Implementation of population-based newborn screening reveals low incidence of spinal muscular atrophy.

PURPOSE: Spinal muscular atrophy (SMA) was added to the Recommended Uniform Screening Panel (RUSP) in July 2018, following FDA approval of the first effective SMA treatment, and demonstration of feasibility of high-throughput newborn screening using a primary molecular assay. SMA newborn screening was implemented in New York State (NYS) on 1 October 2018. METHODS: Screening was conducted using DNA extracted from dried blood spots with a multiplex real-time quantitative polymerase chain reaction (qPCR) assay targeting the recurrent SMN1 exon 7 gene deletion. RESULTS: During the first year, 225,093 infants were tested. Eight screened positive, were referred for follow-up, and confirmed to be homozygous for the deletion. Infants with two or three copies of the SMN2 gene, predicting more severe, earlier-onset SMA, were treated with antisense oligonucleotide and/or gene therapy. One infant with ≥4 copies SMN2 also received gene therapy. CONCLUSION: Newborn screening permits presymptomatic SMA diagnosis, when treatment initiation is most beneficial. At 1 in 28,137 (95% confidence interval [CI]: 1 in 14,259 to 55,525), the NYS SMA incidence is 2.6- to 4.7-fold lower than expected. The low SMA incidence is likely attributable to imprecise and biased estimates, coupled with increased awareness, access to and uptake of carrier screening, genetic counseling, cascade testing, prenatal diagnosis, and advanced reproductive technologies.

Pilot study of population-based newborn screening for spinal muscular atrophy in New York state.

PurposeTo determine feasibility and utility of newborn screening for spinal muscular atrophy (SMA) in New York State.MethodsWe validated a multiplex TaqMan real-time quantitative polymerase chain reaction assay using dried blood spots for SMA. From January 2016 to January 2017, we offered, consented, and screened 3,826 newborns at three hospitals in New York City and tested newborns for the deletion in exon 7 of SMN1.ResultsNinety-three percent of parents opted in for SMA screening. Overall the SMA carrier frequency was 1.5%. We identified one newborn with a homozygous SMN1 deletion and two copies of SMN2, which strongly suggests the severe type 1 SMA phenotype. The infant was enrolled in the NURTURE clinical trial and was first treated with Spinraza at age 15 days. She is now age 12 months, meeting all developmental milestones, and free of any respiratory issues.ConclusionOur pilot study demonstrates the feasibility of population-based screening, the acceptance by families, and the benefit of newborn screening for SMA. We suggest that SMA be considered for addition to the national recommended uniform screening panel.

Clinical Sensitivity of Cystic Fibrosis Mutation Panels in a Diverse Population.

Infants are screened for cystic fibrosis (CF) in New York State (NYS) using an IRT-DNA algorithm. The purpose of this study was to validate and assess clinical validity of the US FDA-cleared Illumina MiSeqDx CF 139-Variant Assay (139-VA) in the diverse NYS CF population. The study included 439 infants with CF identified via newborn screening (NBS) from 2002 to 2012. All had been screened using the Abbott Molecular CF Genotyping Assay or the Hologic InPlex CF Molecular Test. All with CF and zero or one mutation were tested using the 139-VA. DNA extracted from dried blood spots was reliably and accurately genotyped using the 139-VA. Sixty-three additional mutations were identified. Clinical sensitivity of three panels ranged from 76.2% (23 mutations recommended for screening by ACMG/ACOG) to 79.7% (current NYS 39-mutation InPlex panel), up to 86.0% for the 139-VA. For all, sensitivity was highest in Whites and lowest in the Black population. Although the sample size was small, there was a nearly 20% increase in sensitivity for the Black CF population using the 139-VA (68.2%) over the ACMG/ACOG and InPlex panels (both 50.0%). Overall, the 139-VA is more sensitive than other commercially available panels, and could be considered for NBS, clinical, or research laboratories conducting CF screening.

Rare Variants in RPPH1 Real-Time Quantitative PCR Control Assay Binding Sites Result in Incorrect Copy Number Calls.

