United States

### Background 
Quality assessments relying solely on structured EHR data do not capture note-documented explanations for apparent non-adherence to lipid management guidelines. Large language models (LLMs) present a promising approach for extracting this information at scale.

### Purpose 
To evaluate the accuracy and impact of LLMs when applied to assess adherence to lipid management guidelines in CAD. 

### Methods 
We used structured EHR data to analyze LDL-C levels and statin use of patients with ASCVD seen in cardiology clinics at a large academic medical center from 2022-2024. We then applied GPT-4o with zero-shot, chain-of-thought prompting to free-text cardiology clinic notes to (1) identify external LDL-C values documented within the notes of patients without a recent in-system lipid panel and (2) infer reasons for statin non-use among those with LDL-C ≥70 mg/dL. We validated GPT-4o&#39;s accuracy through manual review of 500 randomly selected charts. 

### Results 
Among 8,595 patients (median age 71.0 years, 61.5% male, 70.8% White) initially identified, 53.6% were on high-intensity statins and 38.1% had LDL-C &lt;70 mg/dL. GPT-4o confirmed a CAD diagnosis in 6,534 (76.0%) patients (Figure); within this group, GPT-4o identified external lipid panels in n=403 (6.2%), high-intensity statin use (n=59, 0.7%), and statin intolerance (n=804, 12.3%) documented in notes but not captured in structured fields (PPV 0.91-1.00). Among patients with GPT-4o-confirmed CAD and updated medication/laboratory data had higher measured rates of high-intensity statin use (61.4% vs. 53.6%) and LDL-C &lt;70 mg/dL (44.1% vs. 38.1%). Excluding statin-intolerant patients further increased high-intensity statin use to 68.4%. Among patients with LDL-C ≥70 mg/dL not on high-intensity statins (N=1,164), GPT-4o-extracted reasons for nonuse were clinician decision (13.0%), intolerance (38.5%), patient preference (8.2%), and undocumented (40.4%). GPT-4o classification performance was strong (F1 &gt; 0.71 across all categories), with the highest performance observed for statin intolerance (F1 = 0.96).

### Conclusions
GPT-4o can accurately identify clinical factors not captured in structured EHR data. This approach enables more accurate guideline-based quality assessments and yields actionable insights into reasons for apparent gaps in care.

AMA Research Challenge 2025 

Enhancing the Precision of Lipid Management Quality Assessment in ASCVD Using Large Language Models

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AMA Research Challenge 2022

Welcome to the largest national, multi-specialty research event featuring research from 750 medical students, residents and fellows, and international medical graduates.


### Background 
Quality assessments relying solely on structured EHR data do not capture note-documented explanations for apparent non-adherence to lipid management guidelines. Large language models (LLMs) present a promising approach for extracting this information at scale.

### Purpose 
To evaluate the accuracy and impact of LLMs when applied to assess adherence to lipid management guidelines in CAD. 

### Methods 
We used structured EHR data to analyze LDL-C levels and statin use of patients with ASCVD seen in cardiology clinics at a large academic medical center from 2022-2024. We then applied GPT-4o with zero-shot, chain-of-thought prompting to free-text cardiology clinic notes to (1) identify external LDL-C values documented within the notes of patients without a recent in-system lipid panel and (2) infer reasons for statin non-use among those with LDL-C ≥70 mg/dL. We validated GPT-4o's accuracy through manual review of 500 randomly selected charts. 

### Results 
Among 8,595 patients (median age 71.0 years, 61.5% male, 70.8% White) initially identified, 53.6% were on high-intensity statins and 38.1% had LDL-C <70 mg/dL. GPT-4o confirmed a CAD diagnosis in 6,534 (76.0%) patients (Figure); within this group, GPT-4o identified external lipid panels in n=403 (6.2%), high-intensity statin use (n=59, 0.7%), and statin intolerance (n=804, 12.3%) documented in notes but not captured in structured fields (PPV 0.91-1.00). Among patients with GPT-4o-confirmed CAD and updated medication/laboratory data had higher measured rates of high-intensity statin use (61.4% vs. 53.6%) and LDL-C <70 mg/dL (44.1% vs. 38.1%). Excluding statin-intolerant patients further increased high-intensity statin use to 68.4%. Among patients with LDL-C ≥70 mg/dL not on high-intensity statins (N=1,164), GPT-4o-extracted reasons for nonuse were clinician decision (13.0%), intolerance (38.5%), patient preference (8.2%), and undocumented (40.4%). GPT-4o classification performance was strong (F1 > 0.71 across all categories), with the highest performance observed for statin intolerance (F1 = 0.96).

