Homozygous EPRS1 missense variant causing hypomyelinating leukodystrophy-15 alters variant-distal mRNA m6A site accessibility.

Hypomyelinating leukodystrophy (HLD) is an autosomal recessive disorder characterized by defective central nervous system myelination. Exome sequencing of two siblings with severe cognitive and motor impairment and progressive hypomyelination characteristic of HLD revealed homozygosity for a missense single-nucleotide variant (SNV) in EPRS1 (c.4444 C > A; p.Pro1482Thr), encoding glutamyl-prolyl-tRNA synthetase, consistent with HLD15. Patient lymphoblastoid cell lines express markedly reduced EPRS1 protein due to dual defects in nuclear export and cytoplasmic translation of variant EPRS1 mRNA. Variant mRNA exhibits reduced METTL3 methyltransferase-mediated writing of N6-methyladenosine (m6A) and reduced reading by YTHDC1 and YTHDF1/3 required for efficient mRNA nuclear export and translation, respectively. In contrast to current models, the variant does not alter the sequence of m6A target sites, but instead reduces their accessibility for modification. The defect was rescued by antisense morpholinos predicted to expose m6A sites on target EPRS1 mRNA, or by m6A modification of the mRNA by METTL3-dCas13b, a targeted RNA methylation editor. Our bioinformatic analysis predicts widespread occurrence of SNVs associated with human health and disease that similarly alter accessibility of distal mRNA m6A sites. These results reveal a new RNA-dependent etiologic mechanism by which SNVs can influence gene expression and disease, consequently generating opportunities for personalized, RNA-based therapeutics targeting these disorders.

Absence of iron-responsive element-binding protein 2 causes a novel neurodegenerative syndrome.

Disruption of cellular iron homeostasis can contribute to neurodegeneration. In mammals, two iron-regulatory proteins (IRPs) shape the expression of the iron metabolism proteome. Targeted deletion of Ireb2 in a mouse model causes profoundly disordered iron metabolism, leading to functional iron deficiency, anemia, erythropoietic protoporphyria, and a neurodegenerative movement disorder. Using exome sequencing, we identified the first human with bi-allelic loss-of-function variants in the gene IREB2 leading to an absence of IRP2. This 16-year-old male had neurological and haematological features that emulate those of Ireb2 knockout mice, including neurodegeneration and a treatment-resistant choreoathetoid movement disorder. Cellular phenotyping at the RNA and protein level was performed using patient and control lymphoblastoid cell lines, and established experimental assays. Our studies revealed functional iron deficiency, altered post-transcriptional regulation of iron metabolism genes, and mitochondrial dysfunction, as observed in the mouse model. The patient's cellular abnormalities were reversed by lentiviral-mediated restoration of IRP2 expression. These results confirm that IRP2 is essential for regulation of iron metabolism in humans, and reveal a previously unrecognized subclass of neurodegenerative disease. Greater understanding of how the IRPs mediate cellular iron distribution may ultimately provide new insights into common and rare neurodegenerative processes, and could result in novel therapies.

Triple A syndrome presenting as complicated hereditary spastic paraplegia.

BACKGROUND: Hereditary spastic paraplegia (HSP) is a group of rare disorders characterized by spastic paraparesis and other symptoms. Often, other diseases can mimic HSP, which may delay diagnosis and treatment. METHODS: Whole exome sequencing was performed in families with clinically suspected HSP without a genetic diagnosis. RESULTS: We report three patients from two families who presented with lower limb spasticity, muscular atrophy, and other neurological symptoms, who were clinically diagnosed with complicated HSP. Whole exome sequencing revealed bi-allelic AAAS nonsense mutations; one individual was homozygous for the p.(Arg478*) mutation, and two siblings were homozygous for the p.(Arg286*) mutation, leading to the diagnosis of triple A syndrome. This rare syndrome is typically characterized by a triad of symptoms: achalasia, adrenal insufficiency, and alacrima, and is often accompanied by other neurological abnormalities. CONCLUSIONS: Our findings suggest that triple A syndrome should be suspected in complicated HSP patients without a known genetic cause, especially if at least one of the main triad of triple A syndrome symptoms is present.

