurate, flexible, and can be adapted for high-throughput. Since NBS for biotinidase deficiency is by enzymatic assay, less than optimal quality of the DBS itself can compromise enzyme activity, while the DNA from these samples mostly remains unaffected. This assay warrants evaluation as a viable complement to the biotinidase semi-quantitative colorimetric assay.

High-throughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunodeficiency in population-based newborn screening.

BACKGROUND: Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample. METHODS: We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded. RESULTS: The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR. CONCLUSIONS: Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

A multiplex immunoassay using the Guthrie specimen to detect T-cell deficiencies including severe combined immunodeficiency disease.

BACKGROUND: Severe combined immunodeficiency (SCID) fulfills many of the requirements for addition to a newborn screening panel. Two newborn screening SCID pilot studies are now underway using the T-cell receptor excision circle (TREC) assay, a molecular technique. Here we describe an immunoassay with CD3 as a marker for T cells and CD45 as a marker for total leukocytes that can be used with the Guthrie specimen. METHODS: The multiplexing capabilities of the Luminex platform were used. Antibody pairs were used to capture and detect CD3 and CD45 from a single 3-mm punch of the Guthrie specimen. The assay for each biomarker was developed separately in identical buffers and then combined to create a multiplex assay. RESULTS: Using calibrators made from known amounts of leukocytes, a detection limit of 0.25 x 10(6) cells/mL for CD3 and 0.125 x 10(6) cells/mL for CD45 was obtained. Affinity tests showed no cross-reactivity between the antibodies to CD3 and CD45. The multiplex assay was validated against 8 coded specimens of known clinical status and linked to results from the TREC assay that had identified them. All were correctly identified by the CD345 assay. CONCLUSIONS: The performance parameters of the CD345 assay met the performance characteristics generally accepted for immunoassays. Our assay classifications of positive specimens concur with previous TREC results. This CD345 assay warrants evaluation as a viable alternative or complement to the TREC assay as a primary screening tool for detecting T-cell immunodeficiencies, including SCID, in Guthrie specimens.

Trends in incidence rates of congenital hypothyroidism related to select demographic factors: data from the United States, California, Massachusetts, New York, and Texas.

Primary congenital hypothyroidism (CH) is a common and preventable cause of intellectual disability. The incidence rate of CH has been reported to be increasing in the United States, but the factors behind the observed rate increase are not known. We summarize here the data presented at a workshop on CH, at which factors potentially related to the CH-incidence-rate increase (namely, race, ethnicity, sex, and birth outcomes) were evaluated. Data sources for the analyses included a national data set of newborn-screening results and state-specific data from newborn-screening programs in California, Massachusetts, New York, and Texas. The incidence rate of CH increased in the United States by 3% per year; however, an increase did not occur in all states, at a constant rate, or even at the same rate. Analysis of US data (1991-2000) showed a CH-incidence-rate increase only among white newborns. More recently, in California (2000-2007), the rate was constant in non-Hispanic newborns, but it increased among Hispanic newborns. In the national data, the CH-incidence rate increased similarly among boys and girls, whereas in Texas (1992-2006), the rate among boys increased significantly more than among girls and varied according to race and ethnicity. In Massachusetts (1995-2007), low birth weight newborns or newborns who had a delayed rise in thyrotropin concentration accounted for the majority of the recent rate increase. Race, ethnicity, sex, and pregnancy outcomes have affected the observed increasing incidence rate of CH, although there have been some inconsistencies and regional differences. The association with preterm birth or low birth weight could reflect the misclassification of some cases of transient hypothyroxinemia as true CH. Future studies of risk factors should focus on correct initial identification and reporting of demographic characteristics and pregnancy outcomes for cases of CH. In addition, long-term follow-up data of presumed cases of CH should be ascertained to differentiate true cases of CH from cases of transient hypothyroidism.

Guidelines for the Development of Comprehensive Care Centers for Congenital Adrenal Hyperplasia: Guidance from the CARES Foundation Initiative.

Patients with rare and complex diseases such as congenital adrenal hyperplasia (CAH) often receive fragmented and inadequate care unless efforts are coordinated among providers. Translating the concepts of the medical home and comprehensive health care for individuals with CAH offers many benefits for the affected individuals and their families. This manuscript represents the recommendations of a 1.5 day meeting held in September 2009 to discuss the ideal goals for comprehensive care centers for newborns, infants, children, adolescents, and adults with CAH. Participants included pediatric endocrinologists, internal medicine and reproductive endocrinologists, pediatric urologists, pediatric surgeons, psychologists, and pediatric endocrine nurse educators. One unique aspect of this meeting was the active participation of individuals personally affected by CAH as patients or parents of patients. Representatives of Health Research and Services Administration (HRSA), New York-Mid-Atlantic Consortium for Genetics and Newborn Screening Services (NYMAC), and National Newborn Screening and Genetics Resource Center (NNSGRC) also participated. Thus, this document should serve as a "roadmap" for the development phases of comprehensive care centers (CCC) for individuals and families affected by CAH.

