Thyroid Hormone Function in Small for Gestational Age Term Newborns.

OBJECTIVE: To test the hypothesis that term-born small for gestational age (SGA) neonates have elevated thyroid-stimulating hormone (TSH) concentrations and an increased incidence of congenital hypothyroidism compared with non-SGA term neonates. STUDY DESIGN: This retrospective cohort study included all term neonates screened in Wisconsin in 2015 and 2016. The cohort was divided based on SGA status, defined as birth weight <10th percentile as calculated from the World Health Organization's sex-specific growth charts for age 0-2 years. TSH concentration on first newborn screening performed between birth and 96 hours of life and incidence of congenital hypothyroidism were compared between the SGA and non-SGA groups. RESULTS: A total of 115 466 term neonates, including 11 498 (9.96%) SGA neonates, were included in the study. TSH concentration and incidence of congenital hypothyroidism was significantly higher in the SGA group, but only TSH concentration remained significant when adjusted for potential confounding variables. CONCLUSIONS: Our data do not support a higher incidence of congenital hypothyroidism in term SGA neonates after adjusting for potential confounders. However, TSH concentrations were higher in term SGA neonates compared with term non-SGA neonates. The effects of mild thyroid hormone dysfunction on neurodevelopmental outcomes and development of chronic medical conditions merit long-term study.

Carrier frequency estimation of Zellweger spectrum disorder using ExAC database and bioinformatics tools.

PURPOSE: We aimed to estimate the carrier frequency of Zellweger spectrum disorder (ZSD), a rare autosomal recessive disease, and the associated disease incidence based on data from the Exome Aggregation Consortium (ExAC) of approximately 60,000 individuals. METHODS: We obtained variants from ExAC in 13 PEX genes associated with ZSD. Potentially pathogenic missense variants were identified with computational variant analysis tools according to three stringency levels. Using variants classified as potentially pathogenic, we estimated the carrier frequency and the associated incidence for the entire ExAC population and its subpopulations. We also evaluated variants based on pathogenicity criteria for sequence variant interpretation outlined by the American College of Medical Genetics and Genomics (ACMG) and calculated the carrier frequency and incidence based on those variants. RESULTS: The bioinformatically estimated incidence rate of ZSD in the ExAC population is 1 in 83,841 using our least stringent pathogenicity cutoff. Under clinical guidelines outlined by ACMG, the estimated incidence is 1 in 3,275,751 births. CONCLUSION: We outlined a process for estimating the ZSD disease carrier frequency and incidence in a large consortium using bioinformatics tools. Our results are close to current newborn screening estimates in New York of 1 in 90,000 births, estimated from 1.08 million screenings.

Increased Congenital Hypothyroidism Detection in Preterm Infants with Serial Newborn Screening.

OBJECTIVES: To determine the incidence of congenital hypothyroidism in preterm infants and to identify associated risk factors. STUDY DESIGN: A population-based cohort study was performed in preterm infants born at <32 weeks of gestational age between 2012 and 2016 in Wisconsin. Newborn screening (NBS) results and demographic data were obtained from the Wisconsin State Laboratory of Hygiene. Congenital hypothyroidism was subdivided to early TSH elevation (eTSH) and delayed TSH elevation (dTSH). Multivariate logistic regression analyses were performed to identify demographic factors associated with dTSH. RESULTS: A total of 3137 preterm infants born at 22-31 weeks of gestational age were included in the study. Mean gestational age was 28.4 ± 2.4 weeks and mean birth weight was 1191 ± 399 g. Forty-nine infants were diagnosed with congenital hypothyroidism. The overall incidence of congenital hypothyroidism was 1.56%, including a 0.13% incidence of eTSH and a 1.43% incidence of dTSH. Birth weight <1000 g, multiple gestation, and initial TSH level were identified as independent predictors for dTSH. CONCLUSION: Targeted serial NBS in Wisconsin led to a higher rate of diagnosis of congenital hypothyroidism in preterm infants than has been reported previously. The majority (92%) of congenital hypothyroidism cases were diagnosed with dTSH. Birth weight <1000 g, multiple gestation, and elevated initial TSH level were associated with increased risk for development of dTSH. We recommend obtaining targeted serial NBS in preterm infants (<32 weeks of gestational age) to improve the detection of congenital hypothyroidism.

