Newborn Screening Cards Research Articles

Epigenetic Immune Cell Counting to Analyze Potential Biomarkers in Preterm Infants: A Proof of Principle in Necrotizing Enterocolitis.

Epigenetic immune cell counting is a DNA (de)methylation-based technique which can be used to quantify lymphocyte subsets on dried blood spots (DBS). The foregoing techniques allow for a retrospective investigation of immune cell profiles in newborns. In this study, we used this technique for determining lymphocyte subcounts as a potential biomarker for necrotizing enterocolitis (NEC). We investigated whether this technique can be implemented in the field of neonatology, by testing whether regulatory T cell (Treg) levels are pre-existently low in preterms with NEC. Newborn screening (NBS) cards from 32 preterms with NEC and 32 age- and weight-matched preterm controls, and 60 healthy term newborns, were analyzed. Relative and absolute cell counts were determined for CD3+, CD4+, CD8+, Th17, and Treg T cells. For both relative and absolute cell counts of CD3+, CD4+, CD8+, and Th17 T cells, significant differences were found between healthy term controls and both preterm groups, but not between preterm groups. For Tregs, no significant differences were found in either relative or absolute counts between any of the newborn groups. This study demonstrates the principle of epigenetic immune cell counting to analyze lymphocyte subsets in preterm neonates.

Multi-omics analysis from archival neonatal dried blood spots: limitations and opportunities.

Newborn screening (NBS) programs operate in many countries, processing millions of dried bloodspot (DBS) samples annually. In addition to early identification of various adverse health outcomes, these samples have considerable potential as a resource for population-based research that could address key questions related to child health. The feasibility of archival DBS samples for emerging targeted and untargeted multi-omics analysis has not been previously explored in the literature. This review aims to critically evaluate the latest advances to identify opportunities and challenges of applying omics analyses to NBS cards in a research setting. Medline, Embase and PubMed databases were searched to identify studies utilizing DBS for genomic, proteomic and metabolomic assays. A total of 800 records were identified after removing duplicates, of which 23 records were included in this review. These papers consisted of one combined genomic/metabolomic, four genomic, three epigenomic, four proteomic and 11 metabolomic studies. Together they demonstrate that the increasing sensitivity of multi-omic analytical techniques makes the broad use of NBS samples achievable for large cohort studies. Maintaining the pre-analytical integrity of the DBS sample through storage at temperatures below-20°C will enable this important resource to be fully realized in a research capacity.

Кожные проявления первичных иммунодефицитных состояний

Primary immune deficiency diseases (PIDDs) are a group of severe genetically determined diseases caused by a disorder of the immunity links: humoral and cell-mediated links, phagocytosis, complement system. PIDDs are characterized by severe atypical recurrent infections, as well as autoimmune and oncological processes. In the case of clinical symptoms, skin manifestations in PIDDs allow to suspect the presence of congenital disorders of immunity. There is a technique in laboratory diagnostics of PIDDs, that determines a decrease in the number of T- and B-lymphocytes based on measuring the levels of TREC (T-cell Receptor Excision Circle) and KREC (Kappa-deleting Recombination Excision Circle), which are by-products of recombination of T- and B-cell receptor genes. TREC and KREC levels can be assessed using a reagent kit for quantifying TREC and KREC DNA by PCR in real time. The analysis can be conducted using both whole blood and DNA obtained from dried blood stains collected during the national newborn screening program. Today, quantitative analysis of TREC and KREC in dried blood stains on newborn screening cards is becoming the method of choice for newborn screening of PIDDs. The inclusion of the PIDDs diagnosis in the federal newborn screening program will allow detecting disorders of the immune system before their clinical manifestations, which will allow to apply modern treatment protocols, reduce disability and ensure high patient survival. KEYWORDS: TREC, KREC, PIDDs, newborn screening, PCR, immunity, early diagnosis. FOR CITATION: Churyukina E.V., Kudlay D.A. Skin manifestations of primary immune deficiency diseases. Russian Medical Inquiry. 2022;6(2):98–104 (in Russ.). DOI: 10.32364/2587-6821-2022-6-2-98-104

