Small Molecule Targets TMED9 and Promotes Lysosomal Degradation to Reverse Proteinopathy.

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.

An effective approach for BRAF V600E mutation analysis of routine thyroid fine needle aspirates.

INTRODUCTION: Molecular testing for genetic alterations in thyroid neoplasms, including BRAF V600E (BRAF) mutation, are often applied to thyroid aspirates falling into the Bethesda System for Reporting Thyroid Cytopathology indeterminate categories. Current methods typically use dedicated aspirated material, without morphological determination of containing the cells of interest and may be of elevated cost. We describe our experience with BRAF mutation analysis on material obtained from Papanicolaou (PAP)-stained ThinPrep® (TP) slides. METHODS: Eighty-three cases collected between 2012 and 2019 with more than 100 cells were selected. An electronic record of a whole slide scan was made for each case before testing. The coverslips were removed, and DNA was extracted from material scraped from each slide using the Qiagen QIAamp DNA FFPE Tissue Kit. BRAF testing was performed using a highly sensitive mutation detection assay, either COLD-PCR, castPCR, or droplet digital PCR. RESULTS: Fourteen out of 83 cases had a BRAF mutation. Of these, 8 were classified as atypia of undetermined significance or suspicious for malignancy in which follow-up showed conventional papillary thyroid carcinoma in 5 out of 6 cases. The specificity and positive predictive value were 97% and 91%, respectively. CONCLUSIONS: BRAF mutation analysis can be performed on material obtained from routine clinical PAP-stained TP slides. As a first step, this unconventional effective approach may reduce costs related to the molecular evaluation of thyroid nodule aspirates and provides the opportunity for cytomorphological confirmation that the cells of interest are present in material submitted for BRAF mutation analysis.

Phenotypic Diversity Caused by Differential Expression of SFTPC-Cre-Transgenic Alleles.

Type II alveolar epithelial cells (AEC2s) play an essential role in the function and maintenance of the pulmonary epithelium. Several transgenic mice have been developed to study the function of these cells in vivo by using the human SFTPC promoter to drive expression of Cre recombinase. The precise activity of each of these transgenic alleles has not been studied, and previous reports suggest that their activity can depend on breeding strategies. We bred mice with a conditional allele of the essential telomere capping protein TRF2 with two different SFTPC-Cre-transgenic strains and observed opposite phenotypes (100% lethality vs. 100% viability). We characterized the Cre recombinase activity in these two transgenic lines and found that the contrasting phenotypes were driven by difference in embryonic expression of the two transgenes, likely due to position effects or differences in the transgenic constructs. We also tested if SFTPC-Cre activity was dependent on maternal or paternal inheritance. When paternally inherited, both SFTPC-Cre alleles produced offspring with constitutive reporter activity independent of the inheritance of the Cre allele, suggesting that Cre recombinase was expressed in the male germline before meiosis. Immunohistochemical analysis of the testis showed reporter activity during spermatogenesis. Analysis of single-cell RNA sequencing data from murine and human testis demonstrated SFTPC expression uniquely during human spermatogenesis, suggesting that use of the human promoter in these constructs is responsible for male germline activity. Our data highlight the importance of careful analysis of transgenic allele activity and identify an SFTPC-Cre allele that is useful for panepithelial targeting in the mouse.

Disease detection methodologies in relapsed B-cell acute lymphoblastic leukemia: Opportunities for improvement.

BACKGROUND: Accurate disease detection is integral to risk stratification in B-cell acute lymphoblastic leukemia (ALL). The gold standard used to evaluate response in the United States includes morphologic evaluation and minimal residual disease (MRD) testing of aspirated bone marrow (BM) by flow cytometry (FC). This MRD assessment is usually made on a single aspirate sample that is subject to variability in collection techniques and sampling error. Additionally, central nervous system (CNS) assessments for ALL include evaluations of cytopathology and cell counts, which can miss subclinical involvement. PROCEDURE: We retrospectively compared BM biopsy, aspirate, and FC samples obtained from children and young adults with relapsed/refractory ALL to identify the frequency and degree of disease discrepancies in this population. We also compared CNS FC and cytopathology techniques. RESULTS: Sixty of 410 (14.6%) BM samples had discrepant results, 41 (10%) of which were clinically relevant as they resulted in a change in the assignment of marrow status. Discrepant BM results were found in 28 of 89 (31.5%) patients evaluated. Additionally, cerebrospinal fluid (CSF) FC identified disease in 9.7% of cases where cytopathology was negative. CONCLUSIONS: These results support further investigation of the role of concurrent BM biopsy, with aspirate and FC evaluations, and the addition of FC to CSF evaluations, to fully assess disease status and response, particularly in patients with relapsed/refractory ALL. Prospective studies incorporating more comprehensive analysis to evaluate the impact on clinical outcomes are warranted.

