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.

Rare variants in CAPN2 increase risk for isolated hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) characterized by hypoplasia of the left ventricle and aorta along with stenosis or atresia of the aortic and mitral valves. HLHS represents only ∼4%-8% of all CHDs but accounts for ∼25% of deaths. HLHS is an isolated defect (i.e., iHLHS) in 70% of families, the vast majority of which are simplex. Despite intense investigation, the genetic basis of iHLHS remains largely unknown. We performed exome sequencing on 331 families with iHLHS aggregated from four independent cohorts. A Mendelian-model-based analysis demonstrated that iHLHS was not due to single, large-effect alleles in genes previously reported to underlie iHLHS or CHD in >90% of families in this cohort. Gene-based association testing identified increased risk for iHLHS associated with variation in CAPN2 (p = 1.8 × 10-5), encoding a protein involved in functional adhesion. Functional validation studies in a vertebrate animal model (Xenopus laevis) confirmed CAPN2 is essential for cardiac ventricle morphogenesis and that in vivo loss of calpain function causes hypoplastic ventricle phenotypes and suggest that human CAPN2707C>T and CAPN21112C>T variants, each found in multiple individuals with iHLHS, are hypomorphic alleles. Collectively, our findings show that iHLHS is typically not a Mendelian condition, demonstrate that CAPN2 variants increase risk of iHLHS, and identify a novel pathway involved in HLHS pathogenesis.

Exome-wide assessment of isolated biliary atresia: A report from the National Birth Defects Prevention Study using child-parent trios and a case-control design to identify novel rare variants.

The etiology of biliary atresia (BA) is unknown, but recent studies suggest a role for rare protein-altering variants (PAVs). Exome sequencing data from the National Birth Defects Prevention Study on 54 child-parent trios, one child-mother duo, and 1513 parents of children with other birth defects were analyzed. Most (91%) cases were isolated BA. We performed (1) a trio-based analysis to identify rare de novo, homozygous, and compound heterozygous PAVs and (2) a case-control analysis using a sequence kernel-based association test to identify genes enriched with rare PAVs. While we replicated previous findings on PKD1L1, our results do not suggest that recurrent de novo PAVs play important roles in BA susceptibility. In fact, our finding in NOTCH2, a disease gene associated with Alagille syndrome, highlights the difficulty in BA diagnosis. Notably, IFRD2 has been implicated in other gastrointestinal conditions and warrants additional study. Overall, our findings strengthen the hypothesis that the etiology of BA is complex.

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.

Genetic drivers of Cushing's disease: Frequency and associated phenotypes.

PURPOSE: Cushing's disease (CD) is often explained by a single somatic sequence change. Germline defects, however, often go unrecognized. We aimed to determine the frequency and associated phenotypes of genetic drivers of CD in a large cohort. METHODS: We studied 245 unrelated patients with CD (139 female, 56.7%), including 230 (93.9%) pediatric and 15 (6.1%) adult patients. Germline exome sequencing was performed in 184 patients; tumor exome sequencing was also done in 27 of them. A total of 43 germline samples and 92 tumor samples underwent Sanger sequencing of specific genes. Rare variants of uncertain significance, likely pathogenic (LP), or pathogenic variants in CD-associated genes, were identified. RESULTS: Germline variants (13 variants of uncertain significance, 8 LP, and 11 pathogenic) were found in 8 of 19 patients (42.1%) with positive family history and in 23 of 226 sporadic patients (10.2%). Somatic variants (1 LP and 7 pathogenic) were found in 20 of 119 tested individuals (16.8%); one of them had a coexistent germline defect. Altogether, variants of interest were identified at the germline level in 12.2% of patients, at the somatic level in 7.8%, and coexisting germline and somatic variants in 0.4%, accounting for one-fifth of the cohort. CONCLUSION: We report an estimate of the contribution of multiple germline and somatic genetic defects underlying CD in a single cohort.

Whole Exome Sequencing in Patients With Ectopic Posterior Pituitary.