Association of In Utero Exposure to Polybrominated Diphenyl Ethers With the Risk of Hypospadias.

Importance: Polybrominated diphenyl ethers (PBDEs) are added to many consumer products as flame retardants, and their endocrine-disrupting properties are a growing health concern in pregnancy. Objective: To investigate whether in utero PBDE exposure as measured in maternal hair is associated with increased risk for hypospadias. Design, Setting, and Participants: In this case-control study, the setting was the urology clinic of a tertiary pediatric hospital between January 3, 2011, and April 1, 2013. Participants were children diagnosed as having hypospadias and their mothers and a control group of children without hypospadias and their mothers. Dates of data analysis were September 3, 2017, to December 28, 2017. Exposures: Gestational exposure to 8 PBDEs as measured in the 3-cm segment closest to the skull of maternal hair by gas chromatography-mass spectroscopy as a proxy for in utero exposure. The mothers resided in the same household for the duration of their pregnancy. Main Outcomes and Measures: Difference in total maternal hair PBDE levels between the hypospadias and control groups. Results: Total PBDE levels were significantly higher among mothers of infants with hypospadias (n = 152) (total PBDE level, 51.4 pg/mg; interquartile range, 35.8-78.5 pg/mg) than among controls (n = 64) (total PBDE level, 35.8 pg/mg; interquartile range, 18.1-69.9 pg/mg) (P = .02). Of the 152 women with sufficient hair samples for analysis in the case group, 89 completed a questionnaire and were included in a multivariable analysis, and of the 64 women with sufficient hair samples for analysis in the control group, 54 completed a questionnaire and were included in a multivariable analysis. Adjusting for potential confounders, hypospadias was associated with a relative 48.2% (95% CI, 23.2%-65.4%) higher maternal level of total PBDE levels in the multivariable analysis. Conclusions and Relevance: In this analysis, mothers of children with hypospadias were exposed during pregnancy to significantly higher levels of PBDEs. The results of this study suggest that level of exposure to PBDEs during gestation may have a role in the etiology of hypospadias.

Impact of Mobility Device Use on Quality of Life in Children With Friedreich Ataxia.

OBJECTIVE: To determine how mobility device use impacts quality of life in children with Friedreich ataxia. STUDY DESIGN: Data from 111 pediatric patients with genetically confirmed Friedreich ataxia were collected from a prospective natural history study utilizing standardized clinical evaluations, including health-related quality of life using the Pediatric Quality of Life Inventory (PedsQL) 4.0 Generic Core Module. RESULTS: Mobility device use was associated with worse mean PedsQL total, physical, emotional, social, and academic subscores, after adjusting for gender, age of disease onset, and Friedreich Ataxia Rating Scale score. The magnitude of the difference was greatest for the physical subscore (-19.5 points, 95% CI = -30.00, -8.99, P < .001) and least for the emotional subscore (-10.61 points, 95% CI = -20.21, -1.02, P = .03). Transition to or between mobility devices trended toward worse physical subscore (-16.20 points, 95% CI = -32.07, -0.33, P = .05). CONCLUSIONS: Mobility device use is associated with significant worsening of all domains of quality of life in children with Friedreich ataxia.

Erratum: "A Case-Control Study of Maternal Polybrominated Diphenyl Ether (PBDE) Exposure and Cryptorchidism in Canadian Populations".

[This corrects the article DOI: 10.1289/EHP522.].