### Conclusions
GPT-4o can accurately identify clinical factors not captured in structured EHR data. This approach enables more accurate guideline-based quality assessments and yields actionable insights into reasons for apparent gaps in care.

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### Objective
Multiple sclerosis (MS) is a chronic autoimmune disorder of the central nervous system frequently associated with visual symptoms. This study aimed to characterize the prevalence of ophthalmic conditions in individuals with MS and compare it to matched controls without MS.

### Methods
A matched case-control, cross-sectional study was conducted using data from the All of Us Research Program, a large, nationwide NIH-funded initiative. Individuals with MS (n = 3771) were matched 1:3 to controls without MS (n = 11,313) by age, sex, and race/ethnicity. Four ophthalmic conditions—optic neuritis, diplopia, nystagmus, and uveitis—were examined. Based on electronic health records, diagnosis dates were categorized into ophthalmic conditions occurring before, concurrently with, or after the MS diagnosis. Prevalence rates of ophthalmic conditions in individuals with MS were compared to those of matched controls, through univariate and multivariate logistic regression models, adjusting for sociodemographic factors, lifestyle variables, prior brain injury, family history of MS, and comorbidities.

### Results
All ophthalmic conditions were significantly more prevalent in individuals with MS than controls. Optic neuritis was most common (17.0 % vs. 0.2 %; adjusted odds ratio (aOR) = 31.96; 95 % CI: 22.09–48.20; p < 0.001), followed by diplopia (10.1 % vs. 1.0 %; aOR = 4.88; 95 % CI: 3.87–6.19; p < 0.001), uveitis (3.4 % vs. 0.7 %; aOR = 2.30; 95 % CI: 1.66–3.19; p < 0.001), and nystagmus (2.5 % vs. 0.2 %; aOR = 6.97; 95 % CI: 4.31–11.62; p < 0.001). The temporal analysis revealed that some cases of optic neuritis (31.9 %), nystagmus (21.9 %), diplopia (27.7 %), and uveitis (38.3 %) preceded MS diagnosis. Optic neuritis prior to MS was significantly more prevalent than matched controls (5.4 % vs. 0.2 %; aOR = 7.77; 95 % CI: 5.25, 11.94; p < 0.001).

### Conclusion
This study presents population-based data on the frequency of ophthalmic disease in patients with MS. Ophthalmic conditions, particularly optic neuritis, were significantly more common in individuals with MS compared to matched controls. The study also characterizes the prevalence of uveitis, which is relatively rarer than other ophthalmic conditions in the MS population, yet still more common than matched controls. Some visual conditions occurred before the clinical diagnosis of MS, some concurrently, and others afterward. These findings underscore the importance of recognizing visual disturbances in the diagnosis and management of MS and highlight the need for close collaboration between neurologists and ophthalmologists in the care of patients with MS.

Ophthalmic Conditions in Patients with Multiple Sclerosis

### Background
Idecabtagene vicleucel (ide-cel) is a B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapy, it has emerged as a transformative treatment for relapsed/refractory multiple myeloma (R/RMM). On April 4, 2024, the U.S. Food and Drug Administration (FDA) approved ide-cel for adult patients with R/RMM who have received at least two prior lines of therapy, including an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 monoclonal antibody. This meta-analysis evaluates the efficacy and safety outcomes of ide-cel from current clinical evidence.