Is Low FMR1 CGG Repeat Length in Males Correlated with Family History of BRCA-Associated Cancers? An Exploratory Analysis of Medical Records.

The FMR1 gene has been studied extensively with regard to expansions and premutations, but much less research has focused on potential effects of low CGG repeat length. Previous studies have demonstrated that BRCA1/2 positive women are more likely to have an FMR1 genotype with one low CGG allele, and that women with both FMR1 alleles in the low CGG repeat range are more likely to have had breast cancer compared to women with normal numbers of CGG repeats. However, there has been no research as to whether low CGG repeat length impacts cancer risks in men. Therefore, this study aimed to examine cancer incidence and related risk factors in men with low CGG repeat length in the FMR1 gene. We utilized subject data from the Marshfield Personalized Medicine Research Project to compare cancer-related diagnoses between 878 males with low CGG repeat length (< 24 repeats) and 368 male controls with CGG repeats in the normal range (24 to 40 repeats). We utilized ICD-9 codes to examine various cancer diagnoses, family histories of cancer, other non-malignant neoplasms, cancer surveillance, and genetic susceptibility. Men with low CGG repeats were identified to have significantly higher rates of family history of any cancer type (p = 0.011), family history of any BRCA-associated cancer (p = 0.002), and specifically, family history of prostate cancer (p = 0.007). The mean number of BRCA-associated cancer diagnoses (breast, prostate, pancreatic, and melanoma) per individual in the low CGG group was slightly higher than that of the control group, with this difference trending toward significance (p = 0.091). Additionally, men with low CGG repeats had significantly higher rates of connective/soft tissue neoplasms (p = 0.026). Additional research is needed to replicate the observations reported in this preliminary exploratory study, particularly including verification of ICD-9 codes and family history by a genetic counselor.

Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study.

PURPOSE: Many regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology. METHODS: An NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen-positive samples with one CFTR mutation. RESULTS: The NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified. CONCLUSION: The NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.

Prevalence and mutation analysis of short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) detected on newborn screening in Wisconsin.

Short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD), also called 2-methylbutyryl CoA dehydrogenase deficiency (2-MBCDD), is a disorder of l-isoleucine metabolism of uncertain clinical significance. SBCADD is inadvertently detected on expanded newborn screening by elevated 2-methylbutyrylcarnitine (C5), which has the same mass to charge (m/s) on tandem mass spectrometry (MS/MS) as isovalerylcarnitine (C5), an analyte that is elevated in isovaleric acidemia (IVA), a disorder in leucine metabolism. SBCADD cases identified in the Hmong-American population have been found in association with the c.1165 A>G mutation in the ACADSB gene. The purposes of this study were to: (a) estimate the prevalence of SBCADD and carrier frequency of the c.1165 A>G mutation in the Hmong ethnic group; (b) determine whether the c.1165 A>G mutation is common to all Hmong newborns screening positive for SBCADD; and (c) evaluate C5 acylcarnitine cut-off values to detect and distinguish between SBCADD and IVA diagnoses. During the first 10years of expanded newborn screening using MS/MS in Wisconsin (2001-2011), 97 infants had elevated C5 values (≥0.44µmol/L), of whom five were Caucasian infants confirmed to have IVA. Of the remaining 92 confirmed SBCADD cases, 90 were of Hmong descent. Mutation analysis was completed on an anonymous, random sample of newborn screening cards (n=1139) from Hmong infants. Fifteen infants, including nine who had screened positive for SBCADD based on a C5 acylcarnitine concentration ≥0.44µmol/L, were homozygous for the c.1165 A>G mutation. This corresponds to a prevalence in this ethnic group of being homozygous for the mutation of 1.3% (95% confidence interval 0.8-2.2%) and of being heterozygous for the mutation of 21.8% (95% confidence interval 19.4-24.3%), which is consistent with the Hardy-Weinberg equilibrium. Detection of homozygous individuals who were not identified on newborn screening suggests that the C5 screening cut-off would need to be as low as 0.20µmol/L to detect all infants homozygous for the ACADSB c.1165 A>G mutation. However, lowering the screening cut-off to 0.20 would also result in five "false positive" (non-homozygous) screening results in the Hmong population for every c.1165 A>G homozygote detected. Increasing the cut-off to 0.60µmol/L and requiring elevated C5/C2 (acetylcarnitine) and C5/C3 (propionylcarnitine) ratios to flag a screen as abnormal would reduce the number of infants screening positive, but would still result in an estimated 5 infants with SBCADD per year who would require follow-up and additional biochemical testing to distinguish between SBCADD and IVA diagnoses. Further research is needed to determine the clinical outcomes of SBCADD detected on newborn screening and the c.1165 A>G mutation before knowing whether the optimal screening cut-off would minimize true positives or false negatives for SBCADD associated with this mutation.