GDF15 is an epithelial-derived biomarker of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and devastating of the interstitial lung diseases. Epithelial dysfunction is thought to play a prominent role in disease pathology, and we sought to characterize secreted signals that may contribute to disease pathology. Transcriptional profiling of senescent type II alveolar epithelial cells from mice with epithelial-specific telomere dysfunction identified the transforming growth factor-ß family member, growth and differentiation factor 15 (Gdf15), as the most significantly upregulated secreted protein. Gdf15 expression is induced in response to telomere dysfunction and bleomycin challenge in mice. Gdf15 mRNA is expressed by lung epithelial cells, and protein can be detected in peripheral blood and bronchoalveolar lavage following bleomycin challenge in mice. In patients with IPF, GDF15 mRNA expression in lung tissue is significantly increased and correlates with pulmonary function. Single-cell RNA sequencing of human lungs identifies epithelial cells as the primary source of GDF15, and circulating concentrations of GDF15 are markedly elevated and correlate with disease severity and survival in multiple independent cohorts. Our findings suggest that GDF15 is an epithelial-derived secreted protein that may be a useful biomarker of epithelial stress and identifies IPF patients with poor outcomes.

Composite Graft Pretreatment With Hydrogen Sulfide Delays the Onset of Acute Rejection.

INTRODUCTION: Vascularized composite allotransplantation can reconstruct devastating tissue loss by replacing like-with-like tissues, most commonly in the form of hand or face transplantation. Unresolved technical and ethical challenges have meant that such transplants remain experimental treatments. The most significant barrier to expansion of this field is the requirement for systemic immunosuppression, its toxicity and effect on longevity.Hydrogen sulfide (H2S) has been shown experimentally to ameliorate the ischemia reperfusion injury associated with composite tissue autotransplantation, which has been linked to acute rejection in solid organ transplantation. In this protocol, a large-animal model was used to evaluate the effect of H2S on acute rejection after composite tissue allotransplantation. MATERIALS AND METHODS: A musculocutaneous flap model in SLA-mismatched swine was used to evaluate acute rejection of allotransplants in 2 groups: control animals (n = 8) and a treatment group in which the allografts were pretreated with hydrogen sulfide (n = 8). Neither group was treated with systemic immunosuppression. Acute rejection was graded clinically and histopathologically by an independent, blinded pathologist. Data were analyzed by t tests with correction for multiple comparisons by the Holm-Sídák method. RESULTS: Clinically, H2S-treated tissue composites showed a delay in the onset of rejection that was statistically significant from postoperative day 6. Histopathologically, this difference between groups was also apparent, although evidence of a difference in groups disappeared beyond day 10. CONCLUSIONS: Targeted hydrogen sulfide treatment of vascularized composite allografts immediately before transplantation can delay acute rejection. This may, in turn, reduce or obviate the requirement for systemic immunosuppression.

Iodide Improves Outcome After Acute Myocardial Infarction in Rats and Pigs.

OBJECTIVES: In this study, we tested whether iodide would reduce heart damage in rat and pig models of acute myocardial infarction as a risk analysis for a human trial. DESIGN: Prospective blinded and randomized laboratory animal investigation. SETTING: Animal research laboratories. SUBJECTS: Sexually mature rats and pigs. INTERVENTIONS: Acute myocardial infarction was induced by temporary ligation of the coronary artery followed by reperfusion. Iodide was administered orally in rats or IV in rats and pigs just prior to reperfusion. MEASUREMENTS AND MAIN RESULTS: Damage was assessed by blood cardiac troponin and infarct size; heart function was determined by echocardiography. Blood peroxide scavenging activity was measured enzymatically, and blood thyroid hormone was determined using radioimmune assay. Iodide administration preserved heart function and reduced blood cardiac troponin and infarct size by approximately 45% in pigs and approximately 60% in rats. Iodide administration also increased blood peroxide scavenging activity and maintained thyroid hormone levels. CONCLUSIONS: Iodide administration improved the structure and function of the heart after acute myocardial infarction in rats and pigs.