### Methods
A systematic search of Medline, Embase, Cochrane Library, Web of Science, and Scopus was conducted through July 2025 and supplemented with data from ClinicalTrials.gov. Five relevant clinical trials (n=730 patients) were included. We analyzed outcomes such as overall response rate (ORR), complete response (CR) rate, progression-free survival (PFS), and safety endpoints such as cytokine release syndrome (CRS), neurotoxicity, infections, and treatment-related mortality (TRM). Pooled estimates were calculated using weighted averages based on sample size.

### Results
The pooled ORR was 72.9%, with a CR or better rate of 30.2%. Median PFS across studies was 9.7 months. CRS occurred in 81.8% of patients, with grade ≥3 CRS in 3.7%. Neurotoxicity was reported in 27.1% of patients, including 4.6% with grade ≥3 events. Infections were noted in 44.8% of patients, and TRM was 5.5%, primarily due to infections or immune-mediated toxicity. CRS and neurotoxicity were largely early-onset and manageable with tocilizumab and corticosteroids. Toxicity incidence increased with higher CAR T-cell doses and advanced disease burden.

### Conclusion
Ide-cel demonstrates strong efficacy in heavily pretreated R/RMM patients, with high response rates and a median PFS approaching 10 months. The safety profile is characterized by frequent but manageable CRS and neurotoxicity, and infections remain a key consideration. These findings support ide-cel as a valuable treatment option in R/RMM, in line with its recent FDA approval. It is suggested to maintain ongoing monitoring and long-term follow-up to fully assess durability of benefit and late-onset toxicities or even second primary malignancies.

Efficacy and Safety of Idecabtagene Vicleucel in Relapsed/Refractory Multiple Myeloma

Background 
Chronic pain imposes a significant global burden, contributing to reduced quality of life and increased healthcare costs. Pain's multifaceted nature complicates the development of standardized treatment plans that meet diverse patient needs. Patients increasingly adopt complementary health approaches (CHAs), yet utilization patterns and clinical effects remain understudied. Recently acquired data were used to quantify the association between chronic pain and CHA utilization in U.S. adults, focusing on osteopathic manipulative treatment and other non-traditional modalities, and to determine whether any observed associations persist after adjusting for demographic factors. 
Methods 
Pre-existing data from a cross-sectional online survey of a representative sample of 3,022 U.S. adults were examined to identify patterns of CHA use among individuals diagnosed with chronic pain in the past year. Statistical analysis included Chi-Square tests of bivariate associations of chronic pain and types of CHA use. Logistic regression analyses, with and without adjustment for demographic variables, evaluated the clinical significance of these associations. CHAs were categorized into the five NIH-defined groups: Physical, Psychological, Nutritional, Combined, and System-Based, encompassing 30 distinct interventions. CHA usage, favorable expectation, and favorable experience of these modalities were analyzed. 
Results 
Individuals with chronic pain demonstrated significantly higher overall utilization of CHAs than those without (adjusted odds ratio = 2.36, 95% CI: 1.44–3.87). Osteopathic manipulative treatment (adjusted odds ratio = 3.29, 95% CI: 2.15–5.02) was among many CHA modalities more commonly used by these individuals. Although both groups had low expectancy for positive outcomes, those with chronic pain reported significantly lower expectations for various modalities, such as Any Nutritional CHA use, herbalist, non-RX diet, prayer, mindfulness, meditation, and yoga. Reported favorable experience for Any CHA use among those with chronic pain was 93.5%. Various Physical CHAs, acupuncture, and art, music, or dance therapy were reported to be high in both groups, but favorable experience was higher in chronic pain patients. 
Conclusion 
The preference for CHAs among chronic pain patients demonstrates the unmet needs in conventional care regarding symptom management, functional recovery, and mind-body interactions. While the cross-sectional design limits causal interpretation and self-reporting may introduce bias, these results support further longitudinal research to evaluate efficacy, drivers of utilization, and long-term outcomes.

The Association of Chronic Pain and Complementary Health Approach Usage: A Comprehensive Analysis Adjusting for Demographics

Background
Meniscus injuries are common with approximately 850,000 patients undergoing surgical treatment in the United States yearly. To investigate regenerative strategies for avascular meniscus tears, our lab has been utilizing bovine meniscus explants for investigating regenerative strategies for tears and crosstalk between joint tissues. However, inter-species crosstalk between bovine meniscus and human derived joint tissues remains a concern.

