Pediatric and Adolescent Endocrinology

MON-234 - Differential gene expression in infants with normal and elevated thyroid stimulating hormone levels from stored newborn screen blood spot samples

Monday, June 15, 2026

9:00 AM - 2:00 PM CT

Late-Breaking Poster Presenter(s)

Alexandra Jacqueline Boni, MD (she/her/hers)

Fellow
Icahn School of Medicine at Mount Sinai
New York, New York, United States

Co-Author(s)

SS

Shwetha Hara Sridhar, n/a

Bioinformatician II
Icahn School of Medicine at Mount Sinai
New York, New York, United States
EE

Ethan Ellis, n/a

Research Manager
Icahn School of Medicine at Mount Sinai
New York, United States
SC

Stephen A. Caponetti, n/a

Associate Researcher II
Icahn School of Medicine at Mount Sinai
New York, New York, United States
HS

Hardik Shah

University of Chicago, IL, United States
HS

Hyomin Seo, MS

Bioinformatician
Icahn School of Medicine at Mount Sinai
New York, New York, United States
YW

Ying-Chih Wang, n/a

Biomedical Software Developer
Icahn School of Medicine at Mount Sinai
New York, New York, United States
SP

Sruti Patel, MD

Fellow
Icahn School of Medicine at Mount Sinai
New York, New York, United States
VT

Vanessa Torrice, MD

Fellow
Icahn School of Medicine at Mount Sinai
New York, New York, United States
NT

Norma Tavakoli, PhD

Research Scientist
New York State Department of Health
Albany, New York, United States
RS

Robert Sebra, PhD

Professor
Icahn School of Medicine at Mount Sinai
New York, New York, United States

Senior Author/Primary Investigator(s)

RR

Robert Rapaport, MD

Professor
Icahn School of Medicine at Mount Sinai
New York, United States

Background:

The New York State Newborn Screen (NBS) Program measures thyroid stimulating hormone (TSH) in dried blood spots on filter paper to screen for congenital hypothyroidism. Gene expression studies infrequent in pediatrics are especially rare in newborns.

Aims:

1 Establish whether RNA extracted from archival filter paper can be reliably utilized to measure gene expression

2 Determine if there are significant differences in gene expression between newborns with normal and elevated TSH levels

Methods:

We obtained 96 deidentified NBS stored dried blood spot samples courtesy of the NYS NBS Program: 48 from infants with TSH >100 µU/mL and 48 with TSH < 25 µU/mL. RNA extraction was performed using a custom extraction method. NGS libraries were prepared using the Takara SMART-Seq Total RNA Pico Input with UMIs (ZapR Mammalian). Bulk RNA-seq was then performed on the Illumina NovaSeq6000 platform, targeting 40M reads per sample.

RNA-seq data were processed using the RAPiD Nextflow pipeline, including alignment, quality control, and gene quantification using featureCounts. Differential expression (DE) analysis was performed using DESeq2, which models count data using a negative binomial distribution and incorporates dispersion estimation and log fold-change shrinkage. Downstream analyses included PCA and UMAP for dimensionality reduction, MA and volcano plots for visualization, Gene Ontology (GO) enrichment analysis, and network-based pathway integration. A curated gene set was used for comparative validation.

Results and

Discussion:

There were significant DE signatures between the two sample populations including 248 downregulated and 61 upregulated genes. Key upregulated genes include EGR2, EGR3, NR4A2, CCL3, RGS1, immediate early response and immune-associated genes and metabolic regulators such as G0S2. Several genes involved in transcriptional regulation and cellular metabolism were downregulated, including EGR1, FOS, NAMPT, and RPL22. Enrichment analysis revealed significant involvement of oxidative phosphorylation, mitochondrial ATP synthesis, RNA splicing, and nucleocytoplasmic transport pathways. Network analysis demonstrated tightly connected modules related to metabolic regulation and RNA processing. Comparative analysis with previously published data showed concordance in pathway-level findings, though with contrasting directionality of gene regulation. Evidence showed strong bias towards downregulation in patients with elevated TSH compared to patients with normal TSH.

Conclusion:

1 The study demonstrates, with novel methodology, the ability to extract genetic material from stored NBS blood spots.

2 Preliminary data identified differential gene expression between newborns with elevated TSH and normal TSH. Further investigation of genes and pathways involved in thyroid hormone gland development and hormone production is ongoing.

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