Mei W. Baker

Newborn Screening for Severe Combined Immunodeficiency in 11 Screening Programs in the United States

IMPORTANCE Newborn screening for severe combined immunodeficiency (SCID) using assays to detect T-cell receptor excision circles (TRECs) began in Wisconsin in 2008, and SCID was added to the national recommended uniform panel for newborn screened disorders in 2010. Currently 23 states, the District of Columbia, and the Navajo Nation conduct population-wide newborn screening for SCID. The incidence of SCID is estimated at 1 in 100,000 births. OBJECTIVES To present data from a spectrum of SCID newborn screening programs, establish population-based incidence for SCID and other conditions with T-cell lymphopenia, and document early institution of effective treatments. DESIGN Epidemiological and retrospective observational study. SETTING Representatives in states conducting SCID newborn screening were invited to submit their SCID screening algorithms, test performance data, and deidentified clinical and laboratory information regarding infants screened and cases with nonnormal results. Infants born from the start of each participating program from January 2008 through the most recent evaluable date prior to July 2013 were included. Representatives from 10 states plus the Navajo Area Indian Health Service contributed data from 3,030,083 newborns screened with a TREC test. MAIN OUTCOMES AND MEASURES Infants with SCID and other diagnoses of T-cell lymphopenia were classified. Incidence and, where possible, etiologies were determined. Interventions and survival were tracked. RESULTS Screening detected 52 cases of typical SCID, leaky SCID, and Omenn syndrome, affecting 1 in 58,000 infants (95% CI, 1/46,000-1/80,000). Survival of SCID-affected infants through their diagnosis and immune reconstitution was 87% (45/52), 92% (45/49) for infants who received transplantation, enzyme replacement, and/or gene therapy. Additional interventions for SCID and non-SCID T-cell lymphopenia included immunoglobulin infusions, preventive antibiotics, and avoidance of live vaccines. Variations in definitions and follow-up practices influenced the rates of detection of non-SCID T-cell lymphopenia. CONCLUSIONS AND RELEVANCE Newborn screening in 11 programs in the United States identified SCID in 1 in 58,000 infants, with high survival. The usefulness of detection of non-SCID T-cell lymphopenias by the same screening remains to be determined.

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.

Prevalence of CGG Expansions of the FMR1 Gene in a US Population-Based Sample

The primary goal of this study was to calculate the prevalence of the premutation of the FMR1 gene and of the “gray zone” using a population‐based sample of older adults in Wisconsin (n = 6,747 samples screened). Compared with past research, prevalence was relatively high (1 in 151 females and 1 in 468 males for the premutation and 1 in 35 females and 1 in 42 males for the gray zone as defined by 45–54 CGG repeats). A secondary study goal was to describe characteristics of individuals found to have the premutation (n = 30, 7 males and 23 females). We found that premutation carriers had a significantly higher rate of divorce than controls, as well as higher rates of symptoms that might be indicative of fragile X‐associated tremor ataxia syndrome (FXTAS; numbness, dizziness/faintness) and fragile X primary ovarian insufficiency (FXPOI; age at last menstrual period). Although not statistically significant, premutation carriers were twice as likely to have a child with disability.

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.

Implementing Routine Testing for Severe Combined Immunodeficiency within Wisconsin's Newborn Screening Program

Severe combined immunodeficiency (SCID) is the result of genetic defects that impair normal T-cell development. SCID babies typically appear normal at birth, but acquire multiple life-threatening infections within a few months. Early diagnosis and treatment with a bone-marrow transplant markedly improves long-term outcomes. On January 1, 2008, the newborn screening (NBS) program in Wisconsin became the first in the world to routinely test all newborns for SCID. A realtime quantitative polymerase chain reaction assay measures T-cell receptor excision circles (TRECs), which are formed during the maturation of normal T-cells. A lack or very low number of TRECs is consistent with T-cell lymphopenia. The development and validation of the TREC assay and the results of the first year of screening have been published. This article describes the process used to add SCID to the NBS panel, the establishment of follow-up capacity, and the integration of SCID screening into routine NBS workflows. The development of this expanded NBS program is described so that other states might benefit from the processes used in Wisconsin.

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.

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.Genet Med 18 3, 231–238.