This study was designed to engineer and validate a model of human meniscus for engineered healing of avascular meniscus tears under the crosstalk between meniscus, synovial membrane, and IFP.

Methods
A wedge-shaped scaffold was 3D-printed with Ecoflex F Blend C1200 polymer. Synovial mesenchymal stem/progenitor cells (syMSCs) were isolated from surgically removed synovium of anonymous adult donors as per our previous methods. The scaffolds were then loaded with collagen bioink and 2,000,000 cell /mL syMSCs and then placed into the PDMS molds. Experiment 1: For the first 2 weeks, the tissue constructs were cultured in Fibrogenic induction supplemented (FIS) media with connective tissue growth factor (CTGF). For weeks 3-4, the tissue constructs were cultured in chondrogenic induction supplemented media (CIS) and transforming growth factor beta-3 (TGFβ-3). Experiment 2: For 4 weeks tissue constructs were cultured in FIS with CTGF and CIS with TGFβ-3. No loading was applied. Experiment 3: Tissue constructs were cultured in combined FIS (w/ CTGF) and CIS (w/ TGFβ-3) with no FBS and no loading.

Results
At 4 weeks, histology showed new tissue integration into the pores throughout the scaffolds. Picrosirius Red (PR) and Alcian Blue (AB) staining suggest the de novo tissues in the no loading group are dense fibrocartilaginous tissues. Polarized images of PR-stained section showed densely aligned collagen fibers, reminiscent of native meniscus, comprising circumferential fibers and radial tie fibers. Mechanical loading resulted in less mature fibrous tissue matrix with limited cartilaginous matrix. Combined culture with FIS (w/ CTGF) and CIS (w/ TGFβ-3) showed the most mature and dense fibrocartilage tissue development after 4 weeks. RNA sequencing data show similar expression levels of fibrochondrocyte-specific genes, including COL1A2, ACAN, COMP, and COL1A1, between engineered and native human meniscus tissue.



Conclusion
Our study suggests a reliable and effective approach to engineer human meniscus-like tissues that can serve as an experimental explant model for various types of meniscus explant research. Combined culture with FIS and CIS creates more mature fibrocartilage structure than step-wise differentiation under no physiological loading. These findings may have implications for engineering human meniscus-like tissue for various types of meniscus research.


Stepwise Differentiation of Engineered Human Meniscus Tissues in 3D-Printed Flexible Scaffolds

BACKGROUND

• Listeria monocytogenes is a Gram-positive, facultative
intracellular bacterium found in soil, water, and
contaminated foods (e.g., unpasteurized dairy,
processed meats, raw vegetables, and honey).
• It causes life-threatening infections such as meningitis,
encephalitis, and sepsis.
• Primarily affects neonates, pregnant women, the
elderly, and immunocompromised individuals.
• In immunocompromised adults, infection typically
manifests as a mild, self-limiting gastroenteritis.
• Invasive CNS infections in healthy individuals are
extremely uncommon and can delay diagnosis.
• Transmission occurs mainly through ingestion of
contaminated food products.
• Early recognition and prompt antimicrobial therapy
(usually ampicillin with or without gentamicin) are
crucial for favorable outcomes.
• This case is notable due to its occurrence in a healthy
adult following wild honey ingestion, an unusual
source.

