Genetic counseling access and service delivery in New York State is variable for parents of infants with complex CFTR genotypes conferring uncertain phenotypes.

BACKGROUND: New York State (NYS) utilizes a three-tiered cystic fibrosis newborn screening (CFNBS) algorithm that includes cystic fibrosis transmembrane conductance regulator (CFTR) gene sequencing. Infants with >1 CFTR variant of potential clinical relevance, including variants of uncertain significance or varying clinical consequence are referred for diagnostic evaluation at NYS cystic fibrosis (CF) Specialty Care Centers (SCCs). AIMS: As part of ongoing quality improvement efforts, demographic, screening, diagnostic, and clinical data were evaluated for 289 CFNBS-positive infants identified in NYS between December 2017 and November 2020 who did not meet diagnostic criteria for CF and were classified as either: CFTR-related metabolic syndrome/CF screen positive, inconclusive diagnosis (CRMS/CFSPID) or CF carriers. RESULTS: Overall, 194/289 (67.1%) had CFTR phasing to confirm whether the infant's CFTR variants were in cis or in trans. Eighteen complex alleles were identified in cis; known haplotypes (p.R117H+5T, p.F508del+p.L467F, and p.R74W+p.D1270N) were the most common identified. Thirty-two infants (16.5%) with all variants in cis were reclassified as CF carriers rather than CRMS/CFSPID. Among 263 infants evaluated at an NYS SCC, 70.3% were reported as having received genetic counseling about their results by any provider, with 96/263 (36.5%) counseled by a certified genetic counselor. CONCLUSION: Given the particularly complex genetic interpretation of results generated by CFNBS algorithms including sequencing analysis, additional efforts are needed to ensure families of infants with a positive CFNBS result have CFTR phasing when needed to distinguish carriers from infants with CRMS/CFSPID, and access to genetic counseling to address implications of CFNBS results.

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.

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.

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.

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.

Moonlighting newborn screening markers: the incidental discovery of a second-tier test for Pompe disease.

PURPOSE: To describe a novel biochemical marker in dried blood spots suitable to improve the specificity of newborn screening for Pompe disease. METHODS: The new marker is a ratio calculated between the creatine/creatinine (Cre/Crn) ratio as the numerator and the activity of acid α-glucosidase (GAA) as the denominator. Using Collaborative Laboratory Integrated Reports (CLIR), the new marker was incorporated in a dual scatter plot that can achieve almost complete segregation between Pompe disease and false-positive cases. RESULTS: The (Cre/Crn)/GAA ratio was measured in residual dried blood spots of five Pompe cases and was found to be elevated (range 4.41-13.26; 99%ile of neonatal controls: 1.10). Verification was by analysis of 39 blinded specimens that included 10 controls, 24 samples with a definitive classification (16 Pompe, 8 false positives), and 5 with genotypes of uncertain significance. The CLIR tool showed 100% concordance of classification for the 24 known cases. Of the remaining five cases, three p.V222M homozygotes, a benign variant, were classified by CLIR as false positives; two with genotypes of unknown significance, one likely informative, were categorized as Pompe disease. CONCLUSION: The CLIR tool inclusive of the new ratio could have prevented at least 12 of 13 (92%) false-positive outcomes.

Liquid Chromatography-Tandem Mass Spectrometry Assay of Leukocyte Acid α-Glucosidase for Post-Newborn Screening Evaluation of Pompe Disease.

BACKGROUND: Pompe disease (PD) is the first lysosomal storage disorder to be added to the Recommended Uniform Screening Panel for newborn screening. This condition has a broad phenotypic spectrum, ranging from an infantile form (IOPD), with severe morbidity and mortality in infancy, to a late-onset form (LOPD) with variable onset and progressive weakness and respiratory failure. Because the prognosis and treatment options are different for IOPD and LOPD, it is important to accurately determine an individual's phenotype. To date, no enzyme assay of acid α-glucosidase (GAA) has been described that can differentiate IOPD vs LOPD using blood samples. METHODS: We incubated 10 µL leukocyte lysate and 25 µL GAA substrate and internal standard (IS) assay cocktail for 1 h. The reaction was purified by a liquid-liquid extraction. The extracts were evaporated and reconstituted in 200 µL methanol and analyzed by LC-MS/MS for GAA activity. RESULTS: A 700-fold higher analytical range was observed with the LC-MS/MS assay compared to the fluorometric method. When GAA-null and GAA-containing fibroblast lysates were mixed, GAA activity could be measured accurately even in the range of 0%-1% of normal. The leukocyte GAA activity in IOPD (n = 4) and LOPD (n = 19) was 0.44-1.75 nmol · h-1 · mg-1 and 2.0-6.5 nmol · h-1 · mg-1, respectively, with no overlap. The GAA activity of pseudodeficiency patients ranged from 3.0-28.1 nmol · h-1 · mg-1, showing substantial but incomplete separation from the LOPD group. CONCLUSIONS: This assay allows determination of low residual GAA activity in leukocytes. IOPD, LOPD, and pseudodeficiency patients can be partially differentiated by measuring GAA using blood samples.

