Approaches to solving undiagnosed rare genetic diseases in consanguineous populations

Short-read and long-read sequencing: Yield and limitations

To address the missing heritability left unresolved by short-read WES/WGS (srWES/WGS), long-read WGS (lrWGS) has emerged as a high-utility tool. Applied to several URGD cohorts, lrWGS identified the genetic causes in 7–17% of cases by successfully capturing structural variants, complex repeat expansions, and methylation defects within genomic dark regions.^[23,24] However, its efficacy varies significantly depending on the cohort architecture. For example, in a cohort of 34 exome-negative families suspected of having recessive conditions, lrWGS resolved only 13 families (38%); crucially, a retrospective analysis revealed that six of these families could have been resolved through advanced WES reinterpretation, while seven were detectable via standard srWGS.^[25] This study was relatively small, and while lrWGS yield could improve with larger sample sizes, the differences in yields across studies are heavily explained by the nature of sequenced cohorts (consanguineous vs. non-consanguineous), inheritance patterns, and varying bioinformatic interpretation standards.

Given these infrastructure requirements and the economic parameters of genomic medicine, the utility of advanced sequencing technologies must be governed by a strict, cost-effective clinical tiering system. Standard srWES and CMA remain the foundational, first-line diagnostic modalities due to their low cost, high throughput, and mature automated variant-calling pipelines. Because lrWGS incurs substantially higher financial costs (approximately 5–10-fold higher than WES) and requires specialized bioinformatic assembly expertise, its use is restricted to a second-line diagnostic tier or a research-only escalation framework.^[23,24] The clinical algorithm for deploying lrWGS is restricted to cases that fulfill two strict criteria: (1) Negative first-line testing, where the patient has completed srWES/WGS and CMA with negative or inconclusive results; and (2) Targeted phenotypic suggestion, where the clinical phenotype strongly implicates variant classes inherently poorly characterized by short-read platforms. Specifically, lrWGS is indicated for resolving complex structural variations (such as inversions or balanced translocations), repeat expansion disorders, pseudogene interference across highly homologous regions, phased haplotyping configurations without parental testing, and variants across highly GC-rich loci. By restricting lrWGS to an intentional second-line escalation tier, clinical programs can optimize national economic resources while systematically expanding the diagnostic yield for the remaining unsolved rare-disease cohorts.

To understand why a substantial fraction of clinical testing remains unsolved, it is essential to examine the underlying non-technical and interpretative bottlenecks. Beyond primary sequencing constraints, a study of a large clinical Mendelian cohort of molecularly characterized cases diagnosed using clinical diagnostic genetic testing reported five categories of additional challenges that directly account for these unsolved cases:

1. Phenotyping-related pitfalls.

2. Complex pedigree structures with complex inheritance.

3. Positional mapping-related limitations.

4. Failure to discover novel, uncharacterized candidate genes.

5. Intricate variant interpretation.

When all five of these phenotypic and interpretative challenges are systematically addressed and accounted for, any remaining unsolved fraction is highly likely to reflect true missing heritability. By systematically accounting for these phenotype- and pedigree-based pitfalls, researchers successfully resolved 54% (171 of 314) of URGD cases with previously negative clinical exome or genome reports.^[25,26]

Integrated multi-omics approach: Functional genomics to inform genetics

When genomic sequencing yields negative or inconclusive results, follow-up RNA sequencing (RNASeq) for loss-of-function, splicing, intronic, or regulatory variants has been shown to improve diagnostic yields in URGD cohorts by 13.5%.^[27,28] Concurrently, there have been significant developments and adoption of clinical and research-based untargeted comprehensive metabolomic testing to complement targeted clinical metabolic screenings. This modality confirms the pathogenicity of variants of uncertain significance detected in known or candidate genes across both metabolic and non-metabolic diseases, guiding the selection of appropriate genetic testing and clinical management.^[29-33] Using a multi-omics approach to identify coordinated signals remains vital for validating rare disease genes and variants.^[34] Furthermore, a systematic review reported that bioinformatic reanalysis of legacy next-generation sequencing data after 12–24 months from the first reporting is associated with a steady increase in diagnostic yield when accompanied by complementary research-based functional validation and open data sharing to replicate gene/variant-disease associations.^[35]

Therefore, it is highly probable that the diagnostic yield of the aforementioned study^[26] would be significantly higher if investigated using an integrated clinical and research-based multi-omics approach, validated by functional studies, rather than relying exclusively on diagnostic genetic testing with limited access to research. Research groups or consortia in Saudi Arabia should develop and utilize existing infrastructure to pair state-of-the-art genome (DNA sequencing) and transcriptome (RNASeq) analyses with proteomics, metabolomics, cell biology, and animal models to solve the genetic causes of URGD and reduce the corresponding socioeconomic and healthcare burdens in Saudi Arabia [Figure 1].

