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. 2025 Nov 14;18(1):182.
doi: 10.1186/s12920-025-02227-z.

RNA sequencing provides functional insights and diagnostic resolution in previously unsolved rare disease cases

Robert G Lewis  1   2 , John M O'Shea  1   2 , Lucilla Pizzo  1   2 , Ting Wen  1   2   3 , Makenzie L Fulmer  1   2 , Jian Zhao  1   2 , Jan Verheijen  1   2 , Chaofan Zhang  1   2 , Matt Velinder  4 , Thomas J Nicholas  4 , Steven E Boyden  4 , Alistair Ward  4 , Erin E Baldwin  5 , Ashley Andrews  5 , Joselin Hernandez Ruiz  4 , Marco Marchetti  4 , David Viskochil  5 , John C Carey  5 , Steven B Bleyl  5 , Russell J Butterfield  6 , Vanina Taliercio  5 , Lorenzo D Botto  5 , Rong Mao  1   2 , Pinar Bayrak-Toydemir  7   8 ; Undiagnosed Diseases Network
Affiliations

RNA sequencing provides functional insights and diagnostic resolution in previously unsolved rare disease cases

Robert G Lewis et al. BMC Med Genomics. .

Abstract

Exome and genome sequencing have greatly improved the diagnosis of rare genetic disorders but remain limited in their ability to identify and classify non-coding variants, including intronic variants, cryptic splice-site alterations, and disruptions in regulatory regions. RNA sequencing (RNA-seq) has emerged as a powerful tool to bridge this gap by providing functional insights into genomic variants that disrupt splicing or gene expression, thereby aiding in variant interpretation and classification. We retrospectively reviewed 30 cases from the Utah Penelope Program and the Undiagnosed Diseases Network over a three-year period, in which RNA-seq was performed on whole blood and/or fibroblasts following either negative DNA sequencing or the identification of candidate variants requiring functional assessment. In these cases, RNA-seq identified exon skipping, cryptic splice-site activation, and intron retention, leading to transcript disruption. Additionally, positional enrichment analysis clarified X-inactivation patterns and dosage effects, confirming the pathogenicity of copy number variants. By detecting these transcript-level alterations, RNA-seq provided functional evidence supporting the reclassification of multiple variants of uncertain significance, contributing to diagnostic resolution in selected cases. This study underscores the clinical utility of RNA-seq in detecting splicing and regulatory defects that DNA sequencing and predictive tools alone cannot resolve. Integrating RNA-seq into clinical workflows can support variant classification, aid in diagnostic resolution for selected cases, and provide mechanistic insights into genetic disorders, contributing to patient care and genetic counseling.