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.

Substantial effect of efavirenz monotherapy on bilirubin levels in healthy volunteers.

BACKGROUND: Efavirenz exhibits multiple interactions with drug-metabolizing enzymes and transporters, and for this reason efavirenz-based HIV therapy is associated with altered pharmacokinetics of coadministered drugs. Probably by the same mechanism, efavirenz-based HIV therapy affects the disposition of endogenous compounds, but this effect is difficult to directly link with efavirenz because it is used in combination with other drugs. OBJECTIVES: To explore the effect of efavirenz monotherapy on biochemical laboratory values in a clinical trial of healthy volunteers. METHODS: Men and women (aged 18-49 years) with body mass index ≤32 who were assessed to be healthy based on medical history, physical examination, and standard laboratory screening received a single (600 mg) and multiple doses (600 mg/d for 17 days) of efavirenz orally. This trial was designed to determine the pharmacokinetics and drug interactions of efavirenz. As part of this study, analysis of serum chemistries that were measured at study entry (screening) and 1 week after completion of the multiple dose study (exit) is reported. RESULTS: Data from 60 subjects who fully completed and 13 subjects who partially completed the study are presented. Total bilirubin was substantially reduced at exit (by ~30%, with large intersubject variability) compared with screening values (P < 0.0001). The percent changes were in part explained by the intersubject differences in baseline total bilirubin because there was a significant correlation between baseline (screening) values and percent change at exit (r = 0.50; P < 0.0001). Hemoglobin and absolute neutropenia were also substantially decreased at exit compared with screening, but this may be due to intensive blood sampling rather than direct effect of efavirenz on these parameters. No significant correlation was found between percent change in hemoglobin versus percent change in bilirubin, indicating the effect of efavirenz on bilirubin is independent of its effects on hemoglobin. CONCLUSIONS: Efavirenz monotherapy significantly lowers plasma total bilirubin concentration in healthy volunteers independent of its effect on hemoglobin, probably through its effects on bilirubin metabolism and transport (uptake and efflux). These findings help explain reversal by efavirenz of hyperbilirubinemia induction observed by some protease inhibitor antiretroviral drugs (eg, atazanavir). Besides its well-documented role on drug interactions, efavirenz may alter the disposition of endogenous compounds relevant in physiologic homeostasis through its interaction with drug metabolizing enzymes and/or drug transporters. ClinicalTrials.gov identifier: NCT00668395.

Cost-effective smoke-free multiunit housing media campaigns: connecting with local communities.

Presented are cost-effective paid media strategies to educate Californians to advocate for stronger smoke-free multiunit housing (SF-MUH) policies between 2006 and 2008. Included is a summary of general market and specific ethnic market costs that correspond to SF-MUH attitudes and home smoking bans. Statewide questionnaires indicated that half of the intended general market saw an antitobacco TV ad and half of the intended ethnic markets heard radio ads. Analyses indicated that it cost $0.67 and $0.78 per person to see Caution Tape and Apartment TV ads, respectively. Slightly higher per capita costs corresponded with positive attitudes toward SF-MUH: $0.87 for Caution Tape and $1.00 for Apartment. Lessons learned from this campaign included effectiveness of specific ads in ethnic markets, impact on SF-MUH work plan policy objectives, and the need for collaborations among state and local partners throughout the message development process.

Assessing the relationship between ad volume and awareness of a tobacco education media campaign.