Close

Figure 1: Study design to solve the undiagnosed rare genetic diseases (URGD) and generate a Saudi-based URGD database. The known candidate gene variants produced from the discovery phase should be replicated and validated prior to their submission to the URGD database. Variants in known genes should be reported to the patients and submitted to the URGD database. NGS: Next generation sequencing.

Export to PPT

Proposed diagnostic framework: The need for functional validation and reverse phenotyping

Clinical WES and WGS pipelines yield a diagnostic rate of approximately 50%, leaving roughly 45% of cases completely unaccounted for from a genetic testing perspective.^[12,13] By resolving sequencing- and non-sequencing-related limitations, 13.5% and 54.5% of cases can be progressively addressed, respectively. Furthermore, identifying novel variants without matching functional validation introduces an alarming rate of false-positive assertions if those variants are ultimately categorized as benign. This carries severe clinical consequences for RGD patients, their families, and offspring.

In Saudi Arabia, historical URGD studies have been largely restricted to case reports or small cohorts evaluated exclusively through clinically validated assays, resulting in the omission of the “discovery” or candidate-gene fraction of the regional Mendeliome. To address these limitations and close the knowledge gap, a larger national URGD cohort must be established and equipped to resolve URGD through a structured multi-tier approach:

• Targeted participants: To enrich for the genetic causes of URGD, families with (1) A strong clinical suspicion of genetic disease after excluding non-genetic causes, or (2) A history of URGD in two or more relatives should be the primary focus. Three-generation family information and related samples (parents, brothers) should be recruited as controls for the segregation and functional studies.

• Novel-gene discovery: The comprehensive DNA (WES and WGS with copy number variant calling, complemented by lrWGS) and RNA (RNASeq) sequencing technologies, as well as optical genome mapping, should be utilized along with allele frequency databases, pangenome mapping, in silico pathogenicity prediction tools, and artificial intelligence programs in a stepwise approach [Figure 1] to capture all types of genetic variants in known and candidate genes.^[11,32,35] This cost- and effort-effective approach allows for interrogating the coding and non-coding parts of the human genome and linking them to URGD through homozygosity mapping, segregation, and transcriptomics.

• Reverse phenotyping: Once genetic testing is completed and variants are identified, each URGD case should undergo reverse phenotyping (clinical reevaluation and correlation with the genetic findings) to support decisions regarding pathogenicity and functional characterization of the detected variants.^[36]

• Variant triage and longitudinal care: URGD cases with pathogenic and likely pathogenic variants in known genes (positive results) should be returned to families and submitted to clinical databases by certified molecular geneticists or consultants with relevant credentials. Novel candidate genes and variants (inconclusive results) must be functionally validated and shared (and submitted to local and international databases) as well for replication or exclusion [Figure 2].^[6,9,26,32] Utilizing gene matching databases is critical for connecting independent clinicians and researchers who share an interest in these rare candidate genes. This international collaboration facilitates the rapid identification of additional unrelated cases with similar phenotypic and genotypic profiles. Consequently, these multi-center matches could provide the essential evidence needed to confirm or exclude novel disease-gene associations, thereby resolving previously inconclusive findings. All cases with VUS and no detected variants (negative results) should be bioinformatically re-analyzed every 12 months if new genetic or clinical evidence has emerged in the interim period that could change the interpretation.^[37,38]

Close

Figure 2: The implementation of the undiagnosed rare genetic diseases (URGD) gene panel and database in the clinical diagnostic pipeline. The known and validated candidate gene variants should be incorporated into clinical diagnostic testing to address undiagnosed rare genetic diseases cases in clinical laboratories. The results of the clinical diagnosis will help curate the URGD gene panels and expand the database. WES: Whole exome sequencing; WGS: Whole genome sequencing.

Export to PPT

Functional validation strategies

Depending on the gene’s predicted or known function, the novel candidate genes and variants should be validated and functionally characterized through proteomic, targeted, and untargeted clinical- and research-grade metabolomic analyses, in vitro and in vivo cellular assays, and animal-based platforms. Because of the high cost and longer time associated with functional studies, only candidate genes that pass the discovery and replication stages should undergo functional genomic characterization using patients’ cells (such as skin fibroblasts) and samples, cell-free assays, and cell- and animal-based models. The assay and disease-model system (animal model, patient-derived cells, body fluids such as plasma, urine, or cerebrospinal fluid) should be selected based on the gene function, the pathway involved, and the patient’s phenotype associated with the variant.

Organoids and animal models should be essential tools for deciphering the pathogenesis of orphan Mendelian diseases. Patient-derived organoids from patient-derived cells or reprogrammed induced pluripotent stem cells (iPSCs) can be used to model the development and function of organs such as the skin, liver, and brain. Animal models, such as mice, Xenopus, and zebrafish, should also be developed to study URGD. They have the advantage of being genetically modified to mimic variants that cause human diseases and to better capture the physiological processes that occur at the level of the organism.