Keywords: Genomics; RNA sequencing; Rare disease; Splicing; Variant reclassification.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: This study adhered to the Declaration of Helsinki. This study was reviewed and approved by the Institutional Review Board at the Undiagnosed Diseases Network and the University of Utah. IRB identifiers: 15HG0130 (UDN) and IRB_00129735 (Utah Penelope Program). Clinical and genomic data used in this study were obtained through participation in the Undiagnosed Diseases Network and the Utah Penelope Program. All protocols were reviewed and approved by the appropriate Institutional Review Boards, and informed consent was obtained from all participants or their legal guardians in accordance with institutional and national ethical guidelines. Consent for publication: Written informed consent has been obtained from all the participants and the parents/legal guardians of minors for their personal or clinical details along with any identifying images to be published in this study. Participants were fully briefed on the purpose and use of their contributions and voluntarily agreed to their inclusion. Participant images were reviewed and have written approval by all participants and/or their legal guardians. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Overview of cohort characteristics and case outcomes. A Age distribution of pediatric and adult participants who underwent combined DNA and RNA sequencing. Each dot represents an individual and horizontal lines represent median and 95% confidence intervals (CI). Blue circles represent males and red circles represent females. B Comparison of ages between participants with resolved and unresolved cases. Each dot represents an individual and the horizontal lines indicate the median and 95% CI. Participants with resolved cases were significantly younger than those with cases not resolved participants (two-tailed Student’s t-test, P = 0.0054). Blue circles represent males and red circles represent females. C Sample types used for RNA-seq in resolved and not resolved cases, represented as a bar chart. D Parts-of-a-whole chart illustrating clinical categories of participants, distinguishing between those with case resolution (Dx) and those without (No Dx). E Molecular mechanisms for each variant assessed, represented as a bar chart. F Time to case resolution in 10 participants, showing weeks from initial clinical consultation to final resolution and from RNA-seq analysis to resolution. Horizontal bars represent the median and 95% confidence interval. Case 9 was excluded (Table 1), as RNA-seq was performed concurrently with karyotyping, and the diagnosis was established prior to the return of RNA-seq results
Fig. 2
Fig. 2
Clinical and molecular findings in a participant with a de novo Xq22.1–Xq28 duplication and nested Xq28 triplication. A Clinical photograph of the participant demonstrating non-familial facial features and limb asymmetry. B Cytogenetic analysis showing the abnormal X chromosome (right) with additional material on the long arm compared to a normal X chromosome (left) which was described initially as 46,X,add(X)(q28) and was later clarified as 46,X,dup(X)(q22.1q28). C Cytogenomic microarray results demonstrating a 50.4 Mb duplication spanning Xq22.1–Xq28 and a nested cytogenetically cryptic 3.5 Mb triplication of Xq28. D Interphase and metaphase fluorescence in situ hybridization (FISH) using a red X centromere probe and a green Xq28 probe confirms the gain of the Xq28 region. E RNA-seq analysis from participant-derived fibroblasts. Left: Volcano plot of differentially expressed genes from the OUTRIDER analysis. Upregulated genes are shown in red, downregulated genes in blue, and genes located within the duplicated Xq22.1–Xq28 region in green. Middle: Positional enrichment analysis of upregulated genes by chromosome using DAVID. Right: Positional enrichment of upregulated genes by cytogenetic band, highlighting the duplicated interval. F RNA-seq analysis of whole blood. Left, middle, and right panels show the same volcano plot and positional enrichment analyses as in (E), illustrating consistent overexpression of genes within the duplicated region in whole blood
Fig. 3
Fig. 3
Clinical, radiologic, and transcriptomic findings in a participant with AIFM1-related spondyloepimetaphyseal dysplasia with hypomyelination. A Skeletal survey demonstrating characteristic radiographic abnormalities including metaphyseal flaring and irregular endplates in the lower extremities. B Brain MRI at age 17 demonstrating extensive bilateral symmetric T2/FLAIR hyperintensities with involvement of the deep and subcortical supratentorial white matter. C IGV sashimi plot of RNA-seq data highlighting aberrant splicing of AIFM1 in the proband. Red box indicates exon 6/8 junction reads present in the proband but absent in controls, consistent with exon 7 skipping. D Volcano plot of differential gene expression from RNA-seq of fibroblasts. Upregulated genes are shown in red, downregulated genes in blue, and AIFM1 is labeled as the top expression outlier (P = 6.5*10–6). E OUTRIDER expression outlier analysis showing normalized AIFM1 transcript counts in the proband (pink triangle) and controls (grey circles). Horizontal lines represent median and 95% CI
Fig. 4
Fig. 4
Clinical, neuroimaging, and transcriptomic findings in a proband with Warburg Micro Syndrome 3 due to a homozygous intronic variant in RAB18. A Sagittal, coronal, and axial T1 brain MRI images (left to right) demonstrate diffuse, bilateral, symmetric cerebral polymicrogyria. B Clinical photograph of the proband showing non-familial features including broad nasal root and craniofacial asymmetry. C Sashimi plot from whole blood RNA-seq showing RAB18 exon 3 skipping in the proband and reduced exon 3 usage in both carrier parents. Red boxes highlight exon 2/4 junction reads in the proband and parents. The long red box spanning exon 3 demonstrates complete absence of exon 3 reads in the proband, consistent with exon 3 skipping. D OUTRIDER analysis of RAB18 transcript expression. The proband (pink triangle) shows significant underexpression compared to controls (P = 0.019;) with intermediate reduction observed in the father (blue circle; P = 0.049) and mother (red circle; P = 0.074). Horizontal lines represent median and 95% CI
Fig. 5
Fig. 5
Clinical, radiologic, and transcriptomic findings in a proband with Shwachman-Diamond Syndrome 2 (SDS2) due to compound heterozygous splicing variants in EFL1. A Frontal (left) and lateral (right) clinical photographs of the proband. B Radiograph demonstrating metaphyseal flaring and marked knee abnormalities characteristic of spondyloepimetaphyseal dysplasia (SEMD). C Radiographic image showing abnormally shaped vertebral bodies. D Pelvic radiograph highlighting lacy ilia. E Sashimi plot of EFL1 exon 8 skipping due to the maternally inherited c.932 + 2 T > G variant. Red boxes highlight exon 7/9 junction reads observed in both the proband and mother, absent in the father and controls. F Read coverage plot of EFL1 intron 4 retention associated with the paternally inherited c.245-12A > G variant. Increased read coverage across intron 4 is observed in the proband and father (red boxes) but not in the mother or controls. G OUTRIDER expression outlier analysis showing significantly decreased EFL1 expression in the proband (pink triangle) compared to controls (P = 0.032), consistent with a loss-of-function effect from biallelic splicing disruption. The mother is represented by a red circle and the father by a blue circle. Horizontal lines indicate the median and 95% confidence interval
Fig. 6
Fig. 6
Facial features and transcriptomic analysis of biallelic TBCK variants in two sisters with IHPRF3 and a concurrent NIPBL variant in Sister A. A Front facial photograph of Sister A showing features including synophrys and upturned nose. B Front facial photograph of Sister B with macrocephaly, increased forehead height, low nasal root, and prominent cupid’s bow. C Left: Sashimi plot showing NIPBL splicing in the family and unrelated controls. In Sister A, red box highlights exon 27/29 junction reads consistent with exon 28 skipping; purple box indicates use of an alternative acceptor site. Right: Zoomed-in view of NIPBL exon 28 demonstrating alternative acceptor site usage with increased intronic reads (purple box); position of c.5329-15A > G variant is labeled in red. D Sashimi plot showing skipping of TBCK exon 23 due to the maternally inherited deletion. Red boxes mark exon 22/24 junction reads, while the purple box highlights the deletion breakpoints observed in both sisters and their mother but absent in the father and control samples. E Sashimi plot showing exon 2 skipping due to the paternally inherited TBCK c.193 + 2dup variant. Red boxes highlight exon 1/3 junction reads observed in both sisters (A and B) and their father (F), but not in the mother (M) or controls. F OUTRIDER expression outlier plot showing reduced NIPBL transcript levels in Sister A (green inverted triangle) compared to controls (P = 0.017). Horizontal lines indicate the median and 95% CI. G OUTRIDER expression outlier plot showing significantly reduced TBCK expression in sister A (green inverted triangle; P = 0.049) and sister B (pink triangle; P = 0.046), with intermediate reduction in the mother (red circle; P = 0.095). The father is represented by a blue circle. Horizontal lines indicate the median and 95% CI
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