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Next-Generation Sequencing in Rare Genetic Diseases
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Next-Generation Sequencing in Rare Genetic Diseases

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Human Genomics and Genetic Diseases".

Deadline for manuscript submissions: 10 April 2026 | Viewed by 3437

Special Issue Editors

Dr. Edoardo Errichiello

E-Mail Website
Guest Editor
1. Department of Molecular Medicine, University of Pavia, Pavia, Italy
2. IRCCS Mondino Foundation, Pavia, Italy
Interests: molecular genetics; clinical genetics; next generation sequencing (NGS); molecular karyotyping; prenatal diagnosis; neurogenomics; oncogenomics; bioinformatics
Special Issues, Collections and Topics in MDPI journals
Dr. Paola Dimartino

E-Mail Website
Guest Editor
Department of Molecular Medicine, University of Pavia, Pavia, Italy
Interests: genetics of rare diseases; bioinformatics; NGS data analysis (exome/genome, long-reads, Hi-C) for the identification of structural variations and germline, de novo, post-zygotic variants

Special Issue Information

Dear Colleagues,

Rare genetic diseases (RGDs) affect more than 300–400 million people worldwide. In a large portion of cases, people with an undiagnosed disease incur in the so-called “diagnostic odyssey”. An accurate diagnosis of an RGD can result in better clinical management, the identification of potential therapeutics, and the avoidance of unnecessary treatments that may have non-negligible side effects. In the last decade, the advent of NGS (next-generation sequencing) and omics sciences, such as genomics, transcriptomics, and methylomics, has completely revolutionized the approach to RGDs. In this new scenario, bioinformatics tools, often based on artificial intelligence (AI) approaches, have evolved significantly in parallel with new technological approaches (e.g., long-read sequencing).

This Special Issue aims to highlight the contribution of these novel approaches to unravel the pathogenetic mechanisms, discover novel disease genes and genotype–phenotype associations, and depict the genetic architecture underlying RGDs and driving novel therapeutic approaches. Original articles, case series, reviews, and descriptions of new methodologies in the field of RDs are welcome to contribute to this Special Issue.

Potential topics include, but are not limited to, the following: innovative approaches (NGS-based) to the diagnosis of RGDs (genomic medicine and multiomics data integration), big data and artificial intelligence, disease gene discovery, network analysis and rare disease (epi)signatures, including multilocus and mosaic disorders.

We look forward to receiving your contributions.

Dr. Edoardo Errichiello
Dr. Paola Dimartino
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 250 words) can be sent to the Editorial Office for assessment.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Genes is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • rare genetic diseases
  • next-generation sequencing
  • bioinformatics
  • multiomics data
  • artificial intelligence
  • gene discovery
  • single nucleotide variants
  • structural variants

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Published Papers (6 papers)