Context: Ectopic posterior pituitary (EPP), a condition in which the posterior pituitary gland is displaced due to defective neuronal migration, is frequently associated with hypopituitarism. Genetic variants play a role, but many cases remain unexplained. Objective: A large EPP cohort was studied to explore the importance of genetic variants and how they correlate with clinical findings. Methods: Whole exome sequencing was performed on a discovery sample of 27 cases to identify rare variants. The variants that met the criteria for rarity and biological relevance, or that were previously associated with EPP (ROBO1 and HESX1), were then resequenced in the 27 cases plus a replication sample of 51 cases. Results: We identified 16 different variants in 12 genes in 15 of the 78 cases (19.2%). Complete anterior pituitary deficiency was twice as common in cases with variants of interest compared to cases without variants (9/15 [60%] vs 19/63 [30.1%], respectively; Z test, P = 0.06). Breech presentation was more frequent in the variant positive group (5/15 vs 1/63; Z test, P = 0.003). Four cases had variants in ROBO1 and 1 in HESX1, genes previously associated with EPP. The ROBO1 p.S18* variant has not been reported previously; ROBO1 p.Q1227H has not been associated with EPP previously. Conclusion: EPP cases with variants of interest identified in this study were more likely to present with severe clinical disease. Several variants were identified in genes not previously associated with EPP. Our findings confirm that EPP is a multigenic disorder. Future studies are needed to identify additional genes.

Utility of Newborn Dried Blood Spots to Ascertain Seroprevalence of SARS-CoV-2 Antibodies Among Individuals Giving Birth in New York State, November 2019 to November 2021.

Importance: Serosurveys can be used to monitor population-level dynamics of COVID-19 and vaccination. Dried blood spots (DBSs) collected from infants contain maternal IgG antibodies and are useful for serosurveys of individuals recently giving birth. Objectives: To examine SARS-CoV-2 antibody prevalence in pregnant individuals in New York State, identify associations between SARS-CoV-2 antibody status and maternal and infant characteristics, and detect COVID-19 vaccination among this population. Design, Setting, and Participants: A population-based, repeated cross-sectional study was conducted to detect SARS-CoV-2 nucleocapsid (N) and spike (S) IgG antibodies. Deidentified DBS samples and data submitted to the New York State Newborn Screening Program between November 1, 2019, and November 30, 2021, were analyzed. Exposures: Prenatal exposure to SARS-CoV-2 antibodies. Main Outcomes and Measures: The presence of IgG antibodies to SARS-CoV-2 N and S antigens was measured using a microsphere immunoassay. Data were analyzed by geographic region and compared with reported COVID-19 cases and vaccinations among reproductive-aged females (15-44 years of age). Data were stratified by infant birth weight, gestational age, maternal age, and multiple birth status. Results: Dried blood spot samples from 415 293 infants (median [IQR] age, 1.04 [1.00-1.20] days; 210 805 [51.1%] male) were analyzed for SARS-CoV-2 antibodies. The first known antibody-positive infant in New York State was born on March 29, 2020. SARS-CoV-2 seroprevalence reflected statewide and regional COVID-19 cases among reproductive-aged females in the prevaccine period. From February through November 2021, S seroprevalence was strongly correlated with cumulative vaccinations in each New York State region and in the state overall (rs = 0.92-1.00, P ≤ .001). S and N seroprevalences were significantly lower in newborns with very low birth weight (720 [14.8%] for S and 138 [2.8%] for N, P < .001) and low birth weight (5160 [19.3%] for S and 1233 [4.6%] for N, P = .009) compared with newborns with normal birth weight (77 116 [20.1%] for S and 19 872 [5.2%] for N). Lower N and higher S seroprevalences were observed in multiple births (odds ratio [OR], 0.84; 95% CI, 0.75-0.94; P = .002 for N and OR, 1.24; 95% CI, 1.18-1.31; P < .001 for S) vs single births and for maternal age older than 30 years (OR, 0.87; 95% CI, 0.80-0.94; P < .001 for N and OR, 1.17; 95% CI, 1.11-1.23; P < .001 for S) vs younger than 20 years. Conclusions and Relevance: In this study, seroprevalence in newborn DBS samples reflected COVID-19 case fluctuations and vaccinations among reproductive-aged women during the study period. These results demonstrate the utility of using newborn DBS testing to estimate SARS-CoV-2 seroprevalence in pregnant individuals.