Diagnosis of severe growth hormone deficiency in the newborn

OBJECTIVE Severe neonatal growth hormone deficiency (GHD) can cause recurrent hypoglycaemia. Early diagnosis is warranted. The aim of the study was to analyse the GH content in screening cards of 25 affected and 281 healthy newborns. PATIENTS AND MEASUREMENTS A total of 110 screening cards from ill newborns were sent to us for measuring GH content by a highly sensitive GH ELISA. Clinical information was obtainable in 61 cases. Severe GHD was defined by the presence of recurrent hypoglycaemia with a significant pituitary malformation or two additional pituitary hormone deficiencies. Screening cards from 281 healthy newborns (34.0-37.9 weeks) were prospectively analysed. RESULTS In 25 newborns (5 preterm), the definition of severe GHD was fulfilled based on recurrent hypoglycaemia in combination with malformation of the pituitary or midline structures in 21 cases and combined TSH and ACTH deficiency in four cases. The median GH concentration of those affected with severe GHD was 3.9 µg/L (range: 1.1-11.8), significantly below the previously reported reference range (P < .001). A GH concentration of 7 µg/L was confirmed as the cut-off for term newborns with the best accuracy (90.0% sensitivity and 98.7% specificity). The 95% reference range for healthy preterm newborns (n = 151) was 7.6-47.1 µg/L (median: 20.3 µg/L). CONCLUSIONS A GH content <7.0 µg/L in the newborn screening card confirms severe GHD with high accuracy. In preterm newborns, the lower limit of the reference interval was 0.6 µg/L higher than in term newborns. The newborn screening card is a valuable source for the very early diagnosis of GH deficiency.

Genetic Alterations Precede DNA Methylation Changes in Juvenile Myelomonocytic Leukemia

Introduction Juvenile myelomonocytic leukemia (JMML) is a rare and highly aggressive myeloid malignancy of early childhood. Approximately 95% of patients have at least one mutation that leads to hyperactive RAS signaling. Both the presence of multiple mutations at diagnosis as well as altered DNA methylation are associated with a poor prognosis. Whether altered DNA methylation leads to the acquisition of additional genetic mutations or is a secondary event to genetic mutations is unknown. In this study, we analyzed a total of 33 newborn blood screening (NBS) cards from children who went on to develop JMML later in childhood. Using next generation sequencing to detect both genetic mutations and DNA methylation status we sought to determine the sequence of events that lead to the development of JMML. Patients and Methods We identified 33 patients that were born in the state of California from 1990 to 2017 who were confirmed to have JMML and were previously consented to participate in a JMML tissue bank study. Guthrie cards from these 33 patients as well as 12 healthy controls were obtained from the California Biobank Program. DNA samples (20 ng) were sequenced for mutations using a custom amplicon-based sequencing approach (Paragon Genomics, Hayward, CA) targeting 26 genes that are recurrently mutated in JMML. For DNA methylation profiling, 300ng of genomic DNA was processed for bisulfite conversion using the TrueMethyl oxBS Module (Tecan Genomics Inc., Redwood City, CA.) according to manufacturer's guidelines and next generetation sequencing (NGS) libraries were prepared by targeting 3000 CpG loci using custom probes for Targeted Methyl-Seq assay (Tecan Genomics Inc., Redwood City, CA). Sequence reads were trimmed using Cutadapt to remove adapters and methylation status called using Bismark. Samples were classified into one of three methylation subgroups: Low, Intermediate, or High using 1386 probes according to an international consensus definition. Results At diagnosis of JMML, somatic mutations were identified in 32 of 33 patients. Of the 32 patients with a somatic mutation present upon diagnosis, the same mutations were found in NBS cards in 13 (41%) patients using a mutant allele fraction (MAF) cut-off of 1%. Clonal mutations (MAF &amp;gt;15%) were found in 9 of 32 (28%) patients. Patients who had a somatic mutation detected at birth were significantly younger at diagnosis with a median age of 7.1 months (range 2.5-91.6 months) compared to patients who had none (19.8 months; p-value = 0.042). However, no difference was observed in overall or event-free survival for patients with or without somatic mutations at birth. Methylation profiling classified all NBS cards as having "low" DNA methylation using an international, consensus definition (Figure 1A). Of the 33 patients, ten also had DNA methylation profiling performed on their diagnostic JMML sample which were classified as low (LM), intermediate (IM) or high methylation (HM) (Figure 1B). Amongst patients with both NBS and JMML methylation data available, 5 had mutations present in the NBS card and all 5 had lower methylation (β values) in their NBS card compared to their JMML sample (mean 0.38 vs 0.29, p-value = 0.002). Discussion We identified somatic Ras pathway mutations at birth in 13/32 (41%) of newborns that developed JMML later in their childhood. We found that patients with a somatic mutation detected at birth were significantly younger at diagnosis compared to patients who had no mutations at birth. DNA methylation profiling of NBS cards from children who went on to develop JMML revealed methylation signatures that were similar to normal age-matched controls. All newborn cards were classified as low methylation at birth. Specifically, amongst patients who had genetic mutations present at birth, methylation changes that were eventually detected in JMML samples were not present in the NBS cards. Therefore, we conclude that aberrant DNA methylation is a secondary event to genetic changes in JMML. Figure 1: Panel A: Methylation profiling of JMML NBS and control NBS samples, classified according to the international JMML methylation consensus signature. Panel B: Methylation profiling of paired NBS and diagnosis samples from JMML patients. The star indicates the NBS methylation profile. Heatmaps (A and B) show the beta values of 1386 CpG loci used for methylation classification. Figure 1 Disclosures Loh: Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees.