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Research

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13 pages, 1744 KB  
Article
Distribution of Sequencing Coverage Gaps in Exomes and Genomes: Potential Implications for Diagnostic Accuracy in Neurodevelopmental Disorder Genes
by Emanuela Iovino, Claudia De Masi, Anna Ballestrazzi, Alessandro Mattiaccio, Federica Isidori, Marco Seri and Tommaso Pippucci
Genes 2026, 17(3), 269; https://doi.org/10.3390/genes17030269 - 26 Feb 2026
Viewed by 428
Abstract
Background: Exome (ES) and genome sequencing (GS) are powerful tools for diagnosing neurodevelopmental disorders (NDDs), yet sequencing coverage failures can leave clinically relevant variants undetected. Analyzing the distribution of coverage gaps across sequencing approaches and batches is therefore informative for diagnostic accuracy. Methods: [...] Read more.
Background: Exome (ES) and genome sequencing (GS) are powerful tools for diagnosing neurodevelopmental disorders (NDDs), yet sequencing coverage failures can leave clinically relevant variants undetected. Analyzing the distribution of coverage gaps across sequencing approaches and batches is therefore informative for diagnostic accuracy. Methods: We analyzed sequencing data from 43 NDD patients across four ES runs, including 14 individuals sequenced by both ES (Twist Human-Core-Exome-v1.3) and GS. Low-coverage regions (LCRs) were defined as target intervals with mean depth <20 x, and z-scores < −1.96 were used to identify batch-specific systematic LCRs. LCRs were clinically annotated using OMIM and SysNDD databases. Results: LCR patterns were highly consistent within each ES batch but were characterized by extreme variability between batches. Higher global mean coverage increased intra-batch consistency, but batches sequenced at a commonly accepted yield in clinical sequencing (>100 x mean coverage) showed thousands of batch-specific LCRs. LCR patterns substantially diverged between ES and GS, displaying preferential impact on different genes. Although a restricted group of genes accumulates LCRs disproportionately, most LCRs are broadly dispersed throughout the genome. LCRs were not systematically associated with features such as GC content and genomic location (e.g., exon 1). Interestingly, LCRs affected OMIM/SysNDD genes and occasionally overlapped ClinVar pathogenic variants, indicating potential impact on diagnostic sensitivity. Conclusion: The global distribution of coverage gaps appears strongly influenced by batch-specific effects, making the occurrence of LCRs partly unpredictable even within clinically relevant gene sets. These findings support systematic assessment of LCRs as a component of quality evaluation in diagnostic sequencing workflows. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Rare Genetic Diseases)
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23 pages, 2413 KB  
Article
Next-Generation Sequencing Defines a Molecularly Confirmed ARPKD Core Within the Broader PKHD1-Associated Disease Spectrum
by Paloma Lapunzina-Soler, Amir Shabaka, Ramón Peces, Ángel Alonso, Emilio Cuesta, Rocío Mena, Laura Espinosa-Román, Marta Melgosa, Gema Fernández, Yolanda Muñoz-GᵃPorrero, Jair Tenorio-Castaño, Pablo Lapunzina and Julián Nevado
Genes 2026, 17(2), 229; https://doi.org/10.3390/genes17020229 - 11 Feb 2026
Viewed by 418
Abstract
Background/Objectives: Autosomal recessive polycystic kidney disease (ARPKD) is a severe ciliopathy caused by biallelic pathogenic variants in PKHD1, characterized by variable renal and hepatobiliary involvement. The widespread use of next-generation sequencing (NGS) has revealed a large number of rare PKHD1 variants, [...] Read more.
Background/Objectives: Autosomal recessive polycystic kidney disease (ARPKD) is a severe ciliopathy caused by biallelic pathogenic variants in PKHD1, characterized by variable renal and hepatobiliary involvement. The widespread use of next-generation sequencing (NGS) has revealed a large number of rare PKHD1 variants, creating major challenges in distinguishing molecularly confirmed ARPKD from a broader spectrum of PKHD1-associated disease. Methods: We performed an integrated clinical and molecular analysis of 68 individuals referred for suspected ARPKD. Using phase-aware and family-informed ACMG classification, patients were stratified into three genetically defined groups: 40 with molecularly confirmed ARPKD (biallelic pathogenic, likely pathogenic or segregation-supported VUS-LP variants in trans), 10 with biallelic PKHD1 variants of uncertain pathogenicity, and 18 monoallelic carriers. Genotype–phenotype correlations were restricted to the molecularly confirmed ARPKD group. Results: Among the 40 molecularly confirmed ARPKD patients, 17 (42.5%) carried two loss-of-function (LoF) alleles, 16 (40%) carried one LoF allele, and 7 (17.5%) carried only non-LoF alleles. A strong allele-dose effect was observed. Neonatal or infantile onset occurred in 88% of LoF/LoF patients, compared with 56% of LoF/non-LoF and 29% of non-LoF/non-LoF individuals (p < 0.001). Progression to renal replacement therapy occurred in 65%, 31%, and 0% of patients (p = 0.002). In contrast, hepatobiliary disease was highly prevalent across all genotype classes and showed no significant association with LoF burden. Conclusions: Phase-aware and family-informed interpretation of PKHD1 variants distinguishes a molecularly confirmed ARPKD core from a broader PKHD1 variant spectrum. Within confirmed ARPKD, loss-of-function allele burden is the primary determinant of renal and perinatal severity, whereas hepatic disease is largely independent of truncating allele burden. These findings refine diagnosis, prognosis, and genetic counseling in the genomic era. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Rare Genetic Diseases)
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23 pages, 589 KB  
Article
Molecular Profiling of Polish Pediatric Patients with Epilepsy: A Single-Center Diagnostic Experience Using Next-Generation Sequencing
by Beata Chałupczyńska, Elżbieta Ciara, Paulina Halat-Wolska, Agnieszka Pollak, Piotr Stawiński, Dorota Jurkiewicz, Dorota Piekutowska-Abramczuk, Marzena Gawlik, Justyna Pietrasik, Agata Cieślikowska, Dorota Wicher, Agata Ulatowska, Dominika Jedlińska, Julita Borkowska, Dariusz Chmielewski, Dorota Dunin-Wąsowicz, Katarzyna Kotulska-Jóźwiak, Krystyna Chrzanowska and Agnieszka Madej-Pilarczyk
Genes 2026, 17(2), 133; https://doi.org/10.3390/genes17020133 - 27 Jan 2026
Viewed by 437
Abstract
Introduction: Epilepsy syndromes show marked clinical and genetic heterogeneity, with numerous functionally diverse genes involved in their etiology. Next-generation sequencing (NGS) has facilitated the identification of many monogenic epilepsy syndromes and enables earlier, more accurate diagnosis in pediatric patients. Materials and Methods: This [...] Read more.
Introduction: Epilepsy syndromes show marked clinical and genetic heterogeneity, with numerous functionally diverse genes involved in their etiology. Next-generation sequencing (NGS) has facilitated the identification of many monogenic epilepsy syndromes and enables earlier, more accurate diagnosis in pediatric patients. Materials and Methods: This study analyzes the molecular profiles of 87 pediatric patients with various forms of epilepsy in whom pathogenic or likely pathogenic variants were identified. Next-generation sequencing (NGS) using multi-gene epilepsy panels or whole-exome sequencing (WES) was performed. Results: A total of 88 pathogenic or likely pathogenic variants were detected in 48 epilepsy-related genes; 30 variants occurred de novo. SCN1A and KCNQ2 were the most frequent contributors (12.6% and 9.2%, respectively). The highest percentage of positive diagnoses (48%) was observed in patients with developmental and epileptic encephalopathy (DEE), with variants identified in genes including ALG13, ATP1A2, CACNA1A, CDKL5, CHD2, GABRG2, ITPA, KCNQ2, PCDH19, SCN1A, SCN2A, SCN3A, SCN8A, SMC1A, SPTAN1, STXBP1, and UBA5. Pathogenic variants in ANKRD11 were found in four patients with KBG syndrome, while other genes appeared sporadically. Conclusions: Targeted massively parallel sequencing is an effective diagnostic tool for pediatric epilepsy. The presence of numerous single-case findings highlights the high genetic heterogeneity of epilepsy. This approach enabled more precise diagnoses that would not have been achieved through clinical evaluation alone, underscoring the importance of genetic testing for prognosis and treatment planning in pediatric patients with unexplained epilepsy. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Rare Genetic Diseases)
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15 pages, 1116 KB  
Article
Prenatal Exome Sequencing: When Does Diagnostic Yield Meet Clinical Utility?
by Alessia Carrer, Francesco Maria Crupano, Berardo Rinaldi, Giulietta Scuvera, Claudia Cesaretti, Valeria Nicotra, Silvana Gangi, Lorenzo Colombo, Gabriella Araimo, Matilde Tagliabue, Daniela Marchetti, Laura Pezzoli, Maria Garzo, Veronica Accurti, Grazia Volpe, Simona Boito, Palma Finelli, Monica Fumagalli, Maria Francesca Bedeschi, Maria Iascone, Nicola Persico and Federica Natacciadd Show full author list remove Hide full author list
Genes 2026, 17(1), 37; https://doi.org/10.3390/genes17010037 - 30 Dec 2025
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Abstract
Background/Objectives: Prenatal Exome Sequencing (pES) has revolutionized prenatal diagnosis in fetuses with congenital anomalies. Although its performance is very promising, previous pES studies have mainly focused on diagnostic yield, often without considering the actual impact on ongoing pregnancies. In this study, we aim [...] Read more.
Background/Objectives: Prenatal Exome Sequencing (pES) has revolutionized prenatal diagnosis in fetuses with congenital anomalies. Although its performance is very promising, previous pES studies have mainly focused on diagnostic yield, often without considering the actual impact on ongoing pregnancies. In this study, we aim to (1) assess whether a prenatal molecular diagnosis can reliably predict the clinical features of the unborn child and (2) determine the gestational age (gw) at which ultrasound (US) findings are sufficient to support the pathogenicity of genetic variants detected by pES. Methods: We retrospectively selected 47 cases complicated by US anomalies that underwent Exome Sequencing (ES) and for which complete clinical assessment was available. A blinded reanalysis of ES data was performed, considering only prenatal features. Results: In our cohort, standard ES led to a molecular diagnosis in 43% of cases. The blinded reanalysis revealed that a complete or partial retrospective prenatal diagnosis was achievable in 95% of diagnosed cases. The mean gestational week at which US data would have supported molecular diagnosis was 22 + 5 weeks. The clinical follow-up confirmed a syndromic presentation in 21 out of 23 newborns and in all terminated pregnancies. Conclusions: Our study further confirms that pES is a valuable diagnostic tool for detecting genetic etiology in fetuses with congenital malformations. In most cases, pES results accurately predict the postnatal phenotype. However, the prenatal setting requires specific adjustments and precautions, and a negative pES result cannot be considered reassuring. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Rare Genetic Diseases)
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Review

