SUMMARYGlycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disease caused by deficiency of glycogen debranching enzyme (GDE) in liver and muscle. The disorder is clinically heterogeneous and progressive, and there is no effective treatment. Previously, a naturally occurring dog model for this condition was identified in curly-coated retrievers (CCR). The affected dogs carry a frame-shift mutation in the GDE gene and have no detectable GDE activity in liver and muscle. We characterized in detail the disease expression and progression in eight dogs from age 2 to 16 months. Monthly blood biochemistry revealed elevated and gradually increasing serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities; serum creatine phosphokinase (CPK) activity exceeded normal range after 12 months. Analysis of tissue biopsy specimens at 4, 12 and 16 months revealed abnormally high glycogen contents in liver and muscle of all dogs. Fasting liver glycogen content increased from 4 months to 12 months, but dropped at 16 months possibly caused by extended fibrosis; muscle glycogen content continually increased with age. Light microscopy revealed significant glycogen accumulation in hepatocytes at all ages. Liver histology showed progressive, age-related fibrosis. In muscle, scattered cytoplasmic glycogen deposits were present in most cells at 4 months, but large, lake-like accumulation developed by 12 and 16 months. Disruption of the contractile apparatus and fraying of myofibrils was observed in muscle at 12 and 16 months by electron microscopy. In conclusion, the CCR dogs are an accurate model of GSD IIIa that will improve our understanding of the disease progression and allow opportunities to investigate treatment interventions.

Glycogen storage disease type Ia (GSD-Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), primarily found in liver and kidney, which causes life-threatening hypoglycemia. Dogs with GSD-Ia were treated with double-stranded adeno-associated virus (AAV) vectors encoding human G6Pase. Administration of an AAV9 pseudotyped (AAV2/9) vector to seven consecutive GSD-Ia neonates prevented hypoglycemia during fasting for up to 8 hr; however, efficacy eventually waned between 2 and 30 months of age, and readministration of a new pseudotype was eventually required to maintain control of hypoglycemia. Three of these dogs succumbed to acute hypoglycemia between 7 and 9 weeks of age; however, this demise could have been prevented by earlier readministration an AAV vector, as demonstrated by successful prevention of mortality of three dogs treated earlier in life. Over the course of this study, six out of nine dogs survived after readministration of an AAV vector. Of these, each dog required readministration on average every 9 months. However, two were not retreated until >34 months of age, while one with preexisting antibodies was re-treated three times in 10 months. Glycogen content was normalized in the liver following vector administration, and G6Pase activity was increased in the liver of vector-treated dogs in comparison with GSD-Ia dogs that received only with dietary treatment. G6Pase activity reached approximately 40% of normal in two female dogs following AAV2/9 vector administration. Elevated aspartate transaminase in absence of inflammation indicated that hepatocellular turnover in the liver might drive the loss of vector genomes. Survival was prolonged for up to 60 months in dogs treated by readministration, and all dogs treated by readministration continue to thrive despite the demonstrated risk for recurrent hypoglycemia and mortality from waning efficacy of the AAV2/9 vector. These preclinical data support the further translation of AAV vector-mediated gene therapy in GSD-Ia.

In 1973, Michigan residents were exposed to polybrominated biphenyl (PBB) when it was accidentally added to farm animal feed. Highly exposed individuals and their children have experienced endocrine-related health problems, though the underlying mechanism behind these remains unknown. We investigated whether PBB exposure is associated with variation in DNA methylation in peripheral blood samples from 658 participants of the Michigan PBB registry using the MethylationEPIC BeadChip, as well as investigated what the potential function of the affected regions are and whether these epigenetic marks are known to associate with endocrine system pathways. After multiple test correction (FDR <0.05), 1890 CpG sites associated with total PBB levels. These CpGs were not enriched in any particular biological pathway, but were enriched in enhancer and insulator regions, and depleted in regions near the transcription start site or in CpG islands (p < 0.05). They were also more likely to be in ARNT and ESR2 transcription factor binding sites (p = 3.27e-23 and p = 1.62e-6, respectively), and there was significant overlap between CpGs associated with PBB and CpGs associated with estrogen (p < 2.2e-16). PBBassociated CpGs were also enriched for CpGs known to be associated with gene expression in blood (eQTMs) (p < 0.05). These eQTMs were enriched for pathways related to immune function and endocrine-related autoimmune disease (FDR <0.05). These results indicate that exposure to PBB is associated with differences in epigenetic marks that suggest that it is acting similarly to estrogen and is associated with dysregulated immune system pathways. ARTICLE HISTORY