MicroRNA in dried blood spots from patients with Aagenaes syndrome and evaluation of pre-analytical and analytical factors.

Circulatory miRNAs are promising biomarkers. The feasibility of using miRNA from dried blood spots (DBS) was investigated using newborn screening cards from patients with cholestasis-lymphedema syndrome (Aagenaes syndrome) and controls. Total amount of miRNA and specific miRNAs from DBS were analyzed. miRNA was also obtained from newborn screening cards in patients with cholestasis-lymphedema syndrome/Aagenaes syndrome and in healthy newborns. No differences in miRNA concentrations were found between multispotted samples and samples with one single drop of blood and between central and peripheral punches. Ten repeated freeze-thaw cycles did not significantly change miRNA levels from controls. miR-299 (1.73-fold change, p = 0.034) and miR-365 (1.46-fold change, p = 0.011) were upregulated and miR-30c (0.72-fold change, p = 0.0037), miR-652 (0.85-fold change, p = 0.025), and miR-744 (0.72-fold change, p = 0.0069) were downregulated in patients with Aagenaes syndrome at birth compared to controls. miRNAs were not affected by multispotting or punch location and were stable throughout repeated freeze-thaw cycles. miRNA in dried blood spots could be used to detect differential expression of miRNA in newborns with Aagenaes syndrome and healthy controls in newborn screening cards. Dried blood spots may be a useful source to explore circulating miRNA as biomarkers. Circulating miRNAs can be useful biomarkers. miRNAs from dried blood spots were not affected by multispotting or punch location and were stable throughout repeated freeze-thaw cycles. Discrimination between patients and controls are allowed even with few individuals. Early after birth, patients with cholestasis-lymphedema syndrome exhibit miRNA profiles associated with liver fibrosis. This study demonstrated that newborn screening cards may be a useful source for studying miRNA as the technical variability is smaller than biological variation.

Diagnosis of severe growth hormone deficiency in the newborn.

Severe neonatal growth hormone deficiency (GHD) can cause recurrent hypoglycaemia. Early diagnosis is warranted. The aim of the study was to analyse the GH content in screening cards of 25 affected and 281 healthy newborns. A total of 110 screening cards from ill newborns were sent to us for measuring GH content by a highly sensitive GH ELISA. Clinical information was obtainable in 61 cases. Severe GHD was defined by the presence of recurrent hypoglycaemia with a significant pituitary malformation or two additional pituitary hormone deficiencies. Screening cards from 281 healthy newborns (34.0-37.9weeks) were prospectively analysed. In 25 newborns (5 preterm), the definition of severe GHD was fulfilled based on recurrent hypoglycaemia in combination with malformation of the pituitary or midline structures in 21 cases and combined TSH and ACTH deficiency in four cases. The median GH concentration of those affected with severe GHD was 3.9µg/L (range: 1.1-11.8), significantly below the previously reported reference range (P<.001). A GH concentration of 7µg/L was confirmed as the cut-off for term newborns with the best accuracy (90.0% sensitivity and 98.7% specificity). The 95% reference range for healthy preterm newborns (n=151) was 7.6-47.1µg/L (median: 20.3µg/L). A GH content <7.0µg/L in the newborn screening card confirms severe GHD with high accuracy. In preterm newborns, the lower limit of the reference interval was 0.6µg/L higher than in term newborns. The newborn screening card is a valuable source for the very early diagnosis of GH deficiency.