Comparative analysis of lipid-mediated CRISPR-Cas9 genome editing techniques.

CRISPR-Cas technology has revolutionized genome engineering. While Cas9 was not the first programmable endonuclease identified, its simplicity of use has driven widespread adoption in a short period of time. While CRISPR-Cas genome editing holds enormous potential for clinical applications, its use in laboratory settings for genotype-phenotype studies and genome-wide screens has led to breakthroughs in the understanding of many molecular pathways. Numerous protocols have been described for introducing CRISPR-Cas components into cells, and here we sought to simplify and optimize a protocol for genome editing using readily available and inexpensive tools. We compared plasmid, ribonucleoprotein (RNP), and RNA transfection to determine which was method was most optimal for editing cells in a laboratory setting. We limited our comparison to lipofection-mediated introduction because the reagents are widely available. To facilitate optimization, we developed a novel reporter assay to measure gene disruption and the introduction of a variety of exogenous DNA tags. Each method efficiently disrupted endogenous genes and was able to stimulate the introduction of foreign DNA at specific sites, albeit to varying efficiencies. RNP transfection produced the highest level of gene disruption and was the most rapid and efficient method overall. Finally, we show that very short homology arms of 30 base pairs can mediate site-specific editing. The methods described here should broaden the accessibility of RNP-mediated lipofection for laboratory genome-editing experiments.

Challenges in Complementing Data from Ground-Based Sensors with Satellite-Derived Products to Measure Ecological Changes in Relation to Climate-Lessons from Temperate Wetland-Upland Landscapes.

Assessing climate-related ecological changes across spatiotemporal scales meaningful to resource managers is challenging because no one method reliably produces essential data at both fine and broad scales. We recently confronted such challenges while integrating data from ground- and satellite-based sensors for an assessment of four wetland-rich study areas in the U.S. Midwest. We examined relations between temperature and precipitation and a set of variables measured on the ground at individual wetlands and another set measured via satellite sensors within surrounding 4 km² landscape blocks. At the block scale, we used evapotranspiration and vegetation greenness as remotely sensed proxies for water availability and to estimate seasonal photosynthetic activity. We used sensors on the ground to coincidentally measure surface-water availability and amphibian calling activity at individual wetlands within blocks. Responses of landscape blocks generally paralleled changes in conditions measured on the ground, but the latter were more dynamic, and changes in ecological conditions on the ground that were critical for biota were not always apparent in measurements of related parameters in blocks. Here, we evaluate the effectiveness of decisions and assumptions we made in applying the remotely sensed data for the assessment and the value of integrating observations across scales, sensors, and disciplines.

Impact of maternal prenatal and parental postnatal stress on 1-year-old child development: results from the OTIS antidepressants in pregnancy study.

Perinatal psychological stress has been associated with unfavorable maternal and neonatal outcomes. We aimed to assess the impact of perinatal stress on infant development at 1 year of age. We recruited pregnant women calling North American Teratogen Information Services or attending outpatient clinics at CHU Sainte Justine (Montreal) between 2008 and 2010 and their spouses. To be part of our study, women had to be (1) >18 years of age, (2) <15 weeks of gestational age at recruitment, (3) living within 250-km radius of Montreal, and (4) taking antidepressants or non-teratogenic drugs. Stress was assessed using the telephone-administered four-item perceived stress scale during pregnancy in mothers and at 2 months postpartum in both parents. Child development at 1 year of age was evaluated with the Bayley III scales. Socio-demographic and potential confounders were collected through telephone interviews. Multivariable linear regression models were built to assess the association between perinatal parental stress and child development. Overall, 71 couples and their infants were included. When adjusted for potential confounders, maternal prenatal stress was positively associated with motor development (adjusted ß = 1.85, CI 95 % (0.01, 3.70)). Postpartum maternal and paternal stresses were negatively associated with motor and socio-emotional development, respectively (adjusted ß = -1.54, CI 95 % (-3.07, -0.01) and adjusted ß = -1.67, CI 95 % (-3.25, -0.10), respectively). Maternal and paternal postnatal stress seems to be harmful for the motor and socio-emotional development in 1-year-old children. No association was demonstrated between parental stress and cognitive, language, and adaptive behavioral development. However, prenatal maternal stress appears to improve motor skills.

