Utility of specific amino acid ratios in screening for pyruvate dehydrogenase complex deficiencies and other mitochondrial disorders associated with congenital lactic acidosis and newborn screening prospects.

Pyruvate dehydrogenase complex deficiencies (PDCDs) and other mitochondrial disorders (MtDs) can (a) result in congenital lactic acidosis with elevations of blood alanine (Ala) and proline (Pro), (b) lead to decreased ATP production, and (c) result in high morbidity and mortality. With ~140,000 live births annually in Ohio and ~1 in 9,000 overall prevalence of MtDs, we estimate 2 to 3 newborns will have PDCD and 13 to 14 others likely will have another MtD annually. We compared the sensitivities of plasma amino acids (AA) Alanine (Ala), Alanine:Leucine (Ala:Leu), Alanine:Lysine and the combination of Ala:Leu and Proline:Leucine (Pro:Leu), in subjects with known primary-specific PDCD due to PDHA1 and PDHB mutations vs controls. Furthermore, in collaboration with the Ohio newborn screening (NBS) laboratory, we determined Ala and Pro concentrations in dried blood spot (DBS) specimens using existing NBS analytic approaches and evaluated Ala:Leu and Pro:Leu ratios from DBS specimens of 123,414 Ohio newborns in a 12-month period. We used the combined Ala:Leu ≥4.0 and Pro:Leu ≥3.0 ratio criterion from both DBS and plasma specimens as a screening tool in our retrospective review of newborn data. The screening tool applied on DBS and/or plasma (or serum) AA specimens successfully identified three unrelated females with novel de novo PDHA1 mutations, one male with a novel de novo X-linked HSD17B10 mutation, and a female with VARS2 mutations. This work lays the first step for piloting an NBS protocol in Ohio for identifying newborns at high risk for primary-specific PDCD and other MtDs who might benefit from neonatal diagnosis and early institution of known therapy and/or potential novel therapies for such disorders.

Congenital Hypothyroidism 3-Year Follow-Up Project: Region 4 Midwest Genetics Collaborative Results.

To identify the 3-year follow-up management and education patterns of primary care clinicians and pediatric endocrinologists for children diagnosed with congenital hypothyroidism (CH) through newborn screening programs, the Region 4 Midwest Genetics Collaborative, made up of seven regional states (Illinois, Indiana, Kentucky, Michigan, Minnesota, Ohio, Wisconsin), performed a survey study of parents and physicians caring for children identified with CH. The clinicians and parents of 409 children with CH regionally identified in 2007 were invited to participate in a voluntary survey. Responses relating to treatment, monitoring practices, educational resources, genetic counseling, and services provided/received were collected from 214 clinicians and 77 parents. In total, 99% had undergone a confirmatory test following positive newborn screening and 55% had imaging at diagnosis, but only 50% were identified as having the etiology identified. Thyroid withdrawal challenge testing was the choice method for re-evaluating thyroid function, but the approach varied. Clinician and parent responses to education and genetic counseling also differed. Clinicians report face-to-face education as the most common method, with less than 50% providing handouts to patients. Only 14% of patients were referred to a genetics counselor. Of parents reporting on their educational experience, 86% received face-to-face education from a pediatric endocrinologist and 4% received education from a genetic counselor. Only 65%, however, were satisfied with their education. These survey data suggest a lack of a standardized approach to diagnosis, follow-up, education, and genetic counseling. This collaborative effort provides insight into developing three-year follow-up, education and genetic counseling guidelines for children diagnosed with CH.

Congenital Hypothyroidism Long-Term Follow-up Project: Navigating the Rough Waters of a Multi-Center, Multi-State Public Health Project.

