The genomics of neonatal abstinence syndrome

Significant variability has been observed in the development and severity of neonatal abstinence syndrome (NAS) among neonates exposed to prenatal opioids. Since maternal opioid dose does not appear to correlate directly with neonatal outcome, maternal, placental, and fetal genomic variants may play important roles in NAS. Previous studies in small cohorts have demonstrated associations of variants in maternal and infant genes that encode the μ-opioid receptor (OPRM1), catechol-O-methyltransferase (COMT), and prepronociceptin (PNOC) with a shorter length of hospital stay and less need for treatment in neonates exposed to opioids in utero. Consistently falling genomic sequencing costs and computational approaches to predict variant function will permit unbiased discovery of genomic variants and gene pathways associated with differences in maternal and fetal opioid pharmacokinetics and pharmacodynamics and with placental opioid transport and metabolism. Discovery of pathogenic variants should permit better delineation of the risk of developing more severe forms of NAS. This review provides a summary of the current role of genomic factors in the development of NAS and suggests strategies for further genomic discovery

Patient-specific iPSCs carrying an SFTPC mutation reveal the intrinsic alveolar epithelial dysfunction at the inception of interstitial lung disease

Alveolar epithelial type 2 cell (AEC2) dysfunction is implicated in the pathogenesis of adult and pediatric interstitial lung disease (ILD), including idiopathic pulmonary fibrosis (IPF); however, identification of disease-initiating mechanisms has been impeded by inability to access primary AEC2s early on. Here, we present a human in vitro model permitting investigation of epithelial-intrinsic events culminating in AEC2 dysfunction, using patient-specific induced pluripotent stem cells (iPSCs) carrying an AEC2-exclusive disease-associated variant (SFTP

Sequencing of idiopathic pulmonary fibrosis-related genes reveals independent single gene associations

BACKGROUND: Previous studies investigating a genetic basis for idiopathic pulmonary fibrosis (IPF) have focused on resequencing single genes in IPF kindreds or cohorts to determine the genetic contributions to IPF. None has investigated interactions among the candidate genes. OBJECTIVE: To compare the frequencies and interactions of mutations in six IPF-associated genes in a cohort of 132 individuals with IPF with those of a disease-control cohort of 192 individuals with chronic obstructive pulmonary disease (COPD) and the population represented in the Exome Variant Server. METHODS: We resequenced the genes encoding surfactant proteins A2 (SFTPA2), and C (SFTPC), the ATP binding cassette member A3 (ABCA3), telomerase (TERT), thyroid transcription factor (NKX2-1) and mucin 5B (MUC5B) and compared the collapsed frequencies of rare (minor allele frequency <1%), computationally predicted deleterious variants in each cohort. We also genotyped a common MUC5B promoter variant that is over-represented in individuals with IPF. RESULTS: We found 15 mutations in 14 individuals (11%) in the IPF cohort: (SFTPA2 (n=1), SFTPC (n=5), ABCA3 (n=4) and TERT (n=5)). No individual with IPF had two different mutations, but one individual with IPF was homozygous for p.E292V, the most common ABCA3 disease-causing variant. We did not detect an interaction between any of the mutations and the MUC5B promoter variant. CONCLUSIONS: Rare mutations in SFTPA2, SFTPC and TERT are collectively over-represented in individuals with IPF. Genetic analysis and counselling should be considered as part of the IPF evaluation

Rare recessive loss-of-function methionyl-tRNA synthetase mutations presenting as a multi-organ phenotype

BACKGROUND: Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to its cognate transfer RNA and therefore plays an essential role in protein biosynthesis. METHODS: We used exome sequencing, aminoacylation assays, homology modeling, and immuno-isolation of transfected MARS to identify and characterize mutations in the methionyl-tRNA synthetase gene (MARS) in an infant with an unexplained multi-organ phenotype. RESULTS: We identified compound heterozygous mutations (F370L and I523T) in highly conserved regions of MARS. The parents were each heterozygous for one of the mutations. Aminoacylation assays documented that the F370L and I523T MARS mutants had 18 ± 6% and 16 ± 6%, respectively, of wild-type activity. Homology modeling of the human MARS sequence with the structure of E. coli MARS showed that the F370L and I523T mutations are in close proximity to each other, with residue I523 located in the methionine binding pocket. We found that the F370L and I523T mutations did not affect the association of MARS with the multisynthetase complex. CONCLUSION: This infant expands the catalogue of inherited human diseases caused by mutations in aminoacyl-tRNA synthetase genes

Precise breakpoint detection in a patient with 9p- syndrome

We present a case of 9p- syndrome with a complex chromosomal event originally characterized by the classical karyotype approach as 46,XX,der(9)t(9;13)(p23;q13). We used advanced technologies (Bionano Genomics genome imaging and 10× Genomics sequencing) to characterize the location of the translocation and accompanying deletion on Chromosome 9 and duplication on Chromosome 13 with single-nucleotide breakpoint resolution. The translocation breakpoint was at Chr 9:190938 and Chr 13:50850492, the deletion at Chr 9:1-190938, and the duplication at Chr 13:50850492-114364328. We identified genes in the deletion and duplication regions that are known to be associated with this patient\u27s phenotype (e.g.

CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism

Human pluripotent stem cell modeling of alveolar type 2 cell dysfunction caused by ABCA3 mutations

Mutations in ATP-binding cassette A3 (ABCA3), a phospholipid transporter critical for surfactant homeostasis in pulmonary alveolar type II epithelial cells (AEC2s), are the most common genetic causes of childhood interstitial lung disease (chILD). Treatments for patients with pathological variants of ABCA3 mutations are limited, in part due to a lack of understanding of disease pathogenesis resulting from an inability to access primary AEC2s from affected children. Here, we report the generation of AEC2s from affected patient induced pluripotent stem cells (iPSCs) carrying homozygous versions of multiple ABCA3 mutations. We generated syngeneic CRISPR/Cas9 gene-corrected and uncorrected iPSCs and ABCA3-mutant knockin ABCA3:GFP fusion reporter lines for in vitro disease modeling. We observed an expected decreased capacity for surfactant secretion in ABCA3-mutant iPSC-derived AEC2s (iAEC2s), but we also found an unexpected epithelial-intrinsic aberrant phenotype in mutant iAEC2s, presenting as diminished progenitor potential, increased NFκB signaling, and the production of pro-inflammatory cytokines. The ABCA3:GFP fusion reporter permitted mutant-specific, quantifiable characterization of lamellar body size and ABCA3 protein trafficking, functional features that are perturbed depending on ABCA3 mutation type. Our disease model provides a platform for understanding ABCA3 mutation-mediated mechanisms of alveolar epithelial cell dysfunction that may trigger chILD pathogenesis

From karyotypes to precision genomics in 9p deletion and duplication syndromes

While 9p deletion and duplication syndromes have been studied for several years, small sample sizes and minimal high-resolution data have limited a comprehensive delineation of genotypic and phenotypic characteristics. In this study, we examined genetic data from 719 individuals in the worldwide 9p Network Cohort: a cohort seven to nine times larger than any previous study of 9p. Most breakpoints occur in bands 9p22 and 9p24, accounting for 35% and 38% of all breakpoints, respectively. Bands 9p11 and 9p12 have the fewest breakpoints, with each accounting for 0.6% of all breakpoints. The most common phenotype in 9p deletion and duplication syndromes is developmental delay, and we identified eight known neurodevelopmental disorder genes in 9p22 and 9p24. Since it has been previously reported that some individuals have a secondary structural variant related to the 9p variant, we examined our cohort for these variants and found 97 events. The top secondary variant involved 9q in 14 individuals (1.9%), including ring chromosomes and inversions. We identified a gender bias with significant enrichment for females (p = 0.0006) that may arise from a sex reversal in some individuals with 9p deletions. Genes on 9p were characterized regarding function, constraint metrics, and protein-protein interactions, resulting in a prioritized set of genes for further study. Finally, we achieved precision genomics in one child with a complex 9p structural variation using modern genomic technologies, demonstrating that long-read sequencing will be integral for some cases. Our study is the largest ever on 9p-related syndromes and provides key insights into genetic factors involved in these syndromes

A phase 1/2 open label nonrandomized clinical trial of intravenous 2-hydroxypropyl-β-cyclodextrin for acute liver disease in infants with Niemann-Pick C1

Introduction: Niemann-Pick C (NPC) is an autosomal recessive disease due to defective NPC1 or NPC2 proteins resulting in Methods: Infants received intravenous 2HPBCD twice a week for 6 weeks, followed by monthly infusion for 6-months. Primary outcome measure was reduction of plasma (3β,5α,6β-trihydroxy-cholan-24-oyl) glycine (TCG), a bile acid generated from cholesterol sequestered in lysosome. Results: Three participants completed this protocol. A fourth patient received intravenous 2HPBCD under an emergency investigational new drug study but later expired from her underlying condition. The three protocol patients are living and have improved liver enzymes and TCG. No patient has experienced a drug-related adverse event. Conclusion: Intravenous 2HPBCD was tolerated in three infants with liver disease due to NPC

Exploring the Mode of Action of Bioactive Compounds by Microfluidic Transcriptional Profiling in Mycobacteria

10.1371/journal.pone.0069191PLoS ONE87-POLN