OR10-02 Diagnosis of Severe GH Deficiency in Newborns: New Reference Range for the Preterm and Confirmation of the GH Cut-Off

IntroductionInborn severe GHD is caused by rare disorders of pituitary morphogenesis or function and frequently associated with additional pituitary hormone deficiencies. Affected newborns commonly present with recurrent hypoglycemia; therefore early diagnosis and therapy is warranted. The GH content of the newborn screening card is a reliable indicator of severe neonatal GHD. Here, we studied the GH content in screening cards and the history of 25 newborns with severe GHD. In addition, we determined the reference range of the GH content in screening cards from 282 healthy preterm newborns.Patients and MethodsSince 2010, a total of 110 screening cards from hospitalized ill newborns were sent to our laboratory for measuring GH content. Using a questionnaire we obtained relevant clinical information from senders in 61 cases. Severe GHD was defined by the presence of recurrent neonatal hypoglycemia with either a significant cerebral MRI morphology or two additional pituitary hormone deficiencies. In addition, the GH content of screening cards from 282 healthy newborns born preterm with a gestational age at birth from 34.0 to 37.9 weeks was prospectively analyzed. The GH concentration of the eluate from the screening card was measured by a highly sensitive ELISA (Mediagnost, Germany); the GH serum concentration was calculated.ResultsIn 25 patients, the definition of severe GHD of the newborn was fulfilled; based on recurrent hypoglycemia in combination with ectopia of the neurohypophysis in 17, septum pellucidum agenesis plus opticus hypoplasia in two, severely hypoplastic pituitary gland in two, and combined TSH and ACTH deficiency with no cMRI findings in four newborns. Five newborns with severe GHD were preterm. The median GH concentration of the term newborns with severe GHD (n=20) was 3.9 µg/l (range; 1.1 to 11.8). This was significantly below the previously reported reference data from healthy term newborns (n=269) (median 16.4 µg/l; 95% reference range 7.0 to 39.4) (p<0.001). Using ROC plot analysis a GH serum concentration of 7.0 µg/l was identified as cut-off with the highest accuracy (90.0% sensitivity and 98.7% specificity). The median GH concentration of the 5 preterm newborns with severe GHD was 7.7 µg/l (range; 2.1 to 9.9). The newly determined 95% reference range for healthy newborns born preterm with a gestational age from 34.0 to 37.9 weeks (n=282) spanned from 7.9 to 41.1 µg/l with a median of 20.3 µg/l.ConclusionsA GH content below 7.0 ng/ml in the newborn screening card identified severe GHD with 90% sensitivity and 98.7% specificity. In preterm newborns, the lower limit of the 95% reference interval was by 0.9 µg/l higher than in term newborns. The newborn screening card is a valuable source for the diagnosis of GH deficiency in newborns and young infants.

Tandem mass spectrometry-based multiplex assays for α-mannosidosis and fucosidosis

Multiplex tandem mass spectrometry (MS/MS)-based enzyme activity assays for newborn screening (NBS) and diagnosis of lysosomal storage diseases (LSDs) in newborns, using dried blood spots (DBS) on newborn screening cards, have garnered much attention due to its sensitivity, high precision, and the capability to screen for an unprecedented number of diseases in a single assay. Herein we report the development of MS/MS-based enzyme assays for the diagnosis of α-mannosidosis and fucosidosis. These new protocols are able to distinguish untreated patients from random newborns, carriers and a post-bone marrow transplant patient. We have successfully multiplexed the α-mannosidosis assay with a multiplex MS/MS assay for the screening and diagnosis of other LSDs, namely Fabry, Pompe, MPS I, Gaucher, Niemann-Pick-A/B, and Krabbe diseases. Additionally, we also multiplexed the fucosidosis NBS assay with a 5-plex assay that tests for MPS-II, MPS-IIIB, MPS-IVA, MPS-VI and MPS-VII.

The utility of post-test newborn blood spot screening cards for epigenetic association analyses: association between HIF3A methylation and birth weight-for-gestational age

Identification of disease-associated epigenetic markers in early life might be useful for pre-emptive intervention to prevent diseases. Epigenome-wide association analyses using newborn blood spot screening cards are an anticipated field of research in Japan. Here, in this study, post-test dried blood spot (DBS) samples were anonymized, with only three attributes of gender, gestational age, and birth weight identified. We isolated DNA from DBS (n = 300) archived for more than 3 years. The median DNA yield (ng) per individual was 429 (interquartile range 300–565). In a model epigenetic analysis, we conducted a confirmative study on the known association between birth weight and hypoxia-inducible factor 3A (HIF3A) gene methylation. DNA methylation levels and cis-acting SNP genotypes (rs8102595 and rs3826795) were measured using EpiTYPER and Taqman assays, respectively. HIF3A methylation was positively associated with birth weight-for-gestational age centile (p = 0.021). While HIF3A methylation was associated with cis-genotypes (rs8102595, p = 2.08E−13; rs3826795, p = 3.63E−09), the association with birth weight centile was retained after adjusting for cis-genotypes (p = 0.029). Thus, we successfully reproduced the results reported previously by others, and demonstrated the usefulness of archived DBS in secondary use for epigenetic association analyses.