In 1973, accidental contamination of Michigan livestock with polybrominated biphenyls (PBBs) led to the establishment of a registry of exposed individuals that have been followed for > 40 years. Besides being exposed to PBBs, this cohort has also been exposed to polychlorinated biphenyls (PCBs), a structurally similar class of environmental pollutants, at levels similar to average US exposure. In this study, we examined the association between current serum PCB and PBB levels and various female reproductive health outcomes to build upon previous work and inconsistencies. participation in this cross-sectional study required a blood draw and completion of a detailed health questionnaire. Analysis included only female participants who had participated between 2012 and 2015 (N = 254). Multivariate linear and logistic regression models were used to identify associations between serum PCB and PBB levels with each gynecological and infertility outcome. Additionally, a generalized estimating equation (GEE) model was used to evaluate each pregnancy and birth outcome in order to account for multiple pregnancies per woman. We controlled for age, body mass index, and total lipid levels in all analyses. A p-value of <0.05 was used for statistical significance. Among the women who reported ever being pregnant, there was a significant negative association with higher total PCB levels associating with fewer lifetime pregnancies (β = −0.11, 95% CI = −0.21 to −0.005, p = 0.04). There were no correlations between serum PCB levels and the self-reported gynecological outcomes (pelvic inflammatory disease, endometriosis, polycystic ovarian syndrome, or uterine fibroids). No associations were identified between serum PCB levels and the prevalence of female infertility in women reporting ever having sexual intercourse with a male partner. There were no associations identified between serum PCB levels and pregnancy outcomes (singleton live births or miscarriages) or birth outcomes (preterm birth, birth weight, birth defects, hypertensive disorders of pregnancy, or gestational diabetes). PBB was not associated with any outcome. Further research is needed to determine if and how PCB may reduce pregnancy number. Polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs) are synthetic chemicals that were once used as coolants in electrical equipment and as flame retardants in the manufacturing of plastics and electronics, respectively 1,2. Although the production of PCBs ceased in 1977 and PBBs in 1976, concern over their health effects remains because of their continued persistence in the environment, the pervasiveness of their exposure in

Endocrine-disrupting compounds (EDCs) are a broad class of chemicals present in many residential products that can disrupt hormone signaling and cause health problems in humans. Multigenerational cohorts, like the Michigan polybrominated biphenyl registry, are ideal for studying the effects of intergenerational exposure. Registry participants report hormone-related health problems, particularly in those exposed before puberty or those in the second generation exposed through placental transfer or breastfeeding. However, more research is needed to determine how EDCs cause health problems and the mechanisms underlying intergenerational exposure. Utilizing existing data in this registry, along with genetic and epigenetic approaches, could provide insight to how EDCs cause human disease and help to determine the risk to exposed populations and future generations.

Endocrine-disrupting compounds are associated with altered epigenetic regulation and adverse health outcomes, although inconsistent results suggest that people have varied responses to the same exposure. Interpersonal variation in response to environmental exposures is not identified using standard, population-based methods. However, methods that capture an individual's response, such as analyzing stochastic epigenetic mutations (SEMs), may capture currently missed effects of environmental exposure. To test whether polybrominated biphenyl (PBB) was associated with SEMs, DNA methylation was measured using Illumina's MethylationEPIC array in PBB-exposed individuals, and SEMs were identified. Association was tested using a linear regression with robust sandwich variance estimators, controlling for age, sex, lipids, and cell types. The number of SEMs was variable (range: 119-18,309), and positively associated with age (p = 1.23e-17), but not with sex (p = 0.97). PBBs and SEMs were only positively associated in people who were older when they were exposed (p = 0.02 vs. p = 0.91). Many subjects had SEMs enriched in biological pathways, particularly in pathways involved with xenobiotic metabolism and endocrine function. Higher number of SEMs was also associated with higher age acceleration (intrinsic: p = 1.70e-3; extrinsic: p = 3.59e-11), indicating that SEMs may be associated with age-related health problems. Finding an association between environmental contaminants and higher SEMs may provide insight into individual differences in response to environmental contaminants, as well as into the biological mechanism behind SEM formation. Furthermore, these results suggest that people may be particularly vulnerable to epigenetic dysregulation from environmental exposures as they age.

Background Michigan residents were directly exposed to endocrine-disrupting compounds, polybrominated biphenyl (PBB) and polychlorinated biphenyl (PCB). A growing body of evidence suggests that exposure to certain endocrine-disrupting compounds may affect thyroid function, especially in people exposed as children, but there are conflicting observations. In this study, we extend previous work by examining age of exposure’s effect on the relationship between PBB exposure and thyroid function in a large group of individuals exposed to PBB. Methods Linear regression models were used to test the association between serum measures of thyroid function (total thyroxine (T 4 ), total triiodothyronine (T 3 ), free T 4 , free T 3 , thyroid stimulating hormone (TSH), and free T 3 : free T 4 ratio) and serum PBB and PCB levels in a cross-sectional analysis of 715 participants in the Michigan PBB Registry. Results Higher PBB levels were associated with many thyroid hormones measures, including higher free T 3 ( p = 0.002), lower free T 4 ( p = 0.01), and higher free T 3 : free T 4 ratio ( p = 0.0001). Higher PCB levels were associated with higher free T 4 ( p = 0.0002), and higher free T 3 : free T 4 ratio (p = 0.002). Importantly, the association between PBB and thyroid hormones was dependent on age at exposure. Among people exposed before age 16 ( N = 446), higher PBB exposure was associated with higher total T 3 (p = 0.01) and free T 3 ( p = 0.0003), lower free T 4 ( p = 0.04), and higher free T 3 : free T 4 ratio (p = 0.0001). No significant associations were found among participants who were exposed after age 16. No significant associations were found between TSH and PBB or PCB in any of the analyses conducted. Conclusions This suggests that both PBB and PCB are associated with thyroid function, particularly among those who were exposed as children or prenatally. Electronic supplementary material The online version of this article (10.1186/s12940-019-0509-z) contains supplementary material, which is available to authorized users.

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