CASE PRESENTATION

• His symptoms began two days after consuming wild
Nepali honey mixed with rum, initially resembling a
mild flu-like illness without fever.
• On hospital day two, the headache intensified, and the
patient developed agitation and delirium requiring
haloperidol and promethazine.
• Non-contrast CT of the head was unremarkable.
• By day three, he developed a high-grade fever (103o F)
and was empirically started on piperacillin-tazobactam.
There were no meningeal signs.
• Lumbar puncture was performed.
• CSF sent for BioFire FilmArray Meningitis/Encephalitis
panel identified Listeria monocytogenes, prompting a
switch to IV ampicillin (2g every 4 hours) and
gentamicin (190mg every 12 hours).
• On day four, meningeal signs developed, and
fundoscopy showed early disc edema, managed with
mannitol.
• Although CSF culture remained negative, the patient
developed a transient pleural effusion by day six, which
resolved spontaneously.
• His Consciousness improved by the third day of
targeted therapy.
• Gentamicin was stopped on day 14; ampicillin was
continued for 28 days.
• The patient was discharged on day 30 and made a full
recovery without neurological complications.

DISCUSSION

• This case is significant for its atypical host and
presentation, resulting in delayed diagnosis and
treatment.
• It underscores the need to consider Listeria even in
immunocompetent adults presenting with
unexplained neurological symptoms.
• The unique association with wild honey ingestion
suggests a possible environment or foodborne
source.
• It emphasizes the importance of considering Listeria
in differential diagnoses, especially when standard
risk factors are absent.
• This case illustrates that Listeria monocytogenes can
cause invasive CNS infections even in
immunocompetent adults, particularly following
exposure to potential foodborne sources such as wild
honey.
• Early recognition and appropriate antibiotic therapy
are critical in preventing complications.
• Based on available literature, this appears to be the
first reported case of Listeria meningitis in an
immunocompetent adult linked to wild honey
consumption in Nepal.

Listeria Meningitis in a Healthy Adult FollowingWild Honey Ingestion: A Rare Case Report from Nepal

Background:
CTEPH, a subset of pulmonary hypertension, often goes undiagnosed despite its mortality rates. It arises from pulmonary artery obstruction due to unresolved thromboembolic disease. Symptoms such as dyspnea, fatigue, chest pain, and swelling necessitate a thorough evaluation. Lifelong anticoagulation is crucial to mitigate thrombotic risk. For operable cases, pulmonary endarterectomy offers a potential cure.

Case Presentation:
In August 2023, a 41-year-old male presented to the emergency department with two weeks of worsening shortness of breath, abdominal bloating, and pitting edema. His medical history included pulmonary embolism, right-sided heart failure, and acute renal failure treated with apixaban. Initial tests indicated acute kidney injury, elevated cardiac markers, and significantly increased d-dimer levels. Echocardiogram findings included severe right ventricular dilation and reduced systolic function. Imaging confirmed multiple perfusion defects consistent with CTEPH. During his hospitalization, the patient received furosemide and oxygen therapy, achieving euvolemia. Upon discharge, he continued rivaroxaban and home oxygen. Our team scheduled outpatient follow-up appointments with cardiothoracic surgery, pulmonology, a DOAC clinic, outpatient CTEPH specialists, and a primary care physician. We filled his medications at our facility and scheduled multiple follow-up appointments. Initially, the patient did not understand the gravity of his disease and expressed that his limited transportation would impact his ability to attend his follow-up appointments. We educated our patient and utilized our resources to create a well-executed discharge plan. In Spring 2024, the patient died from CTEPH complications.

Discussion:
As healthcare providers, we can commit to providing equitable care. Suppose we are vigilant in considering the potential poor outcomes that may arise from poor discharge planning and implementation. In that case, we may go that extra mile to ensure that the patient will understand the disease, diagnostics, medications, and the needed follow-up with various specialists and sub-specialists. Many doctors feel pressured to discharge patients quickly; however, safety can sometimes be compromised. Doctors may forgo parts of the discharge education, such as making follow-up appointments for patients with serious conditions, providing proper education on the importance of medication adherence, and ensuring that case management and social workers provide resources for financial assistance and medications. Discharge planning is a very dynamic and challenging aspect of inpatient care. Many factors must be coordinated to ensure the best outcomes. However, despite our efforts, our patient succumbed to his disease, highlighting the need for equitable change at the hospital and systemic level.