Validation of a Fast, Robust, Inexpensive, Two-Tiered Neonatal Screening Test algorithm on Dried Blood Spots for Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is one of the leading genetic causes of infant mortality with an incidence of 1:10,000. The recently-introduced antisense oligonucleotide treatment improves the outcome of this disease, in particular when applied at an early stage of progression. The genetic cause of SMA is, in >95% of cases, a homozygous deletion of the survival motor neuron 1 (SMN1) gene, which makes the low-cost detection of SMA cases as part of newborn screening programs feasible. We developed and validated a new SALSA MC002 melting curve assay that detects the absence of the SMN1 exon 7 DNA sequence without detecting asymptomatic carriers and reliably discriminates SMN1 from its genetic homolog SMN2 using crude extracts from newborn screening cards. Melting curve analysis shows peaks specific for both the SMN1 gene and the disease modifying SMN2 homolog. The detection of the SMN2 homolog, of which the only clinically relevant difference from the SMN1 gene is a single nucleotide in exon 7, was only used to confirm a correct reaction in samples that lacked the SMN1 gene, and not for SMN2 quantification. We retrieved 47 DBS samples from children with genetically-confirmed SMA, after informed consent from parents, and 375 controls from the national archive of the Dutch National Institute for Public Health and the Environment (RIVM). The assay correctly identified all anonymized and randomized SMA and control samples (i.e., sensitivity and specificity of 100%), without the detection of carriers, on the three most commonly-used PCR platforms with melting curve analysis. This test’s concordance with the second-tier ‘golden standard’ P021 SMA MLPA test was 100%. Using the new P021–B1 version, crude extracts from DBS cards could also be used to determine the SMN2 copy number of SMA patients with a high level of accuracy. The MC002 test showed the feasibility and accuracy of SMA screening in a neonatal screening program.

MON-LB047 GH Values in Serum and Blood Spots on Filter Paper Samples in Infants until 60 Days of Life by Electrochemiluminescence (ECLIA)

Congenital Growth Hormone Deficiency (GHD) in newborn is an infrequent condition, which can cause threat to life due mainly to hypoglycemia that begins in the first week of life. Severe neonatal GHD needs a fast diagnosis and the substitution with recombinant human GH because of the risk the morbi-mortality. A GH basal level (whether random or associated with spontaneous hypoglycemia) that distinguishes infants with GHD from those with GH sufficiency in the neonatal period is not conclusive. Few data have been reported about the GH measurements in serum and dried blood spots on filter paper samples in healthy neonates born appropriate for gestational age (AGA). Aims: To compare and correlate the GH values in serum and blood spots on filter paper samples in infants until 60 days of life. To establish GH reference values in healthy newborn until 15 days of life. To analyzed the correlation between GH concentrations in serum and blood spot and with hormones of the hypothalamic-pituitary-gonadal and adrenal axis. Subjects and methods: We analyzed 301 serum and whole blood spots samples obtained from AGA neonates between 2-60 days of life (2-15 days n=216, 16-30 days n=65 and 31-60 days n=20 (F: 154, M: 147). GH concentrations were measured by ECLIA Roche C600, which is calibrated against the 2nd International Standard Code 98/574 in serum as well as eluted from filter paper samples. The mean and SD GH value, P 5.0 and P 95.0 were calculated. The statistical analysis was performed by Spearman correlation coefficient. Results: 2-5 days: serum GH (ng/mL) mean value (SD): 20.81(15.8); P5: 6.30, P95: 42.30; 6-15 days: mean GH value 8.78 (4.34); P5: 3.29, P95: 15.19; 16-30 days: mean GH value: F: 10.55 (4.58), M: 7.72 (3.75) and 31-60 days: mean GH value: F: 4.67 (2.75), M 6.82 (4.57). Blood spots GH (ng/mL) mean value (SD): 2-5 days: 17.58 (9.95); P5: 5.68, P95: 33.60; 6-15 days: mean GH value: 10.31 (5.88), P5: 3.0, P95: 21.02; 16-30 days: mean GH value: F: 10.41 (6.01), M: 10.99 (7.78) and 31-60 days: mean GH value: 7.02 (5.25), M 9.12 (8.08). The Spearman correlation obtained between both techniques in parallel measurements was r2=0.85 (p< 0.0001). Conclusions: In agreement with different reports, our results showed high average GH levels in the first few days of life. Human GH secretion is pulsatile from the very beginning, however, newborn screening card spotted with blood during the first week of life, when neonatal hypersomatotropism is present, provides such high levels that, even at the nadir of GH pulsatility a basal value could contribute to detect GHD accurately. The good correlation obtained between both type of samples would indicate that the measurement of GH in dried blood spot samples is an appropriate and reliable method which can be incorporated in the diagnosis of neonatal GHD. The newborn screening samples may be a valuable resource for retrospectively assessing GH sufficiency if this neonatal window has passed. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Typical characteristics of children with congenital adrenal hyperplasia due to 11β-hydroxylase deficiency: a single-centre experience and review of the literature.