Newborn screening for cystic fibrosis by use of a multiplex immunoassay.

BACKGROUND: Since its beginnings, newborn screening for cystic fibrosis (CF) using an assay for immunoreactive trypsinogen (IRT) has been plagued by a high rate of false-positive results (screen positive, diagnosis negative), despite attempts to reduce this rate by use of altered cutoffs and second-tier DNA testing. IRT exists as 2 isoforms: IRT1 and IRT2, with IRT2 being more closely aligned with pancreatic disease, including CF. Assay standardization between programs is a continuing problem because the IRT assays currently in use variously recognize either 1 or both isoforms. Here we report the development of a multiplexed assay for both forms of IRT simultaneously. METHODS: Using 2 different Luminex bead sets, we developed assays for each IRT isoform separately and then combined them. Using the sum of IRT1 and IRT2 values (IRT1+IRT2), we compared the results with a CF kit currently in use. RESULTS: In a sample set consisting of 16 cases confirmed positive for CF, we established a cutoff at >97 microg/L total IRT. Seven of 8 carriers with 1 CF mutation screen-positive by the standard method were also screen-positive by IRT1+IRT2. Of 32 cases screen-positive by standard IRT, 11 were screen-negative by IRT1+IRT2. None of these 11 cases had CF mutations identified by the screening program. CONCLUSIONS: These data indicate that the multiplex method with specificity for 2 isoforms of IRT has performance comparable to that of a standard IRT method and the advantage of improved standardization by detection of the 2 isoforms.

Update: newborn screening for endocrinopathies.

Congenital hypothyroidism and congenital adrenal hyperplasia are included in many newborn screening (NBS) panels worldwide and in all state-sponsored programs in the United States. Both conditions meet the fundamental prerequisites for NBS: high incidence in the population; biomarkers in the dried blood specimen that are easily detected; and, effective therapies to lessen, if not prevent, the sequelae of late or no treatment. In this review, the history of NBS is discussed for these 2 conditions. The technologies and protocols used in their detection, and related subjects such as genetics, and treatment and outcomes, are also discussed.

Emergency preparedness for newborn screening and genetic services.

Patients identified in newborn screening programs can be among the most vulnerable during a disaster due to their need to have prompt diagnosis and medical management. Recent disasters have challenged the ability of newborn screening programs to maintain the needed continuity during emergency situations. This has significant implications for the newborn screening laboratories, the diagnostic confirmation providers, and the patients who either require diagnosis or maintenance of their therapeutic interventions. In 2007, the National Coordinating Center (NCC) for the Regional Genetics and Newborn Screening Collaboratives (RCs) sponsored a meeting involving representatives of the Regional Genetics and Newborn Screening Collaborative Groups, state newborn screening programs, providers of diagnosis and confirmation services, manufacturers of equipment, medical foods, and other treatments used in patients identified in newborn screening programs, and individuals from agencies involved in disaster response including the National Disaster Medical Service, the Centers for Disease Control and Prevention, the Emergency Management Assistance Compact, the Federal Emergency Management Agency, and others. In addition to developing contingency plans for newborn screening, we have considered other uses of genetics as it is used in DNA-based kinship identification of mass casualties. The meeting resulted in the description of a wide range of issues facing newborn screening programs, provider groups, and patients for which emergency preparedness development is needed in order that appropriate response is enabled.

Perinatal transfer of genetic information: developing an algorithm for reporting cystic fibrosis prenatal test results to the newborn screening program.

PURPOSE: This study explored the feasibility of statewide reporting of cystic fibrosis fetal diagnostic testing results to the newborn screening program through the birth hospital. METHODS: We evaluated trends in offering and documenting cystic fibrosis carrier screening among prenatal care providers through a survey of 100 medical records of patients who gave birth at St. Vincent's Hospital Manhattan. The hospital's protocol for reporting human immunodeficiency virus testing history to the state program was delineated and adapted in developing an algorithm for cystic fibrosis. Feedback from hospital staff with regard to data transcription and the prospect of transferring cystic fibrosis prenatal information was obtained. RESULTS: Of 98 patients who had prenatal records made available to the birth hospital, 62% had cystic fibrosis carrier screening, 14% declined screening, and 24% had no documentation of their screening history. The hospital staff viewed the transcription of information as relatively simple; however, missing information is a common occurrence that delays the process and results in incomplete data transfer. CONCLUSIONS: Perinatal transfer of cystic fibrosis prenatal information modeled on the system used for reporting human immunodeficiency virus testing history is feasible. However, it will require standardized reporting of cystic fibrosis screening and testing history on the mother's prenatal records among prenatal care providers.