Closing the Gap in CTEPH Care: A Case Review of Disparities and Adherence

Sarcoidosis is a systemic disease defined by the formation of non-caseating granulomas in multiple organs, with cutaneous involvement in ~25% of patients. Scalp involvement in cutaneous sarcoidosis is rare and often misdiagnosed as a more common inflammatory condition including lichen simplex chronicus, scalp psoriasis, and discoid lupus erythematosus. This case illustrates the diagnostic challenge and subsequent necessity of considering sarcoidosis in the differential diagnosis of persistent scalp plaques resistant to therapies. Only a handful of cases describing scalp involvement in sarcoidosis have been reported in the literature, magnifying a gap in clinical awareness that deserves greater attention. A 58-year-old Black female with chronic uveitis presented with a 6-month history of an intermittently pruritic, solitary plaque on the left occipital scalp. She was initially treated for presumed tinea capitis with a 4-week course of oral terbinafine, showing mild improvement. Physical exam revealed a thick, scaly plaque with whitish brown crust and no lymphadenopathy. A KOH exam was inconclusive for hyphae, so a fungal culture was obtained, and she was empirically started on another 4-week course of oral terbinafine and ketoconazole 2% shampoo, with minimal improvement. The culture was negative and she was transitioned to topical fluocinolone 0.1% oil, but at her 3-month follow-up, the disease had progressed, with new violaceous, scaly lesions at the left medial canthus and frontal scalp. A scalp biopsy showed granulomatous dermatitis favoring sarcoidosis. AFB, Fite, and Grocott stains were negative. Elevated ACE levels and imaging revealed hilar and paratracheal lymphadenopathy and breast and axillary lymph node biopsies showed noncaseating granulomas, further supporting systemic involvement. She was diagnosed with systemic sarcoidosis and treated with topical clobetasol, tacrolimus, intralesional Kenalog (10 mg/mL), minocycline, and methotrexate, resulting in significant improvement. Scalp sarcoidosis is uncommon and often mimics common inflammatory conditions such as tinea capitis and psoriasis, as seen in this case. These various clinical morphologies make prompt diagnosis difficult and, thus, appropriate treatment is often delayed. This case contributes to clinical practice by showing that clinical findings alone are insufficient for diagnosing cutaneous sarcoidosis and the value of early biopsy in persistent scalp plaques of ambiguous etiology.


A Persistent Scalp Plaque Resistant to Conventional Therapies

Attention-deficit/hyperactivity disorder (ADHD) remains underdiagnosed in women due to the longstanding reliance on male-centered diagnostic criteria. Growing evidence now shows that hormonal fluctuations — particularly in estrogen and progesterone — influence dopamine and GABA systems, which are critical to ADHD symptom regulation and stimulant medication response.

This research explores how hormonal transitions across the female lifespan — including puberty, the menstrual cycle, pregnancy, postpartum, and menopause — can impact ADHD presentation and treatment efficacy. For instance, stimulant response tends to peak during ovulation, when estrogen and luteinizing hormone levels are highest, and diminishes during the luteal phase, when progesterone predominates. These shifts may explain why many women are diagnosed later in life, often during or after significant hormonal changes.

Research on ADHD treatment during pregnancy remains limited due to ethical constraints and the historical exclusion of pregnant individuals from clinical trials. However, existing data indicate that the primary fetal risk associated with stimulant use is a slight reduction in birth weight, with no consistent evidence of congenital abnormalities or long-term developmental harm.

Menopause may also intensify core ADHD symptoms, including emotional dysregulation and cognitive difficulties, leading to new diagnoses in adulthood.

In addition to pharmacologic strategies, Osteopathic Manipulative Treatment (OMT) may serve as a supportive adjunct by targeting autonomic balance, sleep quality, and somatic tension. These findings highlight the need for individualized, hormone-informed, and integrative approaches to ADHD care in women.

Hormonal Modulation of Stimulant Efficacy Across the Menstrual Cycle, Pregnancy, and Menopause

Introduction
The Taliban’s takeover in 2021 warrants attention to Afghanistan’s compromised economy and healthcare system from a constant state of war since Russia’s invasion in 1978. Maternal Mortality (MM), as defined by the World Health Organization (WHO), is “the death of a woman while pregnant or within 42 days of termination of pregnancy” from non-accidental causes. This paper presents a literature review of MM in Afghanistan, along with interventions and prospects for the future.