Background 11β-hydroxylase deficiency (11βOHD) is a rare disease representing the second most common cause of congenital adrenal hyperplasia (CAH) (5-8%) with an incidence of about 1:100,000. In contrast to 21-hydroxylase deficiency (21OHD), 11βOHD is not included in neonatal screening programmes. The objective of this study was to demonstrate the typical features of male patients with 11βOHD. Methods Clinical, biochemical and radiological data of patients with 11βOHD were analysed in this retrospective single-centre analysis. Results Six male patients of four unrelated families with 11βOHD were identified (0.1-13.5 years of chronological age [CA] at diagnosis). The predominant symptoms were arterial hypertension, tall stature and precocious pseudopuberty. Bone ages (BAs) were remarkably advanced at diagnosis in four index patients (median difference BA-CA: 5.5 years, range 1.5-9.2 years). Homozygous mutations were identified in exon 7 (c.1179_1180dupGA [p.Asn394Argfs*37]) and exon 8 (c.1398+2T>C) of the CYP11B1 gene leading both to a complete loss of function. The latter mutation has not yet been described in databases. 11βOHD was identified by the measurement of 11-deoxycortisol in a newborn screening card of one patient retrospectively. Testicular adrenal rest tumours (TARTs) were detected in three patients at 3.7 years, 11 years and 14.4 years. Conclusion The diagnosis of CAH due to 11βOHD is delayed and should be suspected in children with arterial hypertension, tall stature and precocious pseudopuberty. Patients may develop TARTs as early as infancy. 11βOHD should be included in newborn screening programmes, at least in newborns of index families, to allow early diagnosis and the start of treatment to reduce morbidity.

Newborn screening for Prader-Willi syndrome is feasible: Early diagnosis for better outcomes.

Prader-Willi syndrome (PWS), is a complex genetic disease affecting 1/15,000 individuals, characterized by lack of expression of genes on the paternal chromosome 15q11-q13 region. Clinical features include central hypotonia, poor suck, learning and behavior problems, growth hormone deficiency with short stature, hyperphagia, and morbid obesity. Despite significant advances in genetic testing, the mean age for diagnosis in PWS continues to lag behind. Our goal was to perform a pilot feasibility study to confirm the diagnosis utilizing different genetic technologies in a cohort of 34 individuals with genetically confirmed PWS and 16 healthy controls from blood samples spotted and stored on newborn screening (NBS) filter paper cards. DNA was isolated from NBS cards, and PWS testing performed using DNA methylation-specific PCR (mPCR) and the methylation specific-multiplex ligation dependent probe amplification (MS-MLPA) chromosome 15 probe kit followed by DNA fragment analysis for methylation and copy number status. DNA extraction was successful in 30 of 34 PWS patients and 16 controls. PWS methylation testing was able to correctly identify all PWS patients and MS-MLPA was able to differentiate between 15q11-q13 deletion and non-deletion status and correctly identify deletion subtype (i.e., larger Type I or smaller Type II). mPCR can be used to diagnose PWS and MS-MLPA testing to determine both methylation status as well as the type of deletion or non-deletion status from DNA extracted from NBS filter paper. We propose that PWS testing in newborns is possible and could be included in the Recommended Uniform Screening Panel after establishing a validated cost-effective method.