The Region 4 Midwest Genetics Collaborative, made up of seven regional states (Illinois, Indiana, Kentucky, Michigan, Minnesota, Ohio, and Wisconsin), brought together pediatric endocrinologists, state laboratory experts, public health follow-up specialists, and parents of children with congenital hypothyroidism (CH) to identify the three-year follow-up management and education patterns of primary care clinicians and pediatric endocrinologists in the care of children diagnosed with CH by state newborn screening (NBS) programs. Among a number of challenges, each state had different NBS methods, data systems, public health laws, and institutional review board (IRB) requirements. Furthermore, the diagnosis of CH was complicated by the timing of the NBS sample, the gestational age, weight, and co-morbidities at delivery. There were 409 children with CH identified through NBS in 2007 in the seven state region. The clinician of record and the parents of these children were invited to participate in a voluntary survey. Approximately 64 % of clinician surveys were collected with responses to questions relating to treatment, monitoring practices, educational resources, genetic counseling, and services provided to children with confirmed CH and their families. Nearly one-quarter (24 %) of parents surveyed responded to questions relating to treatment, education, genetic counseling, resources, and services they received or would like to receive. De-identified data from six of the seven states were compiled for analysis, with one state being unable to obtain IRB approval within the study timeline. The data from this collaborative effort will improve state follow-up programs and aid in developing three-year follow-up guidelines for children diagnosed with CH. To aid in the facilitation of similar public health studies, this manuscript highlights the challenges faced, and focuses on the pathway to a successful multi-state public health endeavor.

Sequencing from dried blood spots in infants with "false positive" newborn screen for MCAD deficiency.

BACKGROUND: Newborn screening (NBS) for medium chain acyl-CoA dehydrogenase deficiency (MCADD), one of the most common disorders identified, uses measurement of octanoylcarnitine (C8) from dried blood spots. In the state of Ohio, as in many places, primary care providers, with or without consultation from a metabolic specialist, may perform "confirmatory testing", with the final diagnostic decision returned to the state. Confirmatory testing may involve measurement of metabolites, enzyme analysis, mutation screening, or sequencing. We now report sequencing results for infants said to have "false positive" NBS results for MCAD deficiency, or who died before confirmatory testing could be performed. METHODS: Dried blood spots (DBS) were obtained from all 18 available NBS cards identified as "false positive" by NBS for the 3 year period after screening began in Ohio in 2003 (N=20, thus 2 had no DBS available), and from all 6 infants with abnormal screens who died before confirmatory testing could be obtained. DNA extracted from DBS was screened for the common c.985A>G mutation in exon 11 of the ACADM gene, using a specific restriction digest method, followed by sequencing of the 12 exons, intron-exon junctions, and several hundred base pairs of the 5' untranslated region. RESULTS: The NBS cut-off value for C8 used was 0.7 µmol/L. Sequencing of ACADM in six neonates with elevated C8 on NBS who died before confirmatory testing was obtained did not identify any significant variants in the coding region of the gene, suggesting that MCADD was not a contributing factor in these deaths. The mean C8 for the 18 surviving infants labeled as "False Positives" was 0.90 (95%CI 0.77-1.15), much lower than the mean value for confirmed cases. Ten of the 18 were premature births weighing <1200 g, the rest were normal sized and full term. Eight infants, mostly full term with appropriate birth weight, were heterozygous for the common c.985A>G mutation; one of those also has a novel sequence change identified in exon 9 that predicts a PRO to LEU change at residue 258 of the protein. Both the phase and any possible clinical significance of the variant are unknown, but several lines of evidence suggest that it could lead to protein malfunction. That child had an NBS C8 of 2.2, more than double the mean for the False Positive group. Unfortunately, the study design did not provide clinical outcome data, but the child is not known to have presented clinically by age 7 years. CONCLUSIONS: These results suggest that sequencing of ACADM from dried blood spots can be one useful follow-up tool to provide accurate genetic counseling in the situation of an infant with elevated C8 on NBS who dies before confirmatory testing is obtained. Of surviving neonates, there appear to be two populations of infants with false positive NBS C8 values: 1) term AGA infants who are heterozygous for the common c.985A>G mutation, and, 2) premature infants, regardless of carrier status. The finding of two sequence variants in an infant reported to the state as not affected suggests the possibility that some infants with two mutations may be reported as normal at follow-up. State registries may wish to consider asking that metabolic specialists, who are most familiar with the variability of these rare disorders, be involved in the final diagnostic evaluation. Finally, providers may wish to consider ACADM sequencing, or other diagnostic testing, as part of the confirmatory evaluation for infants with NBS C8 concentrations that are significantly above the cut-off value, even if plasma and urine metabolites are not strikingly increased.