BACKGROUND: The relation between aided ad recall and level of television ad placement in a public health setting is not well established. We examine this association by looking back at 8 years of the California's Tobacco Control Program's (CTCP) media campaign. METHODS: Starting in July 2001, California's campaign was continuously monitored using five telephone series of surveys and six web-based series of surveys immediately following a media flight. We used population-based statewide surveys to measure aided recall for advertisements that were placed in each of these media flights. Targeted rating points (TRPs) were used to measure ad placement intensity throughout the state. RESULTS: Cumulative TRPs exhibited a stronger relation with aided ad recall than flight TRPs or TRP density. This association increased after log-transforming cumulative TRP values. We found that a one-unit increase in log-cumulative TRPs led to a 13.6% increase in aided ad recall using web-based survey data, compared to a 5.3% increase in aided ad recall using telephone survey data. CONCLUSIONS: In California, the relation between aided ad recall and cumulative TRPs showed a diminishing return after a large volume of ad placements These findings may be useful in planning future ad placement for CTCP's media campaign.

Use of a smokers' quitline by Asian language speakers: results from 15 years of operation in California.

OBJECTIVES: We examined state quitline utilization by smokers who called Chinese-, Vietnamese-, or Korean-language lines, and compared their usage rates to those of Asians and Whites calling the English-language line. METHODS: Using data from 15 years (1993-2008) of operation of the California quitline (which included data on 22 061 callers to Chinese, Korean, and Vietnamese lines) and from multiple California Health Interview Surveys, we computed the call rates for Whites, English-speaking Asians, and the 3 Asian-language groups. We also examined callers' demographics and where they heard about the quitline. RESULTS: Asian smokers who spoke English were significantly less likely than English-speaking White smokers to call the quitline (odds ratios range from 0.36 to 0.62). Smokers speaking 1 of the 3 Asian languages were no less likely than White smokers to call (odds ratios range from 0.82 to 3.25). More than 80% of those calling the Asian-language lines reported hearing about the quitline through mass media. CONCLUSIONS: Contrary to general expectation, smokers speaking Asian languages were just as likely to call the quitline as English-speaking White smokers. State quitlines should consider adding Asian-language lines to help address disparities in access to cessation services.

Association between polymorphisms in HSD3B1 and UGT2B17 and prostate cancer risk.

OBJECTIVES: Androgens, especially dihydrotestosterone, have been postulated to modify the risk of prostate cancer. 3-Beta-hydroxysteroid dehydrogenase1 (HSD3B1) and uridine diphosphate-glucuronosyltransferase 2B17 (UGT2B17) are enzymes that inactivate dihydrotestosterone in the prostate and may affect dihydrotestosterone concentration in prostatic tissue. The purpose of this study was to determine whether polymorphisms in HSD3B1 and UGT2B17 increase the risk of prostate cancer. METHODS: In a case-control study of 356 patients with incident primary prostate cancer and 363 age-matched controls, the frequencies of HSD3B1 N367T and UGT2B17 null polymorphisms in genomic DNA were compared between the patients and controls. RESULTS: No evidence was found for a main effect of the HSD3B1 codon 367 polymorphism on prostate cancer risk. However, among white men with family history of prostate cancer, the HSD3B1 367Thr allele was positively associated with prostate cancer (odds ratio 3.0, 95% confidence interval 1.0 to 9.2). A significant association was observed between the UGT2B17 null polymorphism and prostate cancer risk (odds ratio 1.7, 95% confidence interval 1.03 to 2.9). An association with the UGT2B17 null polymorphism was further elevated (odds ratio 2.7, 95% confidence interval 1.1 to 6.5) among individuals with HSD3B1 Asn/Asn genotype. CONCLUSIONS: These results suggest that the HSD3B1 N367T and UGT2B17 null polymorphisms may modify the risk of prostate cancer, particularly among men with a family history of the disease.

Changes of attitudes and patronage behaviors in response to a smoke-free bar law.

OBJECTIVES: We examined patron responses to a California smoke-free bar law. METHODS: Three telephone surveys measured attitudes and behavior changes after implementation of the law. RESULTS: Approval of the law rose from 59.8% to 73.2% (odds ratio [OR] = 1.95; 95% confidence interval [CI] = 1.58, 2.40). Self-reported noncompliance decreased from 24.6% to 14.0% (OR = 0.50; 95% CI = 0.30, 0.85). Likelihood of visiting a bar or of not changing bar patronage after the law was implemented increased from 86% to 91% (OR = 1.76; 95% CI = 1.29, 2.40). CONCLUSIONS: California bar patrons increasingly support and comply with the smoke-free bar law.