OPTIMAL DNA TIER FOR THE IRT/DNA ALGORITHM DETERMINED BY CFTR MUTATION RESULTS OVER 14 YEARS OF NEWBORN SCREENING

BACKGROUND There has been great variation and uncertainty about how many and what CFTR mutations to include in cystic fibrosis (CF) newborn screening algorithms, and very little research on this topic using large populations of newborns. METHODS We reviewed Wisconsin screening results for 1994-2008 to identify an ideal panel. RESULTS Upon analyzing approximately 1 million screening results, we found it optimal to use a 23 CFTR mutation panel as a second tier when an immunoreactive trypsinogen (IRT)/DNA algorithm was applied for CF screening. This panel in association with a 96th percentile IRT cutoff gave a sensitivity of 97.3%, but restricting the DNA tier to F508del was associated with 90% (P<.0001). CONCLUSIONS Although CFTR panel selection has been challenging, our data show that a 23 mutation method optimizes sensitivity and is advantageous. The IRT cutoff value, however, is actually more critical than DNA in determining CF newborn screening sensitivity.

Low-normal FMR1 CGG repeat length: phenotypic associations

This population-based study investigates genotype–phenotype correlations of “low- normal” CGG repeats in the fragile X mental retardation 1 (FMR1) gene. FMR1 plays an important role in brain development and function, and encodes FMRP (fragile X mental retardation protein), an RNA-binding protein that regulates protein synthesis impacting activity-dependent synaptic development and plasticity. Most past research has focused on CGG premutation expansions (41–200 CGG repeats) and on fragile X syndrome (200+ CGG repeats), with considerably less attention on the other end of the spectrum of CGG repeats. Using existing data, older adults with 23 or fewer CGG repeats (2 SDs below the mean) were compared with age-peers who have normal numbers of CGGs (24–40) with respect to cognition, mental health, cancer, and having children with disabilities. Men (n = 341 with an allele in the low-normal range) and women (n = 46 with two low-normal alleles) had significantly more difficulty with their memory and ability to solve day to day problems. Women with both FMR1 alleles in the low-normal category had significantly elevated odds of feeling that they need to drink more to get the same effect as in the past. These women also had two and one-half times the odds of having had breast cancer and four times the odds of uterine cancer. Men and women with low-normal CGGs had higher odds of having a child with a disability, either a developmental disability or a mental health condition. These findings are in line with the hypothesis that there is a need for tight neuronal homeostatic control mechanisms for optimal cognitive and behavioral functioning, and more generally that low numbers as well as high numbers of CGG repeats may be problematic for health.

Microarray analysis of retinoid-dependent gene activity during rat embryogenesis: increased collagen fibril production in a model of retinoid insufficiency.

Retinoic acid (RA) is an essential mediator of embryogenesis. Some, but not all, of its targets have been identified. We previously developed a rat model of gestational retinoid deficiency (RAD; Power et al. [ 1999 ] Dev. Dyn. 216:469–480) and generated embryos with developmental impairments that closely resemble genetic and dietary models of retinoid insufficiency. Here, we used microarray analysis and expression profiling to identify 88 transcripts whose abundance was altered under conditions of retinoid insufficiency, as compared with normal embryos. Among these, the induction by RAD of genes involved in collagen I synthesis (COL1A1, IA2 and VA2, prolyl‐4‐hydroxylase‐α1) and protein galactosylation (galactokinase, ABO galactosyltransferase, UDP‐galactose transporter‐related protein) was especially noteworthy because extracellular matrix regulates many developmental events. We also identified several genes involved with stress responses (cathepsin H, UBC2E, IGFBP3, smoothelin). Real‐time polymerase chain reaction analysis of selected candidates revealed excellent agreement with the array findings. Further validation came from the demonstration that these genes were similarly dysregulated in two genetic models of retinoid insufficiency, the retinol binding protein null‐mutant embryo and the Raldh2 null‐mutant embryo. In situ hybridization of RAD embryos found increased collagen IA1 and IGFBP3 mRNA within the connective mesenchyme and vasculature, respectively, and a failure to repress the growth factor midkine within the RAD neural tube. Many of the identified genes were not known previously to respond to retinoid status and will provide new insights to retinoid roles and to the consequences of retinoid insufficiency. Developmental Dynamics 229:886–898, 2004.