Development and assessment of educational materials for spinal muscular atrophy carrier screening in the Plain community.

Spinal muscular atrophy (SMA) has been reported in both Amish and Mennonite (Plain) communities, and a higher incidence has been observed in certain Mennonite communities compared to the general population. There are several therapies for SMA, but all are most effective in pre-symptomatic newborns. To identify couples from the Wisconsin Plain community who are most likely to have a child with SMA, carrier screening is offered via mailed kits with at-home specimen collection. Our survey data about Plain families' perspectives on genetic testing suggest educational materials are needed for individuals providing informed consent with at-home specimen collection. We therefore developed a Plain population-specific educational trifold brochure about SMA carrier screening by incorporating existing medical education strategies and feedback from Plain community members and their health care providers. Along with the brochure, surveys were included in the kits to assess baseline knowledge about SMA carrier screening ("pre-education") as well as improvement in knowledge after reviewing the brochure and cultural appropriateness of the brochure ("post-education"). Fifty-five testing kits were distributed, and 26 survey pairs (pre- and post-education) were returned and analyzed (response rate 47%). Respondents had high baseline knowledge with an average of 5 of 7 questions (71%) answered correctly on the pre-education survey. Knowledge improved after reviewing the brochure as the average score increased to 6.5 of 7 questions (93%) answered correctly. Questions about risks of having an affected child after positive or negative carrier screening showed the most improvement from the pre-education to post-education surveys. Most respondents indicated the brochure was helpful, was easy to understand, and contained the right amount of information. Overall, incorporating elements of existing medical education strategies with feedback from the target population and stakeholders about appropriate language seems to be an effective method for creating beneficial, culturally responsive educational materials for the Plain population.

Newborn Screening for Severe Combined Immunodeficiency: Lessons Learned from Screening and Follow-Up of the Preterm Newborn Population.

Newborn screening (NBS) for Severe Combined Immunodeficiency (SCID) by measurement of T-cell receptor excision circles (TRECs) successfully identifies newborns with SCID and severe T-cell lymphopenia, as intended. At the same time, NBS programs face the challenge of false positive results, with a disproportionately high number in the premature newborn population. This study evaluates TREC values and SCID screening outcomes in premature newborns and elucidates evidence-based SCID screening practices that reduce unnecessary follow-up activities in this population. De-identified individual SCID newborn screening data and aggregate SCID screening data were obtained from seven states across the US for babies born between 2018 and 2020. Relevant statistics were performed on data pooled from these states to quantify screening performance metrics and clinical impact on various birth and gestational age categories of newborns. The data were normalized using multiples-of-the-median (MoM) values to allow for the aggregation of data across states. The aggregation of NBS data across a range of NBS programs highlighted the trajectory of TREC values over time, both between and within newborns, and provides evidence for improved SCID screening recommendations in the premature and low birth weight population.

Mortality in Women across the FMR1 CGG Repeat Range: The Neuroprotective Effect of Higher Education.

Higher education has been shown to have neuroprotective effects, reducing the risk of Alzheimer's and Parkinson's diseases, slowing the rate of age-related cognitive decline, and is associated with lower rates of early mortality. In the present study, the association between higher education, fragile X messenger ribonucleoprotein 1 (FMR1) cytosine-guanine-guanine (CGG) repeat number, and mortality before life expectancy was investigated in a population cohort of women born in 1939. The findings revealed a significant interaction between years of higher education and CGG repeat number. Counter to the study's hypothesis, the effects of higher education became more pronounced as the number of CGG repeats increased. There was no effect of years of higher education on early mortality for women who had 25 repeats, while each year of higher education decreased the hazard of early mortality by 8% for women who had 30 repeats. For women with 41 repeats, the hazard was decreased by 14% for each additional year of higher education. The interaction remained significant after controlling for IQ and family socioeconomic status (SES) measured during high school, as well as factors measured during adulthood (family, psychosocial, health, and financial factors). The results are interpreted in the context of differential sensitivity to the environment, a conceptualization that posits that some people are more reactive to both negative and positive environmental conditions. Expansions in CGG repeats have been shown in previous FMR1 research to manifest such a differential sensitivity pattern.

FMR1 CGG Repeats and Stress Influence Self-Reported Cognitive Functioning in Mothers.