Novel mutation in TP63 associated with ectrodactyly ectodermal dysplasia and clefting syndrome and T cell lymphopenia.

A male child with clinical features consistent with EEC/EECUT plus syndrome (ectrodactyly, ectodermal dysplasia, clefting, urinary tract abnormalities, and thymic abnormalities) including mild ectodermal abnormalities, ectrodactyly of hands and feet, cleft palate, bilateral hydronephrosis, and T cell lymphopenia is reported. He was noted to have T cell receptor excision circle (TREC) analysis below the cutoff for normal on newborn screening and T cell lymphopenia on further immunologic evaluation. A novel, presumably pathogenic de novo 3 bp deletion in exon 7 of TP63 (c.970_972delATT; NCBI Reference Sequence NM_003722.4) was identified. This observation provides supporting evidence for the association between TP63 mutations and EECUT plus syndrome. Clinicians caring for infants presenting with EEC spectrum disorders in the newborn period should also consider the possibility of T cell lymphopenia.

FMR1 CGG expansions: prevalence and sex ratios.

We have estimated the prevalence of FMR1 premutation and gray zone CGG repeat expansions in a population-based sample of 19,996 male and female adults in Wisconsin and compared the observed sex ratios of the prevalence of FMR1 CGG premutation and gray zone expansions to theoretical sex ratios. The female premutation prevalence was 1 in 148 and comparable to past research, but the male premutation prevalence of 1 in 290 is somewhat higher than most previous estimates. The female:male premutation prevalence ratio is in line with the theoretically predicted sex ratio. The prevalence of CGG repeats in the gray zone (45-54 repeats) was 1 in 33 females and 1 in 62 males. The prevalence of the "expanded" gray zone (defined here as 41-54 CGG repeats) was 1 in 14 females and 1 in 22 males, leading to a female:male ratio of 1.62 (95% confidence interval 1.39-1.90). This female:male ratio was significantly lower than the expected ratio of 2.0. We examined results from three previously published FMR1 prevalence studies and found similar female:male ratios for CGG repeats in this "expanded" gray zone range (pooled female:male ratio across all four studies 1.66, 95% confidence interval 1.51-1.82). Further research is needed to understand the apparent excess prevalence of males with CGG repeats in this range.

Newborn screening for severe combined immunodeficiency; the Wisconsin experience (2008-2011).

Severe combined immunodeficiency is a life-threatening primary immune deficiency characterized by low numbers of naïve T cells. Early diagnosis and treatment of this disease decreases mortality. In 2008, Wisconsin began newborn screening of infants for severe combined immunodeficiency and other forms of T-cell lymphopenia by the T-cell receptor excision circle assay. In total, 207,696 infants were screened. Seventy-two infants had an abnormal assay. T-cell numbers were normal in 38 infants, abnormal in 33 infants, and not performed in one infant, giving a positive predictive value for T-cell lymphopenia of any cause of 45.83% and a specificity of 99.98%. Five infants with severe combined immunodeficiency/severe T-cell lymphopenia requiring hematopoietic stem cell transplantation or other therapy were detected. In summary, the T-cell receptor excision circle assay is a sensitive and specific test to identify infants with severe combined immunodeficiency and severe T-cell lymphopenia that leads to life-saving therapies such as hematopoietic stem cell transplantation prior to the acquisition of severe infections.