Aquaporins-2 and -4 regulate glycogen metabolism and survival during hyposmotic-anoxic stress in Caenorhabditis elegans.

Periods of oxygen deprivation can lead to ion and water imbalances in affected tissues that manifest as swelling (edema). Although oxygen deprivation-induced edema is a major contributor to injury in clinical ischemic diseases such as heart attack and stroke, the pathophysiology of this process is incompletely understood. In the present study we investigate the impact of aquaporin-mediated water transport on survival in a Caenorhabditis elegans model of edema formation during complete oxygen deprivation (anoxia). We find that nematodes lacking aquaporin water channels in tissues that interface with the surrounding environment display decreased edema formation and improved survival rates in anoxia. We also find that these animals have significantly reduced demand for glycogen as an energetic substrate during anoxia. Together, our data suggest that reductions in membrane water permeability may be sufficient to induce a hypometabolic state during oxygen deprivation that reduces injury and extends survival limits.

WU polyomavirus in respiratory epithelial cells from lung transplant patient with Job syndrome.

We detected WU polyomavirus (WUPyV) in a bronchoalveolar lavage sample from lungs transplanted into a recipient with Job syndrome by using immunoassays specific for the WUPyV viral protein 1. Co-staining for an epithelial cell marker identified most WUPyV viral protein 1-positive cells as respiratory epithelial cells.

Selenide Targets to Reperfusing Tissue and Protects It From Injury.

OBJECTIVES: Since blood selenium levels decrease after ischemia and reperfusion injury, and low blood selenium correlates with negative outcome, we designed and performed experiments to determine how selenium distribution is affected by ischemia reperfusion injury. Furthermore, we tested whether different chemical forms of selenium would affect outcome after ischemia and reperfusion injury. We also examined the metabolic effects of selenide administration. DESIGN: Laboratory investigation. SETTING: Animal research laboratory. SUBJECTS: Adult male C57BL/6 mice. INTERVENTIONS: To determine selenium localization, we administered tracer doses of radioactive selenium 75 in the form of selenite or selenide and measured blood and tissue selenium levels after ischemia and reperfusion injury. Anesthetized mice were subjected to myocardial ischemia reperfusion injury (coronary artery occlusion for 60 min followed by 5 min of reperfusion after occlusion was removed) or hindlimb ischemia reperfusion injury (left leg tourniquet for 90 min followed by 5 min reperfusion after tourniquet removal). To determine whether exogenous selenium administration could reduce ischemia reperfusion injury, we synthesized and administered sodium hydroselenide and sodium selenite solutions (0.05-2.4 mg/kg). Solutions were administered at the end of coronary artery occlusion but before reperfusion. In order to determine the metabolic effects of selenide administration, we exposed mice to hydrogen selenide gas (0-5 ppm) mixed into air (20.95% oxygen) for up to 3 hours. MEASUREMENTS AND MAIN RESULTS: In targeting assays, we measured blood and tissue selenium levels. We observed that blood selenium decreases after myocardial ischemia reperfusion and displays an inverse correlation with injury severity; selenium accumulation in heart correlates directly with injury severity. We also measured whether oxidized selenium, selenite, and reduced selenium, selenide, would target to injured heart tissue in myocardial ischemia reperfusion and injured leg muscle in a hindlimb model of ischemia reperfusion. Only selenide targets to injured tissue. We also measured damage after myocardial ischemia reperfusion injury using morphometry, neutrophil accumulation, blood cardiac troponin levels, and echocardiography and observed in all assays that selenide reduced damage to the heart; selenite was not effective. And finally, to assay metabolism, we measured oxygen consumption, carbon dioxide production, and body core temperature before, during, and after hydrogen selenide administration. All measurements indicate that selenide decreases metabolism. CONCLUSIONS: Selenide targets to reperfusing tissue and reduces reperfusion injury perhaps by affecting oxygen metabolism.

Hydrogen sulfide mitigates reperfusion injury in a porcine model of vascularized composite autotransplantation.