A Case-Control Study of Maternal Polybrominated Diphenyl Ether (PBDE) Exposure and Cryptorchidism in Canadian Populations.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are flame retardants found in North American household products during the past four decades. These chemicals leach out in dust as products age, exposing individuals daily through inhalation and ingestion. Animal studies suggest that PBDEs disrupt sex hormones and adversely affect development of the reproductive system. OBJECTIVES: In the present study, we examined whether there is a link between maternal hair PBDE concentrations and the risk of cryptorchidism (undescended testes) in male infants; testis descent is known to be dependent on androgens. METHODS: Full-term male infants were recruited through clinics in Montreal, Toronto, and London, Canada. Boys with cryptorchidism at 3-18 months of age (n=137) were identified by pediatric urologists and surgeons; similar-aged controls (n=158) had no genitourinary abnormalities as assessed by pediatricians. Eight BDE congeners (BDE-28, -47, -99, -100, -153, -154, -183, -209) were measured by GC-MS (gas chromatography-mass spectrometry) in maternal hair samples collected at the time of recruitment. RESULTS: The ∑PBDE geometric mean for maternal hair was 45.35 pg/mg for controls and 50.27 pg/mg for cases; the concentrations of three BDEs (BDE-99, -100, and -154) were significantly higher in cases than controls in unadjusted models. In adjusted models, every 10-fold increase in the concentration of maternal hair BDE-99 [OR=2.53 (95% CI: 1.29, 4.95) or BDE-100 [OR=2.45 (95% CI: 1.31, 4.56)] was associated with more than a doubling in the risk of cryptorchidism. BDE-154 [OR=1.88 (95% CI: 1.08, 3.28) was also significant. CONCLUSIONS: Our results suggest that maternal exposure to BDE-99, -100, and -154 may be associated with abnormal migration of testes in the male fetus. This may be due to the anti-androgenic properties of the PBDEs. https://doi.org/10.1289/EHP522.

Evaluating external contamination of polybrominated diphenyl ethers in human hair: clinical and research implications.

BACKGROUND: Human hair is a well-validated matrix for detecting a variety of xenobiotics, including drugs of abuse (cocaine, tetrahydrocannabinol, and morphine) and fatty acid ethyl ethers. Recent studies have shown that hair can also be useful in determining an individual's exposure to polybrominated diphenyl ethers (PBDEs), flame retardants that contaminate the dust in our daily environment. Hair processing before assay varies with each analyte; in particular, the wash protocol must be optimized to remove external contaminants while not affecting levels of the chemical of interest. The aim of this study was to determine whether hair needs to be washed before analysis for PBDEs, and if so, which protocol is most effective to ensure that the level of PBDEs is neither overestimated nor underestimated. METHOD: Individual hair samples from 10 adults (5 men and 5 women) were subjected to 4 different wash protocols: (1) no wash, (2) water, (3) 10% sodium dodecyl sulfate (SDS), and (4) hexane. Both the washes and hair were analyzed for 8 PBDEs by gas chromatography/mass spectrometry. RESULTS: The sum of PBDEs (ΣPBDEs) in the washes was (1) no wash: 0 pg/mg, (2) water: 0.39 ± 0.19 (mean ± SEM), (3) 10% SDS: 1.34 ± 0.68, and (4) hexane: 1.92 ± 0.87. The ΣPBDEs in the hair were: (1) no wash: 20.32 ± 3.05, (2) water: 20.30 ± 2.41, (3) 10% SDS: 19.27 ± 1.87, and (4) hexane: 16.91 ± 2.89. Washing with water, 10% SDS, and hexane decreased the PBDE levels by 1.9%, 7%, and 11.4%, respectively (P < 0.05). CONCLUSIONS: Thus, of the washes evaluated, water is the wash that had the least effect on total PBDE concentrations, providing the best evaluation of an individual's exposure to PBDEs.

Hair as a biomarker of systemic exposure to polybrominated diphenyl ethers.

The efficacy of using hair as a biomarker for exposure to polybrominated diphenyl ether (PBDE) flame retardants was assessed in humans and an animal model. Paired human hair and serum samples were obtained from adult men and women (n = 50). In parallel, hair, serum, liver, and fat were collected from adult male Sprague-Dawley rats exposed to increasing doses of the PBDE mixture found in house dust for 70 days via the diet. All samples were analyzed by GC-MS for eight common PBDEs: BDE-28, -47, -99, -100, -153, -154, -183, and -209. Paired human hair and serum samples had five congeners (BDE-28, -47, -99, -100, and -154) with significant individual correlations (0.345-0.566). In rat samples, BDE-28 and BDE-183 were frequently below the level of detection. Significant correlations were observed for BDE-47, -99, -100, -153, -154, and -209 in rat hair, serum, liver, and fat across doses, with r values ranging from 0.803 to 0.988; weaker correlations were observed between hair and other tissues when data from the lowest dose group or for BDE-209 were analyzed. Thus, human and rat hair PBDE measurements correlate strongly with those in alternative matrices, validating the use of hair as a noninvasive biomarker of long-term PBDE exposure.