However, the practical execution of these functional genomics workflows presents major operational, financial, and biological bottlenecks that must be factored into any national diagnostic network. Establishing patient-derived primary cell lines, culturing organoids, and generating stable transgenic or knockout animal models are highly resource-intensive and frequently require substantial funding and development timelines of 1–3 years per model.^[15,31] Furthermore, not all human candidate genes are biologically amenable to functional validation in standard model systems. Evolutionary divergence, distinct gene duplication events across species, or severe embryonic lethality can mask human-specific pathogenic mechanisms or prevent the generation of viable models in zebrafish, Xenopus, or mice.^[31] In addition, while iPSC-derived platforms and organoids offer powerful humanized cellular contexts, they are subject to pronounced line-to-line genetic and epigenetic variability. Differentiated cells within these models also typically retain an immature, embryonic- or fetal-like molecular profile, posing a significant hurdle to recapitulating or studying adult-onset neurodegenerative or metabolic phenotypes.^[31] Consequently, enforcing a strict computational triage and replication phase ensures that only exceptionally high-confidence candidate variants with clear phenotypic segregation are channeled into these lengthy, high-cost downstream functional validation pipelines.^[34]

Ethical approval, considerations, data protection and long-term governance

Before recruitment, all patients should receive pre-test genetic counseling regarding the study, expected results (including secondary findings), and the potential impact of these findings on the diagnosis, management, and reproductive options. All recruited members of the URGD families should sign the proper consent forms for participating in such studies to provide samples, publish patient photos, receive clinical and research genetic reports (including variants in candidate genes and secondary findings), share and submit their anonymized genetic data to local or international databases, as per approved institutional and national protocols. Genetic testing for pediatric patients (minors) should be performed in accordance with the ACMG guidelines.^[11] All samples should be anonymized and made untraceable to patients to protect patients’ identities and privacy. All patients must have the right to withdraw from the study at any time and to opt out of receiving any results. All patients should receive post-test genetic counseling to explain their genetic results, particularly in the context of reproductive options/planning to avoid RGD recurrence, and to enable information sharing with other family members for familial variant screening. Relatives of the patients should be offered the option of getting tested for the same variants. Data sharing and transfers should be conducted through the data protection and safe-sharing systems adopted by the participating institutions, in compliance with the Saudi Regulations of the Central Data Bank for Genomic Data by King Abdulaziz City for Science and Technology (KACST) and the data regulations of the Saudi Data and AI Authority (SDAIA).

Given the highly consanguineous architecture and close-knit familial structures of the Saudi population, these clinical and data-governance steps intersect deeply with specific psychosocial and cultural dynamics. The disclosure of an autosomal recessive condition implicitly exposes both parents as obligate carriers. To manage the potential psychosocial burdens of this disclosure, such as parental guilt or anxiety regarding extended family stigma, post-test counseling must utilize a culture-sensitive approach that emphasizes family empowerment and proactive future reproductive planning, such as pre-implantation genetic diagnosis. Because close familial relationships can challenge absolute genetic anonymity during pedigree analysis, strict adherence to SDAIA and KACST local data localization on national sovereign servers is paramount to prevent re-identification.

Furthermore, these initiatives operate in complete alignment with Islamic bioethics. Local religious frameworks and fatwas from the Islamic Jurisprudence Council strongly support the use of genomic medicine for disease prevention, early diagnosis, and alleviating human suffering, provided that medical confidentiality is maintained and familial lineage is safeguarded. Rather than hindering marriage prospects or social standing, framing the framework’s discoveries as an extension of the Kingdom’s highly institutionalized national premarital screening program normalizes genetic awareness, protecting family health structures while advancing genomic discovery.

Gaps and opportunities in urgd in Saudi Arabia

Addressing the missing heritability in highly consanguineous populations requires a paradigm shift from rigid, panel-based clinical testing to integrated multi-omics research frameworks. By combining deep clinical phenotyping, advanced short- and long-read sequencing technologies, transcriptomics, and untargeted metabolomics, clinical research teams can systematically uncover structural and non-coding variants that escape standard diagnostics. Crucially, pairing this discovery pipeline with reverse phenotyping and rigorous functional validation across human cellular lines in vitro and animal models in vivo can improve confidence in variant interpretation and reduce the likelihood of inappropriate clinical translation. This prevents misdirected treatments and provides families with accurate diagnostic resolution. Implementing this coordinated approach within Saudi Arabia will optimize clinical care pathways, streamline surgical planning for rare syndromic disorders, and drive the expansion of the global Mendeliome.