Variation in the FMR1 gene may affect aspects of cognition, such as executive function and memory. Environmental factors, such as stress, may also negatively impact cognitive functioning. Participants included 1,053 mothers of children with and without developmental disabilities. Participants completed self-report measures of executive function, memory, and stress (i.e., life events, parenting status), and provided DNA to determine CGG repeat length (ranging from 7 to 192 CGGs). Stress exposure significantly predicted greater self-reported difficulties in executive function and the likelihood of memory problems. Cubic CGG effects independently predicted executive function and memory difficulties, suggesting effects of both genetic variation and environmental stress exposure on cognitive functioning.

Refinement of newborn screening for cystic fibrosis with next generation sequencing.

BACKGROUND: Newborn screening for cystic fibrosis (CF) has been underway universally in the United States for more than a decade, as well in most European countries, and algorithms have been evolving throughout this period with quality improvement projects as immunoreactive trypsinogen (IRT) determinations alone have been transformed to a 2-tier strategy with DNA analyses. OBJECTIVE: To apply next generation sequencing (NGS) as a screening method to expand the DNA tier and identify substantially more variants in the CF transmembrane conductance regulator (CFTR) gene to enhance sensitivity and equity while minimizing incidental findings. DESIGN: Sequential evaluation and improvement plan in three phases using algorithm modifications coupled to statewide follow up and analysis of screening outcomes. RESULTS: After demonstrating feasibility in the first phase, we studied an IRT/NGS algorithm that included CFTR Variants with Varying Clinical Consequences (VVCCs). This revealed a high identification of CF patients with 2-variants detected through screening, but for every CF case there were 1.4 with CF metabolic syndrome/CF screen positive, inconclusive diagnosis (CRMS/CFSPID). This led us to a third phase of improvement in which the VVCCs were eliminated except for R117H, resulting in 94% 2-variant detection of patients and 0.44:1 ratio of CRMS/CFSPID to CF. CONCLUSION: NGS can be used with IRT as an effective method of identifying infants at risk for CF without an appreciable increase in detection of carriers. Its potential added value includes facilitating equity, enhancing sensitivity and detecting more CF patients with 2-variants during the screening process.

Cross-sectional survey on genetic testing utilization and perceptions in Wisconsin Amish and Mennonite communities.

Amish and Mennonite (Plain) communities have increased prevalence of many recessively inherited disorders due to founder variants that can be identified using next-generation sequencing (NGS). We assessed newborn screening (NBS) utilization, prior genetic testing, and perceptions of genetic testing among Wisconsin Plain communities to guide implementation and utilization of a population-specific NGS gene panel testing. A mailed paper survey (N = 959) of demographics, NBS utilization, prior genetic testing, and preferences for categorical genetic disorder and defined clinical context testing was developed. Overall response rate was 39% (N = 378; 183 Amish, 193 Mennonite; 2 not Amish/Mennonite). Mennonites were more likely to respond in favor of carrier screening for metabolic disorders and other surgical conditions and less likely to respond in favor of asymptomatic testing for neurologic disorders and lethal disorders compared to Amish. Reported utilization of NBS was positively associated with stated interest in genetic testing for an asymptomatic child. Reported prior genetic testing was positively associated with stated interest in carrier screening and negatively associated with testing a symptomatic child. Although Plain community members share many common outward characteristics, our survey responses suggest diversity in their views of genetic testing and support laboratory methods that can be flexible to varied needs of individuals.

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.

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.

Common Challenges and Identified Solutions for State Newborn Screening Programs during COVID-19 Pandemic.

During the COVID-19 pandemic, state newborn screening programs faced challenges to ensure this essential public health program continued to function at a high level. In December 2020, the EveryLife Foundation for Rare Diseases held a workshop to discuss these common challenges and solutions. Newborn screening officials described challenges including short staffing across the entire program, collection and transport of specimens, interrupted follow-up activities, and pilot study recruitment. To address these challenges, state programs implemented a wide variety of solutions to maintain the high standards of newborn screening. To address staffing issues, newborn screening programs, public health laboratories, and hospitals all cross-trained personnel, worked to manage staff stress, and established essential functions. Other solutions included working with courier companies to ensure the timely pick-up of specimen, creating educational materials for hospital staff, and the creation of hybrid recruitment models for pilot studies. Implementing the lessons discussed throughout this paper can help to prepare for the next public health emergencies to ensure that a program that interacts with millions of families every year and saves the lives of thousands of children every year is minimally impacted.