Real-time quantitative PCR (qPCR) using RPPH1 as a reference gene is a standard method for assessment and validation of genomic copy number variations. However, variants in the reference amplicon may cause errors, which was investigated herein. While conducting copy number variation validations for birth defects research studies, 13 of 1634 specimens with multiple loci that appeared to be present as three copies were unexpectedly detected. This apparent trisomy was hypothesized to be an amplification artifact caused by a variant in the RPPH1 amplicon. Sequencing revealed all 13 individuals carried one of the four different variants within the RPPH1 amplicon. These variants could produce allelic dropout or altered reaction efficiency, causing an inaccurate measurement of copy number. Additional genotyping predicted a low frequency of the most common variant (rs3093876; 14/3562 alleles; minor allele frequency, 0.39%). Laboratories should recognize the potential for inaccurate results when using a single qPCR control assay. Overestimated CFTR and SMN2 copy numbers identified during newborn screening that otherwise would have been incorrectly called were also detected. Variants in reference loci may produce false-negative normal results for test loci when real deletions are present. For clinical laboratories screening for heterozygous deletions for diagnostic testing or prenatal/carrier screening via qPCR, the most cost-effective solution to maximize sensitivity is to run triplex reactions targeting the region of interest with two control genes.

Newborn Screening for Spinal Muscular Atrophy in New York State: Clinical Outcomes From the First 3 Years.

BACKGROUND AND OBJECTIVES: Spinal muscular atrophy (SMA) was added to the Recommended Uniform Screening Panel (RUSP) in July 2018, largely on the basis of the availability and efficacy of newly-approved disease modifying therapies. New York State (NYS) started universal newborn screening for SMA in October 2018. The authors report the findings from the first 3 years of screening. METHODS: Statewide neonatal screening was conducted using DNA extracted from dried blood spots using a real-time quantitative polymerase chain reaction (qPCR) assay. Retrospective follow-up data were collected from 9 referral centers across the state on 34 infants. RESULTS: In the first three years since statewide implementation, nearly 650,000 infants have been screened for SMA. 34 babies screened positive and were referred to a neuromuscular specialty care center. The incidence remains lower than previously predicted. The majority (94%), including all infants with 2-3 copies of SMN2, have received treatment. Among treated infants, the overwhelming majority (97%; 29/30) have received gene replacement. All infants in this cohort with 3 copies of SMN2 are clinically asymptomatic post-treatment based on early clinical follow-up data. Infants with 2 copies of SMN2 are more variable in their outcomes. Electrodiagnostic outcomes data from a subgroup of patients (n=11) for whom pre- and post-treatment data demonstrated either improvement or no change in CMAP amplitude at last clinical follow-up compared to pre-treatment baseline. Most infants were treated before 6 weeks of age (median = 34.5 DOL; range 11-180). Delays and barriers to treatment identified by treating clinicians followed two broad themes: medical and non-medical. Medical delays most commonly reported were presence of AAV9 antibodies and elevated troponin I levels. Non-medical barriers included delays in obtaining insurance as well as insurance policies regarding specific treatment modalities. DISCUSSION: The findings from the NYS cohort of newborn screen-identified infants are consistent with other reports of improved outcomes from early diagnosis and treatment. Additional biomarkers of motor neuron health including electromyography can potentially be helpful in detecting pre-clinical decline.

Validation of a Custom Next-Generation Sequencing Assay for Cystic Fibrosis Newborn Screening.

Newborn screening (NBS) for Cystic Fibrosis (CF) is associated with improved outcomes. All US states screen for CF; however, CF NBS algorithms have high false positive (FP) rates. In New York State (NYS), the positive predictive value of CF NBS improved from 3.7% to 25.2% following the implementation of a three-tier IRT-DNA-SEQ approach using commercially available tests. Here we describe a modification of the NYS CF NBS algorithm via transition to a new custom next-generation sequencing (NGS) platform for more comprehensive cystic fibrosis transmembrane conductance regulator (CFTR) gene analysis. After full gene sequencing, a tiered strategy is used to first analyze only a specific panel of 338 clinically relevant CFTR variants (second-tier), followed by unblinding of all sequence variants and bioinformatic assessment of deletions/duplications in a subset of samples requiring third-tier analysis. We demonstrate the analytical and clinical validity of the assay and the feasibility of use in the NBS setting. The custom assay has streamlined our molecular workflow, increased throughput, and allows for bioinformatic customization of second-tier variant panel content. NBS aims to identify those infants with the highest disease risk. Technological molecular improvements can be applied to NBS algorithms to reduce the burden of FP referrals without loss of sensitivity.