Investigating the use of hair to assess polybrominated diphenyl ether exposure retrospectively.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are chemicals that are added to a variety of consumer products as flame-retardants and have been classified as emerging endocrine disruptors. They are persistent and have been detected in humans. Previous studies have suggested that hair is a suitable matrix for examining human exposure to organic pollutants such as PBDEs. It is believed that the majority of exposure is from our indoor environment. The aim of this study was to investigate the changes in PBDE patterns and levels along the hair shaft, by using segmental analysis to retrospectively assess long-term exposure over a 1-year period. METHODS: Questionnaires and hair samples from 65 women were collected at the Hospital for Sick Children, in Toronto, as part of a larger study. To assess long-term stability, hair samples were separated into 4- and 3-cm segments representing a 1-year period. Hair segments were analyzed for levels of 8 PBDE congeners, BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183, and BDE-209 on gas chromatography-mass spectrometry (MS). A Friedman test was used to detect the differences in exposure among segments, and factors such as dietary habits, hair care routine, and site of residence were investigated to determine if they might affect hair levels. RESULTS: A significant increase (P < 0.0001) in total PBDEs was seen among segments moving from proximal (root end) to distal along the hair shaft (median in pg/mg): first (33.3), second (43.0), third (61.6), and fourth (75.5) segments. Significantly lower levels of PBDEs were observed in artificially colored hair samples (P = 0.032), and a significant increase in PBDE levels was observed in women who consumed meat on a daily basis as opposed to weekly consumption (P = 0.040). CONCLUSIONS: The increase in PBDEs along the hair shaft suggests that hair PBDEs may be influenced by diet and artificial coloring. More work is needed to validate the use of PBDEs in hair as a biomarker of long-term exposure.

Detection of polybrominated biphenyl ethers (PBDEs) in pediatric hair as a tool for determining in utero exposure.

INTRODUCTION: Cryptorchidism, or undescended/maldescended testis, is the most common birth defect of male genitalia. Its prevalence has been increasing over the past few decades. This may be due to an increase in the prevalence of anti-androgenic chemicals such as polychlorinated biphenyls, organochloride pesticides, plasticizers and fungicides. A newer group of chemicals, brominated flame retardants (BFRs), are being implicated as endocrine-disrupting chemicals. These chemicals are used worldwide in polymers that are incorporated into a variety of consumer products (e.g., textile, computers and televisions, insulating foam, electrical equipment and kitchen appliances). In order to quantify BFRs we introduce the use of hair levels of polybrominated diphenyl esters (PBDEs) as biomarkers of systemic exposure. This approach will allow for the estimation of in utero BFR exposure, in the process of evaluating the potential link between the incidence of cryptorchidism in newborn males and level of exposure of the pregnant mother to environmentally relevant BFRs. For that end we have developed a GC/MS assay in which children's hair is analyzed for the presence of polybrominated biphenyl ethers (PBDEs). METHODS: In this pilot, 10-40mg of hair from 24 children (12 newborn and 12 from children 1 to 15 years) was extracted overnight at 40°C with 4N HCl and hexane (4:1). The samples were eluted from 2g NaSO(4):2g Florisil SPE columns with 8mL hexane. Dried samples are reconstituted with anhydrous isooctane and injected onto a GC/MS and analyzed for BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183 and BDE-209. RESULTS: PBDEs were detected in all of the newborn and child hair. The ΣPBDE ranged from 0.038 to 1.01pg/mg newborn hair and from 0.208 to 2.695ng/mg child hair. The most abundant PBDE in newborn hair was BDE-153 while in child hair the variable PBDEs were BDE-47 and BDE-99. The highest molecular weight congener BDE-209 was detected in 10/24 pediatric hair samples. The LOQ is 0.0625pg/mg (BDE-209 0.625pg/mg) and the efficiency of extraction was between 70 and 90%. CONCLUSION: This GC/MS method is sufficiently sensitive to detect the presence of all 8 PBDE congeners tested in as little as 10mg of pediatric hair. The results show that PBDEs are present in newborn hair, making this matrix useful in examining in utero exposure to PBDEs and linking it to cryptorchidism.