BACKGROUND: Devastating extremity injuries are prevalent but often survivable on the modern battlefield. These complex injuries require advanced methods of reconstruction, involving prolonged ischemic periods and reperfusion injury. Using our group's validated porcine model of gracilis myocutaneous flap transplantation, this study demonstrates that an interim perfusion of hydrogen sulfide (H2S) mitigates the effects of reperfusion injury in the setting of delayed restoration of blood flow. METHODS: A gracilis myocutaneous flap (200-400 g; surface area, 250 cm²) was procured from the hind limb of a Yorkshire swine (70-90 kg, n=16). The right external carotid artery and the internal jugular vein are the recipient axis. Group 1 (control, n = 6) underwent delayed anastomosis with a 3-hour ischemic period. Group 2 (n=10) underwent a similar delayed anastomosis with an interim perfusion of H2S during the ischemic period. The animals survived for 14 days. Systemic biomarker assays for skeletal muscle tissue injury (creatine kinase, lactate dehydrogenase, and aspartate transaminase) and proinflammatory markers (tumor necrosis factor α and interleukin 6) provide assessment of reperfusion injury at the cellular level. RESULTS: The control animals (3 hours of ischemia with an interim perfusion of heparinized saline) demonstrated increased levels of injury biomarkers and proinflammatory cytokines compared with the animals receiving H2S infusion and identical ischemic interval. The control flaps had a mean creatine kinase level of 280³×10 U/L (±80×10³), compared with the H2S group, which had a mean of 99×10³ U/L (±14×10³; P=0.0007 at postoperative day 2). lactate dehydrogenase levels (mean) were 26×10³ U/L (±8×10³) versus 9×10³ U/L (±3×10³; P=0.0004) and aspartate transaminase levels (mean) were 1651 U/L (±324) versus (873 U/L [±279]; P=0.0013) for the control and treatment groups, respectively. Similarly, an intergroup difference in IL-6 was found, although not statistically significant. Tumor necrosis factor α levels (mean) were 93 pg/mL (±14) versus 39 pg/mL (±4; P=0.0013) for the control and treatment groups, respectively. CONCLUSIONS: This study demonstrated the mitigating properties of H2S on reperfusion injury. Interim perfusion with H2S resulted in diminution of ischemia-dependent biomarkers after 3 hours of ischemia. Follow-up studies will translate these findings as an evolving method for reconstructing previously unreconstructable injuries.

Exploring Iodide and Hydrogen Sulfide as ROS Scavengers to Delay Acute Rejection in MHC-Defined Vascularized Composite Allografts.

Vascularized composite allografts (VCA) face ischemic challenges due to their limited availability. Reperfusion following ischemia triggers oxidative stress and immune reactions, and scavenger molecules could mitigate ischemia-reperfusion injuries and, therefore, immune rejection. We compared two scavengers in a myocutaneous flap VCA model. In total, 18 myocutaneous flap transplants were performed in Major histocompatibility complex (MHC)-defined miniature swine. In the MATCH group (n = 9), donors and recipients had minor antigen mismatch, while the animals were fully mismatched in the MISMATCH group (n = 9). Grafts were pretreated with saline, sodium iodide (NaI), or hydrogen sulfide (H2S), stored at 4 °C for 3 h, and then transplanted. Flaps were monitored until clinical rejection without immunosuppression. In the MATCH group, flap survival did not significantly differ between the saline and hydrogen sulfide treatments (p = 0.483) but was reduced with the sodium iodide treatment (p = 0.007). In the MISMATCH group, survival was similar between the saline and hydrogen sulfide treatments (p = 0.483) but decreased with the sodium iodide treatment (p = 0.007). Rhabdomyolysis markers showed lower but non-significant levels in the experimental subgroups for both the MATCH and MISMATCH animals. This study provides insightful data for the field of antioxidant-based approaches in VCA and transplantation.

Measuring telomere length for the early detection of precursor lesions of esophageal squamous cell carcinoma.