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.

Exome sequencing identifies variants in infants with sacral agenesis.

BACKGROUND: Sacral agenesis (SA) consists of partial or complete absence of the caudal end of the spine and often presents with additional birth defects. Several studies have examined gene variants for syndromic forms of SA, but only one has examined exomes of children with non-syndromic SA. METHODS: Using buccal cell specimens from families of children with non-syndromic SA, exomes of 28 child-parent trios (eight with and 20 without a maternal diagnosis of pregestational diabetes) and two child-father duos (neither with diagnosis of maternal pregestational diabetes) were exome sequenced. RESULTS: Three children had heterozygous missense variants in ID1 (Inhibitor of DNA Binding 1), with CADD scores >20 (top 1% of deleterious variants in the genome); two children inherited the variant from their fathers and one from the child's mother. Rare missense variants were also detected in PDZD2 (PDZ Domain Containing 2; N = 1) and SPTBN5 (Spectrin Beta, Non-erythrocytic 5; N = 2), two genes previously suggested to be associated with SA etiology. Examination of variants with autosomal recessive and X-linked recessive inheritance identified five and two missense variants, respectively. Compound heterozygous variants were identified in several genes. In addition, 12 de novo variants were identified, all in different genes in different children. CONCLUSIONS: To our knowledge, this is the first study reporting a possible association between ID1 and non-syndromic SA. Although maternal pregestational diabetes has been strongly associated with SA, the missense variants in ID1 identified in two of three children were paternally inherited. These findings add to the knowledge of gene variants associated with non-syndromic SA and provide data for future studies.

Probing the functional consequence and clinical relevance of CD320 p.E88del, a variant in the transcobalamin receptor gene.

The biological and clinical significance of the p.E88del variant in the transcobalamin receptor, CD320, is unknown. This allele is annotated in ClinVar as likely benign, pathogenic, and of uncertain significance. To determine functional consequence and clinical relevance of this allele, we employed cell culture and genetic association studies. Fibroblasts from 16 CD320 p.E88del homozygotes exhibited reduced binding and uptake of cobalamin. Complete ascertainment of newborns with transiently elevated C3 (propionylcarnitine) in New York State demonstrated that homozygosity for CD320 p.E88del was over-represented (7/348, p < 6 × 10-5 ). Using population data, we estimate that ~85% of the p.E88del homozygotes born in the same period did not have elevated C3, suggesting that cobalamin metabolism in the majority of these infants with this genotype is unaffected. Clinical follow-up of 4/9 homozygous individuals uncovered neuropsychological findings, mostly in speech and language development. None of these nine individuals exhibited perturbation of cobalamin metabolism beyond the newborn stage even during periods of acute illness. Newborns homozygous for this allele in the absence of other factors are at low risk of requiring clinical intervention, although more studies are required to clarify the natural history of various CD320 variants across patient populations.

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.

Exome sequencing of child-parent trios with bladder exstrophy: Findings in 26 children.

Bladder exstrophy (BE) is a rare, lower ventral midline defect with the bladder and part of the urethra exposed. The etiology of BE is unknown but thought to be influenced by genetic variation with more recent studies suggesting a role for rare variants. As such, we conducted paired-end exome sequencing in 26 child/mother/father trios. Three children had rare (allele frequency ≤ 0.0001 in several public databases) inherited variants in TSPAN4, one with a loss-of-function variant and two with missense variants. Two children had loss-of-function variants in TUBE1. Four children had rare missense or nonsense variants (one per child) in WNT3, CRKL, MYH9, or LZTR1, genes previously associated with BE. We detected 17 de novo missense variants in 13 children and three de novo loss-of-function variants (AKR1C2, PRRX1, PPM1D) in three children (one per child). We also detected rare compound heterozygous loss-of-function variants in PLCH2 and CLEC4M and rare inherited missense or loss-of-function variants in additional genes applying autosomal recessive (three genes) and X-linked recessive inheritance models (13 genes). Variants in two genes identified may implicate disruption in cell migration (TUBE1) and adhesion (TSPAN4) processes, mechanisms proposed for BE, and provide additional evidence for rare variants in the development of this defect.