PCP14 - Early Check: A Voluntary Newborn Screening Program and Research Registry to Facilitate Clinical Trials in Pre-Symptomatic Infants with Rare Diseases

Newborn screening (NBS) is designed for pre-symptomatic identification of conditions for which there are effective treatments that must begin early. Central to NBS policy is evidence that pre-symptomatic treatment is more effective than treatment after symptoms appear. Unfortunately, such evidence is difficult to amass because most conditions are rare and not diagnosed or treated early. Researchers and advocates thus find themselves in a classic “Catch 22” situation—NBS for a condition will not happen without sufficient evidence, but gathering this evidence requires large-scale population screening. This causes a formidable barrier to translational research. The purpose of this session will be to describe a statewide, voluntary NBS program in North Carolina which will screen for spinal muscular atrophy and fragile x syndrome. Our goal is to offer voluntary screening to all 120,000 families who give birth in North Carolina each year, starting in 2018. Parents may give permission for participation either before their child’s birth or up to one month after birth. Permission will be obtained through an online electronic module. An extra sample of blood will be taken on the standard NBS card and sent to the North Carolina State Laboratory of Public Health for screening. Upon a positive screen, families will be contacted and diagnostic testing will be conducted. Confirmed cases will be provided genetic counseling and baseline medical and developmental assessments will be conducted. Families will be asked to join the research registry which will be used to facilitate access to clinical trials and other treatment options and to recruit for natural history studies. Our ultimate goal is to create a sustainable infrastructure, available to other researchers, in which voluntary NBS screening is offered in a way that benefits children, supports families, provides early access to treatment options, and generates evidence that contributes to rational policy decisions.

Analysis of MicroRNA Expression in Newborns with Differential Birth Weight Using Newborn Screening Cards.

Birth weight is an early predictor for metabolic diseases and microRNAs (miRNAs) are proposed as fetal programming participants. To evaluate the use of dried blood spots (DBS) on newborn screening cards (NSC) as a source of analyzable miRNAs, we optimized a commercial protocol to recover total miRNA from normal birth weight (NBW, n = 17–20), low birth weight (LBW, n = 17–20) and high birth weight (macrosomia, n = 17–20) newborns and analyzed the relative expression of selected miRNAs by stem-loop RT-qPCR. The possible role of miRNAs on the fetal programming of metabolic diseases was explored by bioinformatic tools. The optimized extraction of RNA resulted in a 1.2-fold enrichment of miRNAs respect to the commercial kit. miR-33b and miR-375 were overexpressed in macrosomia 9.8-fold (p < 0.001) and 1.7-fold, (p < 0.05), respectively and miR-454-3p was overexpressed in both LBW and macrosomia (19.7-fold, p < 0.001 and 10.8-fold, p < 0.001, respectively), as compared to NBW. Potential target genes for these miRNAs are associated to cyclic-guanosine monophosphate (cGMP)-dependent protein kinase (PKG), mitogen-activated protein kinase (MAPK), type 2 diabetes, transforming growth factor-β (TGF-β)and Forkhead box O protein (FoxO) pathways. In summary, we improved a protocol for analyzing miRNAs from NSC and provide the first evidence that birth weight modifies the expression of miRNAs associated to adult metabolic dysfunctions. Our work suggests archived NSC are an invaluable resource in the search for fetal programming biomarkers.

Next generation sequencing as a follow-up test in an expanded newborn screening programme

ObjectivesContrary to many western European countries, most south-eastern European countries do not have an expanded newborn screening (NBS) program using tandem mass spectrometry. This study would represent one of the first expanded NBS studies in south-eastern Europe and will enable the estimation of the incidences of IEM in Slovenia. We proposed an expanded NBS approach including next-generation sequencing (NGS) as a confirmational analysis. Design & methodsWe conducted a pilot study of expanded NBS for selected inborn errors of metabolism (IEM) in Slovenia including 10,048 NBS cards. We used an approach including tandem mass spectrometry followed by second tier tests including NGS. Based on the NBS results, 85 children were evaluated at a metabolic follow-up; 80 of them were analyzed using NGS. ResultsAltogether, glutaric acidemia type 1 was confirmed in one patient who was a compound heterozygote for two known causative GCDH variants. A patient with suspected very long-chain acyl-CoA dehydrogenase deficiency had negative metabolic follow-up tests, but had two heterozygous ACADVL variants; one known disease-causing variant and one indel, namely c.205-8_205-7delinsGC, that is predicted to be causative. Nine participants had elevated metabolites characteristic of 3-methylcrotonyl-CoA carboxylase deficiency, 2 of them had known causative homozygous variants in MCCC1. The other seven were heterozygous; two had a novel genetic variant c.149_151dupCCA (p.Thr50dup). Cumulative incidences of IEM in Slovenia were similar to other European countries. ConclusionsNGS proved to be valuable in explaining the abnormal metabolite concentrations in NBS as it enabled the differentiation between affected patients and mere heterozygotes, and it improved the turnaround time of genetic analysis. The results of this study will be instrumental in the routine implementation of expanded NBS in Slovenia.

