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

Gene therapy potential for genetic disorders of surfactant dysfunction

Pulmonary surfactant is critically important to prevent atelectasis by lowering the surface tension of the alveolar lining liquid. While respiratory distress syndrome (RDS) is common in premature infants, severe RDS in term and late preterm infants suggests an underlying genetic etiology. Pathogenic variants in the genes encoding key components of pulmonary surfactant including surfactant protein B (SP-B

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

Congenital acute myeloid leukemia with unique translocation t(11;19)(q23;p13.3)

Congenital leukemia is rarely encountered in clinical practice, even in tertiary children's hospitals. Leukemia may cause significant coagulopathy, putting the patient at risk of intracranial hemorrhage. In this case, the authors present a female infant with a unique mixed phenotypic congenital acute myeloid leukemia showing mixed-lineage leukemia (MLL) rearrangement and severe coagulopathy resulting in a large subdural hematoma. Despite the fatal outcome in this case, neurosurgical treatment of patients with acute myeloid leukemia should be considered if coagulopathy and the clinical scenario allow

Whole-genome and long-read sequencing identify a novel mechanism in RFC1 resulting in CANVAS syndrome

OBJECTIVES: Cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) results from biallelic intronic pentanucleotide repeats in METHODS: We performed whole-genome sequencing (WGS) on peripheral blood DNA samples from the proband and his unaffected mother. We performed DNA long-read sequencing and synthesized complementary DNA from RNA using peripheral blood from the proband. RESULTS: WGS confirmed the maternally inherited DISCUSSION: We report an adult with CANVAS due to compound heterozygous pathogeni

Discovery of a novel CHD7 CHARGE syndrome variant by integrated omics analyses

Chromodomain helicase DNA-binding protein 7 (CHD7) pathogenic variants are identified in more than 90% of infants and children with CHARGE (Coloboma of the iris, retina, and/or optic disk; congenital Heart defects, choanal Atresia, Retardation of growth and development, Genital hypoplasia, and characteristic outer and inner Ear anomalies and deafness) syndrome. Approximately, 10% of cases have no known genetic cause identified. We report a male child with clinical features of CHARGE syndrome and nondiagnostic genetic testing that included chromosomal microarray, CHD7 sequencing and deletion/duplication analysis, SEMA3E sequencing, and trio exome and whole-genome sequencing (WGS). We used a comprehensive clinical assessment, genome-wide methylation analysis (GMA), reanalysis of WGS data, and CHD7 RNA studies to discover a novel variant that causes CHD7 haploinsufficiency. The 7-year-old Hispanic male proband has typical phenotypic features of CHARGE syndrome. GMA revealed a CHD7-associated epigenetic signature. Reanalysis of the WGS data with focused bioinformatic analysis of CHD7 detected a novel, de novo 15 base pair deletion in Intron 4 of CHD7 (c.2239-20_2239-6delGTCTTGGGTTTTTGT [NM_017780.3]). Using proband RNA, we confirmed that this novel deletion causes CHD7 haploinsufficiency by disrupting the canonical 3′ splice site and introducing a premature stop codon. Integrated genomic, epigenomic, and transcriptome analyses discovered a novel CHD7 variant that causes CHARGE syndrome

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

Novel parent-of-origin-specific differentially methylated loci on chromosome 16

BACKGROUND: Congenital malformations associated with maternal uniparental disomy of chromosome 16, upd(16)mat, resemble those observed in newborns with the lethal developmental lung disease, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interestingly, ACDMPV-causative deletions, involving FOXF1 or its lung-specific upstream enhancer at 16q24.1, arise almost exclusively on the maternally inherited chromosome 16. Given the phenotypic similarities between upd(16)mat and ACDMPV, together with parental allelic bias in ACDMPV, we hypothesized that there may be unknown imprinted loci mapping to chromosome 16 that become functionally unmasked by chromosomal structural variants. RESULTS: To identify parent-of-origin biased DNA methylation, we performed high-resolution bisulfite sequencing of chromosome 16 on peripheral blood and cultured skin fibroblasts from individuals with maternal or paternal upd(16) as well as lung tissue from patients with ACDMPV-causative 16q24.1 deletions and a normal control. We identified 22 differentially methylated regions (DMRs) with ≥ 5 consecutive CpG methylation sites and varying tissue-specificity, including the known DMRs associated with the established imprinted gene ZNF597 and DMRs supporting maternal methylation of PRR25, thought to be paternally expressed in lymphoblastoid cells. Lastly, we found evidence of paternal methylation on 16q24.1 near LINC01082 mapping to the FOXF1 enhancer. CONCLUSIONS: Using high-resolution bisulfite sequencing to evaluate DNA methylation across chromosome 16, we found evidence for novel candidate imprinted loci on chromosome 16 that would not be evident in array-based assays and could contribute to the birth defects observed in patients with upd(16)mat or in ACDMPV

A qualitative investigation into knowledge, beliefs, and practices surrounding mastitis in sub-Saharan Africa: what implications for vertical transmission of HIV?

BACKGROUND: Mastitis constitutes an important risk factor in HIV vertical transmission. Very little, however, is known on how women in sub-Saharan Africa conceptualise health problems related to breastfeeding, such as mastitis, and how they act when sick. We aimed at filling this gap in knowledge, by documenting the indigenous nosography of mastitis, health seeking behaviour, and remedies for prophylaxis and treatment in rural sub-Saharan Africa. METHODS: The study was conducted in the Nouna Health District, rural Burkina Faso. We employed a combination of in-depth individual interviews and focus group discussions reaching both women and guérisseuers. All material was transcribed, translated, and analysed inductively, applying data and analyst triangulation. RESULTS: Respondents perceived breast problems related to lactation to be highly prevalent and described a sequence of symptoms which resembles the biomedical understanding of pathologies related to breastfeeding, ranging from breast engorgement (stasis) to inflammation (mastitis) and infection (breast abscess). The aetiology of disease, however, differed from biomedical notions as both women and guerisseurs distinguished between "natural" and "unnatural" causes of health problems related to breastfeeding. To prevent and treat such pathologies, women used a combination of traditional and biomedical therapies, depending on the perceived cause of illness. In general, however, a marked preference for traditional systems of care was observed. CONCLUSION: Health problems related to breastfeeding are perceived to be very common in rural Burkina Faso. Further epidemiological research to assess the actual prevalence of such pathologies is urgently needed to inform the design of adequate control measures, especially given the impact of mastitis on HIV vertical transmission. Our investigation into local illness concepts and health care seeking behaviour is useful to ensure that such measures be culturally sensitive. Further research into the efficacy of local customs and traditional healing methods and their effect on viral load in breast milk is also urgently needed

Survival among children with "Lethal" congenital contracture syndrome 11 caused by novel mutations in the gliomedin gene (GLDN).

Biallelic GLDN mutations have recently been identified among infants with lethal congenital contracture syndrome 11 (LCCS11). GLDN encodes gliomedin, a protein required for the formation of the nodes of Ranvier and development of the human peripheral nervous system. We report six infants and children from four unrelated families with biallelic GLDN mutations, four of whom survived beyond the neonatal period into infancy, childhood, and late adolescence with intensive care and chronic respiratory and nutritional support. Our findings expand the genotypic and phenotypic spectrum of LCCS11 and demonstrate that the condition may not necessarily be lethal in the neonatal period