Prospective identification by neonatal screening of patients with guanidinoacetate methyltransferase deficiency.

INTRODUCTION: Guanidinoacetate methyltransferase (GAMT) deficiency is an inherited metabolic disorder that impairs the synthesis of creatine (CRE). Lack of CRE in the brain can cause intellectual disability, autistic-like behavior, seizures, and movement disorders. Identification at birth and immediate therapy can prevent intellectual disability and seizures. Here we report the first two cases of GAMT deficiency identified at birth by newborn screening (NBS) in Utah and New York. METHODS: NBS dried blood spots were analyzed by tandem mass spectrometry (MS/MS) using either derivatized or non-derivatized assays to detect guanidinoacetate (GUAC) and CRE. For any positive samples, a second-tier test using a more selective method, ultra-performance liquid chromatography (UPLC) combined with MS/MS, was performed to separate GUAC from potential isobaric interferences. RESULTS: NBS for GAMT deficiency began in Utah on June 1, 2015 using a derivatized method for the detection of GUAC and CRE. In May 2019, the laboratory and method transitioned to a non-derivatized method. GAMT screening was added to the New York State NBS panel on October 1, 2018 using a derivatized method. In New York, a total of 537,408 babies were screened, 23 infants were referred and one newborn was identified with GAMT deficiency. In Utah, a total of 273,902 infants were screened (195,425 with the derivatized method, 78,477 with the non-derivatized method), three infants referred and one was identified with GAMT deficiency. Mean levels of GUAC and CRE were similar between methods (Utah derivatized: GUAC = 1.20 ± 0.43 µmol/L, CRE = 238 ± 96 µmol/L; Utah non-derivatized: GUAC = 1.23 ± 0.61 µmol/L, CRE = 344 ± 150 µmol/L, New York derivatized: GUAC = 1.34 ± 0.57 µmol/L, CRE = 569 ± 155 µmol/L). With either Utah method, similar concentrations of GUAC are observed in first (collected around 1 day of age) and the second NBS specimens (routinely collected at 7-16 days of age), while CRE concentrations decreased in the second NBS specimens. Both infants identified with GAMT deficiency started therapy by 2 weeks of age and are growing and developing normally at 7 (Utah) and 4 (New York) months of age. CONCLUSIONS: Newborn screening allows for the prospective identification of GAMT deficiency utilizing elevated GUAC concentration as a marker. First-tier screening may be incorporated into existing methods for amino acids and acylcarnitines without the need for new equipment or staff. Newborn screening performed by either derivatized or non-derivatized methods and coupled with second-tier testing, has a very low false positive rate and can prospectively identify affected children. SummaryCerebral creatine deficiency syndromes caused by defects in creatine synthesis can result in intellectual disability, and are preventable if therapy is initiated early in life. This manuscript reports the identification of two infants with GAMT deficiency (one of the cerebral creatine deficiency syndromes) by newborn screening and demonstrates NBS feasibility using a variety of methods.

Rare Germline DICER1 Variants in Pediatric Patients With Cushing's Disease: What Is Their Role?

Context: The DICER1 syndrome is a multiple neoplasia disorder caused by germline mutations in the DICER1 gene. In DICER1 patients, aggressive congenital pituitary tumors lead to neonatal Cushing's disease (CD). The role of DICER1 in other corticotropinomas, however, remains unknown. Objective: To perform a comprehensive screening for DICER1 variants in a large cohort of CD patients, and to analyze their possible contribution to the phenotype. Design, setting, patients, and interventions: We included 192 CD cases: ten young-onset (age <30 years at diagnosis) patients were studied using a next generation sequencing panel, and 182 patients (170 pediatric and 12 adults) were screened via whole-exome sequencing. In seven cases, tumor samples were analyzed by Sanger sequencing. Results: Rare germline DICER1 variants were found in seven pediatric patients with no other known disease-associated germline defects or somatic DICER1 second hits. By immunohistochemistry, DICER1 showed nuclear localization in 5/6 patients. Variant transmission from one of the parents was confirmed in 5/7 cases. One patient had a multinodular goiter; another had a family history of melanoma; no other patients had a history of neoplasms. Conclusions: Our findings suggest that DICER1 gene variants may contribute to the pathogenesis of non-syndromic corticotropinomas. Clarifying whether DICER1 loss-of-function is disease-causative or a mere disease-modifier in this setting, requires further studies. Clinical trial registration: ClinicalTrials.gov: NCT00001595.