Newborn screening for severe combined immunodeficiency: Evaluation of a commercial T-cell receptor excision circle-based method in Victorian dried blood spots.

Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiency and is fatal in infancy if untreated. As early diagnosis is associated with improved outcomes, SCID is an ideal condition to consider for inclusion in a newborn screening (NBS) programme in Australia. In this feasibility study, we evaluated the EnLite Neonatal TREC kit for detection of T-cell receptor excision circles (TRECs) from NBS dried blood spots for the identification of known SCID patients in Victoria. TREC copies/μL were measured retrospectively in 14 children diagnosed with SCID or complete DiGeorge syndrome (CDGS) from 2005 to 2015 at the Royal Children's Hospital, Melbourne. In addition, TREC copies/μL were measured for 501 prospective de-identified NBS cards. Of 14 known SCID or CDGS samples, 11 were correctly identified as presumptive positive samples with low or undetectable TREC on duplicate testing. The remaining three samples also had low or undetectable TREC on duplicate testing but were considered invalid due to insufficient β-actin DNA amplification. Of the 501 prospective NBS samples, none were identified as presumptive positive samples on duplicate testing. The EnLite Neonatal TREC kit correctly identified known SCID or CDGS patients as presumptive positive samples, and initial cut-offs for TREC and β-actin in the Victorian NBS population were determined. A larger pilot study is required to confirm these proposed cut-offs and to evaluate the cost and implementation of this screening programme in Victoria, Australia. Overall, this study provides preliminary data to support the introduction of this assay to the NBS programme in Victoria.

Mass Spectrometry but Not Fluorimetry Distinguishes Affected and Pseudodeficiency Patients in Newborn Screening for Pompe Disease.

Deficiency of the lysosomal enzyme acid α-glucosidase (GAA) causes Pompe disease. Newborn screening for Pompe disease is ongoing, and improved methods for distinguishing affected patients from those with pseudodeficiency, especially in the Asian population, would substantially reduce the number of patient referrals for clinical follow-up. We measured the enzymatic activity of GAA in dried blood spots on newborn screening cards (DBS) using a tandem mass spectrometry (MS/MS) assay. The assay displayed a relatively large analytical range compared to the fluorimetric assay with 4-methylumbelliferyl-α-glucoside. DBS from newborns confirmed to have infantile-onset Pompe disease (IOPD, n = 11) or late-onset Pompe disease (LOPD) (n = 12) and those from patients bearing pseudodeficiency alleles with or without Pompe mutations, or Pompe disease carriers (n = 230) were studied. With use of the MS/MS GAA assay in DBS, 96% of the pseudodeficiency newborns and all of the Pompe disease carriers were well separated from the IOPD and LOPD newborns. The fluorimetric assay separated <10% of the pseudodeficiencies from the IOPD/LOPD group. The relatively large analytical range MS/MS GAA assay but not the fluorimetric assay in DBS provides a robust approach to reduce the number of referrals and should dramatically facilitate newborn screening of Pompe disease.

Newborn Screening for Severe Combined Immunodeficiency-A History of the TREC Assay

Infants born with T cell lymphopenias, especially severe combined immunodeficiency (SCID) are at risk for serious, often fatal infections without intervention within the first year or two of life. The majority of these disorders can be detected through the use of the T cell recombination excision circle assay (TREC assay.) The TREC assay detects the presence of non-replicating, episomal DNA that is formed during T cell development. This assay initially developed to measure thymic output during aging and HIV infection, has undergone modifications for the purpose of newborn screening (NBS) for SCID. To meet the requirements for inclusion on NBS panels, the assay needed to utilize blood from dried blood spots on NBS cards, and be both sensitive and specific, avoiding the costs of false positives. Currently, the assay relies upon real time, quantitative PCR (RT-qPCR) to detect TRECs in punches taken from dried blood spots. This review seeks to highlight some of the early work leading up to the initial implementation of the TREC assay for SCID detection, and the subsequent revisions made to optimize the assay.