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15 pages, 619 KB  
Review
From Genomic Diagnosis to Personalized RNA Medicine: Advances in Next-Generation Sequencing and N-of-1 Antisense Oligonucleotide Therapies for Rare Genetic Diseases
by Paris Rodriguez Carstens, Hidenori Moriyama and Toshifumi Yokota
Genes 2026, 17(3), 318; https://doi.org/10.3390/genes17030318 - 15 Mar 2026
Viewed by 322
Abstract
Next-generation sequencing (NGS) and antisense oligonucleotide (ASO) technologies are converging to transform the diagnosis and treatment of rare monogenic disorders. NGS enables comprehensive, single-test molecular diagnoses through targeted panels, whole-exome sequencing, and whole-genome sequencing, which together reveal pathogenic variants across coding, intronic, and [...] Read more.
Next-generation sequencing (NGS) and antisense oligonucleotide (ASO) technologies are converging to transform the diagnosis and treatment of rare monogenic disorders. NGS enables comprehensive, single-test molecular diagnoses through targeted panels, whole-exome sequencing, and whole-genome sequencing, which together reveal pathogenic variants across coding, intronic, and structural domains. Integration with transcriptomic analyses, including RNA sequencing, further refines genotype–phenotype correlations and identifies splicing aberrations amenable to correction by ASOs. Therapeutic advances now span RNase H1-dependent gapmers for transcript knockdown, splice-modulating phosphorodiamidate morpholino oligomers (PMOs), and peptide/antibody-conjugated PMOs that enhance muscle and cardiac delivery. These platforms underpin the rise in N-of-1 ASO therapies—customized drugs developed for individual patients with unique pathogenic variants. Landmark cases such as Milasen and Atipeksen illustrate the clinical feasibility and ethical complexities of personalized RNA therapeutics, while updated FDA guidance supports expedited, patient-specific investigational pathways. Despite progress, challenges persist in delivery efficiency, long-term efficacy, and equitable access. Emerging approaches—including long-read sequencing, AI-driven oligo design, and improved delivery—promise to extend ASO precision and reach. This review synthesizes current advances linking genomic diagnosis to individualized RNA-targeted interventions, outlining how integrated NGS-ASO pipelines are reshaping the therapeutic landscape for rare genetic diseases. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Rare Genetic Diseases)
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Other