Newborn Screen for X-Linked Adrenoleukodystrophy Using Flow Injection Tandem Mass Spectrometry in Negative Ion Mode.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder caused by pathogenic variants in the ATP-binding cassette subfamily D member 1 gene (ABCD1) that encodes the adrenoleukodystrophy protein (ALDP). Defects in ALDP result in elevated cerotic acid, and lead to C26:0-lysophosphatidylcholine (C26:0-LPC) accumulation, which is the primary biomarker used in newborn screening (NBS) for X-ALD. C26:0-LPC levels were measured in dried blood spot (DBS) NBS specimens using a flow injection analysis (FIA) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) performed in negative ion mode. The method was validated by assessing and confirming linearity, accuracy, and precision. We have also established C26:0-LPC cutoff values that identify newborns at risk for X-ALD. The mean concentration of C26:0-LPC in 5881 de-identified residual routine NBS specimens was 0.07 ± 0.02 µM (mean + 1 standard deviation (SD)). All tested true X-ALD positive and negative samples were correctly identified based on C26:0-LPC cutoff concentrations for borderline between 0.15 µM and 0.22 µM (mean + 4 SD) and presumptive screening positive at ≥0.23 µM (mean + 8 SD). The presented FIA method shortens analysis run-time to 1.7 min, while maintaining the previously established advantage of utilizing negative mode MS to eliminate isobaric interferences that could lead to screening false positives.

Newborn screening for spinal muscular atrophy: The Wisconsin first year experience.

Spinal muscular atrophy was recently added to the Wisconsin newborn screening panel. Here we report our screening methods, algorithm, and outcomes. A multiplex real-time PCR assay was used to identify newborns with homozygous SMN1 exon 7 deletion, and those newborns' specimens further underwent a droplet digital PCR assay for SMN2 copy number assessment. An independent dried blood spot specimen was collected and tested to confirm the initial screening results for SMN1 and SMN2. From October 15, 2019 to October 14, 2020, a total of 60,984 newborns were screened for spinal muscular atrophy. Six newborns screened positive for and were confirmed to have spinal muscular atrophy, making the Wisconsin spinal muscular atrophy birth prevalence 1 in 10,164. Of these six infants, two have two copies of SMN2, two have three copies of SMN2, and two have four copies of SMN2. Five newborns received Zolgensma therapy, and one newborn received Spinraza therapy. Our screening method's positive predictive value is 100%. This comprehensive approach, providing both timely SMN2 information and SMN1 and SMN2 confirmation as parts of the algorithm for spinal muscular atrophy newborn screening, facilitated timely clinical follow-up, family counseling, and treatment planning.

Missed Cystic Fibrosis Newborn Screening Cases due to Immunoreactive Trypsinogen Levels below Program Cutoffs: A National Survey of Risk Factors.

Testing immunoreactive trypsinogen (IRT) is the first step in cystic fibrosis (CF) newborn screening. While high IRT is associated with CF, some cases are missed. This survey aimed to find factors associated with missed CF cases due to IRT levels below program cutoffs. Twenty-nine states responded to a U.S-wide survey and 13 supplied program-related data for low IRT false screen negative cases (CFFN) and CF true screen positive cases (CFTP) for analysis. Rates of missed CF cases and odds ratios were derived for each factor in CFFNs, and two CFFN subgroups, IRT above ("high") and below ("low") the CFFN median (39 ng/mL) compared to CFTPs for this entire sample set. Factors associated with "high" CFFN subgroup were Black race, higher IRT cutoff, fixed IRT cutoff, genotypes without two known CF-causing variants, and meconium ileus. Factors associated with "low" CFFN subgroup were older age at specimen collection, Saturday birth, hotter season of newborn dried blood spot collection, maximum ≥ 3 days laboratories could be closed, preterm birth, and formula feeding newborns. Lowering IRT cutoffs may reduce "high" IRT CFFNs. Addressing hospital and laboratory factors (like training staff in collection of blood spots, using insulated containers during transport and reducing consecutive days screening laboratories are closed) may reduce "low" IRT CFFNs.