Use of newborn screening program blood spots for exposure assessment: declining levels of perluorinated compounds in New York State infants.

Temporal biomonitoring studies can assess changes in population exposures to contaminants, but collection of biological specimens with adequate representation and sufficient temporal resolution can be resource-intensive. Newborn Screening Programs (NSPs) collect blood as dried spots on filter paper from nearly all infants born in the United States (U.S.). In this study, we investigated the use of NSP blood spots for temporal biomonitoring by analyzing perfluorooctane sulfonate (PFOS), perfluorooctane sulfonamide (PFOSA), perfluorohexane sulfonate (PFHxS), perfluorooctanoic acid (PFOA), and perfluorononanoic acid (PFNA) in 110 New York State (NYS) NSP blood spot composite specimens collected between 1997 and 2007, representing a total of 2640 infants. All analytes were detected in > or =90% of the specimens. Concentrations of PFOS, PFOSA, PFHxS, and PFOA exhibited significant exponential declines after the year 2000, coinciding with the phase-out in PFOS production in the U.S. Calculated disappearance half-lives for PFOS, PFHxS, and PFOA (4.4, 8.2, and 4.1 years, respectively) were similar to biological half-lives reported for retired fluorochemical workers. Our results suggest sharp decreases in perinatal exposure of NYS infants to PFOS, PFOSA, PFHxS, and PFOA and demonstrate, for the first time, the utility of NSP blood spots for assessment of temporal trends in exposure.

Novel mutations causing medium chain acyl-CoA dehydrogenase deficiency: under-representation of the common c.985 A > G mutation in the New York state population.

Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is one of the most common fatty acid oxidation disorders. A subpopulation of children with MCADD present with metabolic crisis induced by fasting or illness, become lethargic, and can experience seizures or coma, culminating in a 20% mortality rate during the first episode. The frequency of these metabolic crises can be reduced with early diagnosis and treatment. The prevalence of MCADD in the United States is estimated to be 1 per 15,000 with p.K304E (c.985A > G) accounting for 90% of mutant alleles. In an 18-month period after initiating screening, the New York State Newborn Screening Mass Spectrometry Laboratory screened 385,893 newborns and referred 511 samples with elevated (>or=0.3 micromol/L) octanoylcarnitine (C8) levels for molecular testing. Of these referrals, six p.K304E homozygotes and 154 heterozygotes were identified. Twenty infants were biochemically confirmed with MCADD, per report from the child's pediatrician and/or treatment center. In these 20 cases, p.K304E accounted for only 47.5% of the mutant alleles. Further testing showed a second variant, p.Y42H, accounted for 7.5% of mutant alleles while the remaining 45% were unknown. Samples from all diagnosed non-p.K304E homozygous infants, and samples with C8 levels >or=1.0 micromol/L were sequenced (n = 16). Six novel and seven previously reported mutations were detected. These results suggest that p.K304E has a far lower representation in New York newborns with MCADD than current literature estimates and its full mutational spectrum is still unknown.

Increase in congenital hypothyroidism in New York State and in the United States.

Mandated screening of newborns for congenital hypothyroidism (CH) in NYS was initiated in l978. Currently, every newborn screening program in the U.S. includes CH in its panel. Between 1978 and 2005, 7.4 million newborns were screened for CH in NYS. In NYS, between 1978 and 2005, the incidence of CH has increased by 138%. Nationwide (excluding NYS data), with nearly 58 million infants screened between 1987 and 2002, the incidence has increased 73% between 1987 and 2002. These data and possible reasons for the increases are discussed, though no definitive causes are identified.

Neonatal screening by DNA microarray: spots and chips.

Newborn screening (NBS) is a public-health genetic screening programme aimed at early detection and treatment of pre-symptomatic children affected by specific disorders. It currently involves protein-based assays and PCR to confirm abnormal results. We propose that DNA microarray technology might be an improvement over protein assays in the first stage of NBS. This approach has important advantages, such as multiplex analysis, but also has disadvantages, which include a high initial cost and the analysis/storage of large data sets. Determining the optimal technology for NBS will require that technical, public health and ethical considerations are made for the collection and extent of analysis of paediatric genomic data, for privacy and for parental consent.