Methods
A comprehensive literature search was conducted across PubMed, Google Scholar, UNICEF, and WHO to identify relevant studies and data using keywords such as "maternal mortality," "Afghanistan," "healthcare," "women’s health," and "Taliban". Studies were excluded if not available in full text or irrelevant to the topic of focus.

Results 
Despite reaching a record low MM in 2023, and an overall downward MMR trend since 2000, Afghanistan’s MM still far exceeds that of neighboring countries such as Pakistan and Iran. Contributing factors were found to be destruction of economy and infrastructure from war, a woman’s education level, the presence of skilled birth attendants, and the Taliban’s stringent rules for women. 

Conclusion 
Multiple solutions have been implemented and are recommended. Introduced in 2003, the Basic Package of Health Services (BPHS) increased health facilities across Afghanistan in 2011 and resulted in an overall increase in health facility utilization. Maternal mortality ratios decreased significantly from 2002 to 2010, however, women in rural areas remain especially vulnerable. A program designed like the NGO named MEDlife, which recruits volunteers from Western countries to provide sustainable education and healthcare in rural, impoverished regions of Latin America, is recommended to be created and used in Afghanistan.

Maternal Mortality in Afghanistan: A Systemic Review

Background: Dysregulation of mRNA localization to neuronal dendrites and loss of neuronal activity-induced localized mRNA translation at synapses is a common cause of neurodevelopmental disorders such as Fragile X syndrome (FXS). FXS is the most prevalent cause of inherited autism and learning disability, caused by the mutation of an RNA-binding protein, Fragile X messenger ribonucleoprotein (FMRP). FXS is characterized by widespread disruptions in hippocampal memory circuitry as well as abnormalities in dendritic spine (where synapses are formed) density and morphology. FMRP binds to and regulates ~15-20% of mRNAs crucial for synaptic remodeling and neurodevelopment. It is not clearly understood how FMRP target synaptic mRNAs are regulated across different cell types in the brain that might explain the wide range of neuro-cognitive deficits in FXS. We hypothesize that, in the absence of FMRP, cell-specific dysregulation of FMRP target mRNAs underlie the wide range of functional implications in FXS pathophysiology in FMRP-null mice (Fmr1-/Y). 

Methods: We have shortlisted 12 high-confidence putative FMRP-target mRNAs identified previously by high-throughput sequencing. These 12 mRNAs are localized to the dendrite-enriched hippocampal neuropil layer, are differentially expressed in hippocampal cell types and encode proteins critical for synaptic function. To examine the spatial organization of these FMRP mRNA targets in vivo, we have used highly multiplexed single molecule in situ hybridization (HiPlex smFISH) and high-resolution microscopy in juvenile WT and Fmr1-/Y mouse hippocampus. We have quantified mRNA abundance, fluorescent puncta area distribution (proxy measure of copy number state) and their co-distribution (proxy of RNA-RNA coassembly) in CA1 and CA2 neuronal dendrites, two anatomically & functionally distinct cell types relevant to FXS phenotypes. To investigate the dendritic spine abnormality in CA1 and CA2 dendrites, we used golgi-cox impregnation. 

Results: Our preliminary data analyzing a subset of three FMRP-target mRNAs (Adcy1, Psd and Dlg4) in CA1 and CA2 dendrites show a trending decrease in mRNA abundance in CA1 but not in CA2 dendrites of Fmr1-/Y compared to WT. Further analysis is ongoing for the rest of the dataset. Interestingly, these findings also correlate with our ongoing analysis of spine density that shows no significant difference in CA2 dendrites (WT vs FMRP KO) and an observed increased spine density phenotype in FMRP KO CA1 dendrites compared to WT mice.

Conclusion: Integrated with the recent evidence, these data suggest that FMRP deficiency has cell-specific cellular and molecular alterations that would possibly correlate with the functional implications associated with these circuits in FXS pathology.

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