Cause of death in neonates with inconclusive or abnormal T-cell receptor excision circle assays on newborn screening.

INTRODUCTION: During the first 2 years of newborn screening (NBS) for severe combined immunodeficiency (SCID), 39 infants with an abnormal or inconclusive newborn screening test for SCID died prior to assessment of immune function. We investigated if SCID or primary T-cell lymphopenia likely contributed to the death of these neonates. METHODS: This study is a detailed retrospective chart review. RESULTS: Medical records were available in all 39 infants. Three neonates were full-term infants whose deaths were due to congenital anomalies. Thirty-three infants were born <33 weeks estimated gestational age, and the majority of these infants died from complications of prematurity. Six infants died from sepsis: two due to maternal chorioamnionitis, two due to necrotizing enterocolitis, one due to early onset group B strep sepsis, and one from a likely nosocomial infection. CONCLUSIONS: There was no evidence that SCID contributed to the cause of death in neonates with an abnormal of inconclusive NBS test for SCID.

Correction: Furnier et al. Translating Molecular Technologies into Routine Newborn Screening Practice. Int. J. Neonatal Screen. 2020, 6, 80.

In the original article [...].

Newborn Screening for Severe Combined Immunodeficiency: Do Preterm Infants Require Special Consideration?

The Wisconsin Newborn Screening (NBS) Program began screening for severe combined immunodeficiency (SCID) in 2008, using real-time PCR to quantitate T-cell receptor excision circles (TRECs) in DNA isolated from dried blood NBS specimens. Prompted by the observation that there were disproportionately more screening-positive cases in premature infants, we performed a study to assess whether there is a difference in TRECs between full-term and preterm newborns. Based on de-identified SCID data from 1 January to 30 June 2008, we evaluated the TRECs from 2510 preterm newborns (gestational age, 23-36 weeks) whose specimens were collected ≤72 h after birth. The TRECs from 5020 full-term newborns were included as controls. The relationship between TRECs and gestational age in weeks was estimated using linear regression analysis. The estimated increase in TRECs for every additional week of gestation is 9.60%. The 95% confidence interval is 8.95% to 10.25% (p ≤ 0.0001). Our data suggest that TRECs increase at a steady rate as gestational age increases. These results provide rationale for Wisconsin's existing premature infant screening procedure of recommending repeat NBS following an SCID screening positive in a premature infant instead of the flow cytometry confirmatory testing for SCID screening positives in full-term infants.

Newborn Screening for Severe Combined Immunodeficiency Using the Multiple of the Median Values of T-Cell Receptor Excision Circles.

All newborn screening programs screen for severe combined immunodeficiency by measurement of T-cell receptor excision circles (TRECs). Herein, we report our experience of reporting TREC assay results as multiple of the median (MoM) rather than using conventional copy numbers. This modification simplifies the assay by eliminating the need for standards with known TREC copy numbers. Furthermore, since MoM is a measure of how far an individual test result deviates from the median, it allows normalization of TREC assay data from different laboratories, so that individual test results can be compared regardless of the particular method, assay, or reagents used.

Thyroid-stimulating hormone reference ranges for moderate-to-late preterm infants.