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.

Germline CDKN1B Loss-of-Function Variants Cause Pediatric Cushing's Disease With or Without an MEN4 Phenotype.

CONTEXT: Germline loss-of-function CDKN1B gene variants cause the autosomal dominant syndrome of multiple endocrine neoplasia type 4 (MEN4). Even though pituitary neuroendocrine tumors are a well-known component of the syndrome, only 2 cases of Cushing's disease (CD) have so far been described in this setting. AIM: To screen a large cohort of CD patients for CDKN1B gene defects and to determine their functional effects. PATIENTS: We screened 211 CD patients (94.3% pediatric) by germline whole-exome sequencing (WES) only (n = 157), germline and tumor WES (n = 27), Sanger sequencing (n = 6), and/or germline copy number variant (CNV) analysis (n = 194). Sixty cases were previously unpublished. Variant segregation was investigated in the patients' families, and putative pathogenic variants were functionally characterized. RESULTS: Five variants of interest were found in 1 patient each: 1 truncating (p.Q107Rfs*12) and 4 nontruncating variants, including 3 missense changes affecting the CDKN1B protein scatter domain (p.I119T, p.E126Q, and p.D136G) and one 5' untranslated region (UTR) deletion (c.-29_-26delAGAG). No CNVs were found. All cases presented early (10.5 ± 1.3 years) and apparently sporadically. Aside from colon adenocarcinoma in 1 carrier, no additional neoplasms were detected in the probands or their families. In vitro assays demonstrated protein instability and disruption of the scatter domain of CDKN1B for all variants tested. CONCLUSIONS: Five patients with CD and germline CDKN1B variants of uncertain significance (n = 2) or pathogenic/likely pathogenic (n = 3) were identified, accounting for 2.6% of the patients screened. Our finding that germline CDKN1B loss-of-function may present as apparently sporadic, isolated pediatric CD has important implications for clinical screening and genetic counselling.

Exome sequencing of family trios from the National Birth Defects Prevention Study: Tapping into a rich resource of genetic and environmental data.

BACKGROUND: The National Birth Defects Prevention Study (NBDPS) is a multisite, population-based, case-control study of genetic and nongenetic risk factors for major structural birth defects. Eligible women had a pregnancy affected by a birth defect or a liveborn child without a birth defect between 1997 and 2011. They were invited to complete a telephone interview to collect pregnancy exposure data and were mailed buccal cell collection kits to collect specimens from themselves, their child (if living), and their child's father. Over 23,000 families representing more than 30 major structural birth defects provided DNA specimens. METHODS: To evaluate their utility for exome sequencing (ES), specimens from 20 children with colonic atresia were studied. Evaluations were conducted on specimens collected using cytobrushes stored and transported in open versus closed packaging, on native genomic DNA (gDNA) versus whole genome amplified (WGA) products and on a library preparation protocol adapted to low amounts of DNA. RESULTS: The DNA extracted from brushes in open packaging yielded higher quality sequence data than DNA from brushes in closed packaging. Quality metrics of sequenced gDNA were consistently higher than metrics from corresponding WGA products and were consistently high when using a low input protocol. CONCLUSIONS: This proof-of-principle study established conditions under which ES can be applied to NBDPS specimens. Successful sequencing of exomes from well-characterized NBDPS families indicated that this unique collection can be used to investigate the roles of genetic variation and gene-environment interaction effects in birth defect etiologies, providing a valuable resource for birth defect researchers.