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.

Surveillance for the Rare Condition of Sickle Cell Disease in Wisconsin.

INTRODUCTION: Despite universal newborn screening, there is no comprehensive surveillance system to understand the sickle cell disease population in Wisconsin. METHODS: We initiated the development of a sickle cell disease surveillance system by linking newborn screening data and electronic health records from 2 large tertiary health care institutions in Wisconsin: Children's Wisconsin and Froedtert Hospital. RESULTS: There were 1478 individuals within the 3 data sources. One hundred thirty-two (82%) of 159 identified by newborn screening from 2013 through 2019 received care at Children's Wisconsin. The majority of individuals with sickle cell disease at Children's Wisconsin and Froedtert Hospital resided in Milwaukee County. DISCUSSION: The new surveillance program will increase our understanding of the sickle cell disease population in Wisconsin and help improve quality of care and health outcomes.

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

In the original article [...].

Is FMR1 CGG Repeat Number Polymorphism Associated With Phenotypic Variation in the General Population? Report From a Cohort of 5,499 Adults.

FMR1 CGG repeat length was assayed in 5499 research participants (2637 men and 2862 women) in the Wisconsin Longitudinal Study (WLS), a population-based cohort. Most past research has focused on clinically-ascertained individuals with expansions in CGG repeats, either those with fragile X syndrome (> 200 CGG repeats), the FMR1 premutation (55-200 repeats), or in the gray zone (variously defined as 45-54 or 41-54 repeats). In contrast, the WLS is a unique source of data that was obtained from an unselected cohort of individuals from the general population for whom FMR1 CGG repeat length was assayed. The WLS is a random sample of one-third of all high school seniors in the state of Wisconsin in 1957. The most recent round of data collection was in 2011; thus, the study spanned over 50 years. Saliva samples were obtained from 69% of surviving members of the cohort in 2008 and 2011, from which CGG repeats were assayed. With one exception, the CGG repeat length of all members of this cohort was below 100 (ranging from 7 to 84). The present study evaluated the genotype-phenotype associations of CGG repeat number and IQ, college graduation, age at menopause, number of biological children, having a child with intellectual or developmental disabilities, and the likelihood of experiencing an episode of depression during adulthood. Linear and curvilinear effects were probed. Although effect sizes were small, significant associations were found between CGG repeat length and high school IQ score, college graduation, number of biological children, age at menopause, and the likelihood of having an episode of depression. However, there was no significant association between repeat length and having a child diagnosed with an IDD condition. This study demonstrates a continuum of phenotype effects with FMR1 repeat lengths and illustrates how research inspired by a rare genetic condition (such as fragile X syndrome) can be used to probe genotype-phenotype associations in the general population.

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.

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.

Newborn Screening for Severe Combined Immunodeficiency: 10-Year Experience at a Single Referral Center (2009-2018).

In 2008, newborn screening (NBS) for severe combined immunodeficiency (SCID) began as a pilot study in Wisconsin and has recently been added to every state's newborn screen panel. The incidence of SCID is estimated at 1 per 58,000 births which may suggest infrequent NBS SCID screen positive results in states with low annual birth rates. In this study, we report our center's experience with NBS positive SCID screen referrals over a 10-year period. A total of 68 full-term newborns were referred to our center for confirmatory testing. Of these referrals, 50% were false positives, 12% were SCID diagnoses, 20% syndromic T cell lymphopenia (TCL) disorders, and 18% non-SCID, non-syndromic TCL. Through collaboration with our newborn screening lab, second-tier targeted gene sequencing was performed for newborns with SCID screen positive results from communities with known founder pathogenic variants and provided rapid genetic confirmation of SCID and non-SCID TCL disorders. Despite extensive genetic testing, two of the eight (25%) identified newborns with SCID diagnoses lacked a definable genetic defect. Additionally, our referrals included ten newborns who were otherwise healthy newborns with idiopathic TCL and varied CD3+ T cell number longitudinal trajectories. Collectively, referrals to our single site over a 10-year period describe a broad spectrum of medically actionable and idiopathic TCL disorders which highlight the importance of clinical immunology expertise in all states, demonstrate efficiencies and challenges for second-tier genetic testing, and further emphasize the need to development standardized evaluation algorithms for non-SCID TCL.

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.

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.