BACKGROUND: Identifying congenital hypothyroidism through newborn screening (NBS) is higher among moderate-to-late preterm (MLPT) infants. Currently, the same thyroid-stimulating hormone (TSH) cutoffs are used for term and preterm infants. TSH reference ranges for MLPT infants are not currently available. OBJECTIVE: To determine TSH reference ranges for MLPT infants. METHODS: We analyzed 10,987 TSH levels on NBS samples performed on 8499 MLPT infants born between 32 and 36 weeks gestation. RESULTS: TSH median, 5th, 25th, 75th, 95th, and 99th percentiles were defined from day 1 until day 14 of life. TSH levels gradually decreased after birth and reached a plateau around day 6. CONCLUSION: Using a state-wide cohort, we constructed TSH reference charts from day 1 until day 14 for MLPT infants. Relationship between age-adjusted TSH percentiles and long-term neurodevelopmental outcomes should be determined in future studies to define optimal TSH cutoffs for MLPT infants.

Development of a routine newborn screening protocol for severe combined immunodeficiency.

BACKGROUND: Severe combined immunodeficiency (SCID) is characterized by the absence of functional T cells and B cells. Without early diagnosis and treatment, infants with SCID die from severe infections within the first year of life. OBJECTIVE: To determined the feasibility of detecting SCID in newborns by quantitating T-cell receptor excision circles (TRECs) from dried blood spots (DBSs) on newborn screening (NBS) cards. METHODS: DNA was extracted from DBSs on deidentified NBS cards, and real-time quantitative PCR (RT-qPCR) was used to determine the number of TRECs. Positive controls consisted of DBS from a 1-week-old T(-)B(-)NK(+) patient with SCID and whole blood specimens selectively depleted of naive T cells. RESULTS: The mean and median numbers of TRECs from 5766 deidentified DBSs were 827 and 708, respectively, per 3.2-mm punch ( approximately 3 muL whole blood). Ten samples failed to amplify TRECs on initial analysis; all but 1 demonstrated normal TRECs and beta-actin amplification on retesting. No TRECs were detected in either the SCID or naive T-cell-depleted samples, despite the presence of normal levels of beta-actin. CONCLUSIONS: The use of RT-qPCR to quantitate TRECs from DNA extracted from newborn DBSs is a highly sensitive and specific screening test for SCID. This assay is currently being used in Wisconsin for routine screening infants for SCID.

Translating Molecular Technologies into Routine Newborn Screening Practice.

As biotechnologies advance and better treatment regimens emerge, there is a trend toward applying more advanced technologies and adding more conditions to the newborn screening (NBS) panel. In the current Recommended Uniform Screening Panel (RUSP), all conditions but one, congenital hypothyroidism, have well-defined genes and inheritance patterns, so it is beneficial to incorporate molecular testing in NBS when it is necessary and appropriate. Indeed, the applications of molecular technologies have taken NBS to previously uncharted territory. In this paper, based on our own program experience and what has been reported in the literature, we describe current practices regarding the applications of molecular technologies in routine NBS practice in the era of genomic and precision medicine.

The Impact of the CFTR Gene Discovery on Cystic Fibrosis Diagnosis, Counseling, and Preventive Therapy.

Discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was the long-awaited scientific advance that dramatically improved the diagnosis and treatment of cystic fibrosis (CF). The combination of a first-tier biomarker, immunoreactive trypsinogen (IRT), and, if high, DNA analysis for CF-causing variants, has enabled regions where CF is prevalent to screen neonates and achieve diagnoses within 1-2 weeks of birth when most patients are asymptomatic. In addition, IRT/DNA (CFTR) screening protocols simultaneously contribute important genetic data to determine genotype, prognosticate, and plan preventive therapies such as CFTR modulator selection. As the genomics era proceeds with affordable biotechnologies, the potential added value of whole genome sequencing will probably enhance personalized, precision care that can begin during infancy. Issues remain, however, about the optimal size of CFTR panels in genetically diverse regions and how best to deal with incidental findings. Because prospects for a primary DNA screening test are on the horizon, the debate about detecting heterozygote carriers will likely intensify, especially as we learn more about this relatively common genotype. Perhaps, at that time, concerns about CF heterozygote carrier detection will subside, and it will become recognized as beneficial. We share new perspectives on that issue in this article.