The effects of gestational age and birth weight on false-positive newborn-screening rates.

OBJECTIVE: Newborn-screening false-positive rates (FPRs) are disproportionately increased in preterm infants. The objective of this study was to determine variation in newborn screening FPRs according to birth weight and gestational age. Our secondary objective was to examine the effect of postnatal age on FPRs in preterm infants. METHODS: The Ohio State Newborn Screening Program Database was analyzed to determine the overall and birth weight-specific FPRs for 18 analytes. Data were stratified into birth weight categories (<1000 g, 1000-1499 g, 1500-2499 g, 2500-3999 g, and >4000 g). In addition, to examine the effect of postnatal age on FPRs, we examined the 2 analytes with the highest FPRs, thyrotropin with back-up thyroxine and 17-hydroxyprogesterone, in infants whose gestational age was <32 weeks, determined on the basis of postnatal age at screening. RESULTS: Data from 448 766 neonates were reviewed. Infants with very low birth weight (VLBW) comprised 1.9% of the study cohort, but accounted for 18% of false-positive results. For 14 of 18 analytes studied, FPRs increased with decreasing birth weight/gestational age and were significantly increased in infants with VLBW compared with infants who weighed 2500 to 3999 g (P < .001). Thyrotropin/back-up thyroxine and 17-hydroxyprogesterone accounted for 62% of total false-positive results in VLBW infants. When blood specimens were collected at a postnatal age of ≥ 48 hours in infants born at <32 weeks, a 44% relative reduction in 17-hydroxyprogesterone false-positive results was detected. CONCLUSIONS: False-positive newborn-screening rates are disproportionately increased in VLBW infants. FPRs may be reduced by delaying screening of <32 weeks' gestation, preterm infants until 24 to 48 hours' postnatal age.

Finding blunders in thyroid testing: experience in newborns.

We evaluated thyroxin (T4) and thyroid-stimulating hormone (TSH) data along with clinical information from 600,000 newborns. We looked for certain combinations of tests and clinical data that were questionable and possibly mistaken. Our approach suggests that certain combinations of test results, especially the presence of missing results deserved further evaluation for possible blunders. We found that missing tests were frequently the result of oversight. The laboratory used the well-known standard blood-spot-on-filter paper methods for TSH and T4. For quantitation of TSH and T4, we used the time-resolved fluoroimmunoassay available from Perkin Elmer. We found 56 babies with confirmed primary congenital hypothyroidism (PCH) in a total of 600,000 patients. We also found 18 sets of results in the same 600,000 babies that gave inconsistent findings, had missing values, and (or) possible misinterpretations of the clinical and (or) laboratory data. What is an acceptable mistake rate? All mistakes are unacceptable, but there is likely some irreducible mistake rate, and efforts to reduce the mistake or blunder rate still further may not be cost-effective. What can be done is to study the mistake rate per 600,000 babies from year to year; the mistake rate should be decreasing or not changing. This assumes a stable cohort of babies; an assumption that may be acceptable. We applied a form of pattern recognition to identify cases of possible blunders and missing values in either the laboratory or clinical data. What is clear is that we apparently identified some blunders. The 18 mistakes per 600,000 babies may be "very low" and acceptable. We recommend that seeking ever decreasing mistakes is the way to go, and the level of monitoring the data should be very intense given the serious consequences of mis-diagnosed thyroid disorders.