BACKGROUND: Esophageal cancer is the sixth leading cause of cancer death worldwide; current early detection screening tests are inadequate. Esophageal balloon cytology successfully retrieves exfoliated and scraped superficial esophageal epithelial cells, but cytologic reading of these cells has poor sensitivity and specificity for detecting esophageal squamous dysplasia (ESD), the precursor lesion of esophageal squamous cell carcinoma (ESCC). Measuring telomere length, a marker for chromosomal instability, may improve the utility of balloon cytology for detecting ESD and early ESCC. METHODS: We examined balloon cytology specimens from 89 asymptomatic cases of ESD (37 low-grade and 52 high-grade) and 92 age- and sex-matched normal controls from an esophageal cancer early detection screening study. All subjects also underwent endoscopy and biopsy, and ESD was diagnosed histopathologically. DNA was extracted from the balloon cytology cells, and telomere length was measured by quantitative PCR. A receiver operating characteristic (ROC) curve was plotted for telomere length as a diagnostic marker for high-grade dysplasia. RESULTS: Telomere lengths were comparable among the low- and high-grade dysplasia cases and controls, with means of 0.96, 0.96, and 0.92, respectively. The area under the ROC curve was 0.55 for telomere length as a diagnostic marker for high-grade dysplasia. Further adjustment for subject characteristics, including sex, age, smoking, drinking, hypertension, and body mass index did not improve the use of telomere length as a marker for ESD. CONCLUSIONS: Telomere length of esophageal balloon cytology cells was not associated with ESCC precursor lesions. Therefore, telomere length shows little promise as an early detection marker for ESCC in esophageal balloon samples.

Reliability and validity of the 4-item perceived stress scale among pregnant women: results from the OTIS antidepressants study.

We aimed to estimate the reliability of the 4-item Perceived Stress Scale (PSS) and its validity in predicting maternal depression and quality of life (QoL). Data regarding stress, depression and QoL were collected during pregnancy among a sub-sample from the Organization of Teratology Information Specialists Antidepressants in Pregnancy Cohort. The 4-item PSS demonstrated acceptable internal consistency (Cronbach's alpha coefficient = .79), alternate forms stability reliability with the 10-item PSS (Pearson correlation coefficient r = .63; p < .001), convergent validity with the Edinburgh Postnatal Depression Scale (r = .67; p < .001), and concurrent validity with the mental health component of the Short-Form-12 (r = -.62; p < .001) as a measure of QoL. The 4-item PSS is a valid and useful tool for assessing maternal stress during pregnancy.

The feasibility and utility of hair follicle sampling to measure FMRP and FMR1 mRNA in children with or without fragile X syndrome: a pilot study.

BACKGROUND: Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability in males and the most common single gene cause of autism. This X-linked disorder is caused by an expansion of a trinucleotide CGG repeat (> 200 base pairs) on the promotor region of the fragile X messenger ribonucleoprotein 1 gene (FMR1). This leads to the deficiency or absence of the encoded protein, fragile X messenger ribonucleoprotein 1 (FMRP). FMRP has a central role in the translation of mRNAs involved in synaptic connections and plasticity. Recent studies have demonstrated the benefit of therapeutics focused on reactivation of the FMR1 locus towards improving key clinical phenotypes via restoration of FMRP and ultimately disease modification. A key step in future studies directed towards this effort is the establishment of proof of concept (POC) for FMRP reactivation in individuals with FXS. For this, it is key to determine the feasibility of repeated collection of tissues or fluids to measure FMR1 mRNA and FMRP. METHODS: Individuals, ages 3 to 22 years of age, with FXS and those who were typically developing participated in this single-site pilot clinical biomarker study. The repeated collection of hair follicles was compared with the collection of blood and buccal swabs for detection of FMR1 mRNA and FMRP and related molecules. RESULTS: There were n = 15 participants, of whom 10 had a diagnosis of FXS (7.0 ± 3.56 years) and 5 were typically developing (8.2 ± 2.77 years). Absolute levels of FMRP and FMR1 mRNA were substantially higher in healthy participants compared to full mutation and mosaic FXS participants and lowest in the FXS boys. Measurement of FMR1 mRNA and FMRP levels by any method did not show any notable variation by collection location at home versus office across the various sample collection methodologies of hair follicle, blood sample, and buccal swab. CONCLUSION: Findings demonstrated that repeated sampling of hair follicles in individuals with FXS, in both, home, and office settings, is feasible, repeatable, and can be used for measurement of FMR1 mRNA and FMRP in longitudinal studies.