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14 pages, 2427 KB  
Case Report
A Complex Case of Retinoblastoma Solved by the Combined Approach of Humor/Plasma cfDNA-NGS and LR-WGS
by Simona Innamorato, Simona L. Basso, Omaima Belakhdar, Mirella Bruttini, Chiara Fallerini, Heyran Huseynli, Giulia Caccialupi, Elena Pasquinelli, Mariarosaria Adduci, Giorgio Signori, Felice Arcuri, Valeria Malagnino, Maria Chiara Siciliano, Stefano Lazzi, Simone Pesaresi, Daniela Galimberti, Paolo Galluzzi, Sonia De Francesco, Theodora Hadijstillanou, Anna Maria Pinto, Alessandra Renieri and Francesca Arianiadd Show full author list remove Hide full author list
Genes 2025, 16(12), 1399; https://doi.org/10.3390/genes16121399 - 22 Nov 2025
Viewed by 934
Abstract
Background: Complex cases of retinoblastoma (RB) often require integrative molecular approaches to define tumor etiology and guide clinical management. Purpose: Our aim was to evaluate the usefulness of combining aqueous humor (AH)/plasma cell-free DNA next-generation sequencing (cfDNA-NGS) and long-read–whole-genome sequencing (LR-WGS) to resolve [...] Read more.
Background: Complex cases of retinoblastoma (RB) often require integrative molecular approaches to define tumor etiology and guide clinical management. Purpose: Our aim was to evaluate the usefulness of combining aqueous humor (AH)/plasma cell-free DNA next-generation sequencing (cfDNA-NGS) and long-read–whole-genome sequencing (LR-WGS) to resolve diagnostically challenging RB cases. Case Description: We report the case of a 3-year-old Caucasian girl, conceived by heterologous assisted reproductive technology (ART), presenting with unilateral, widely infiltrative RB in the right eye. She exhibited limited verbal communication, a glabellar angioma extending to the nasal bridge and philtrum, and mild hypertelorism. Standard blood testing revealed no pathogenic SNVs, CNVs, or methylation abnormalities in the RB1 gene. Targeted cfDNA analysis using the Illumina TruSight Oncology 500 (TSO500) panel on AH and plasma identified a somatic RB1 splice-site variant (c.1498+2T>C) with a variant allele frequency (VAF) of 98.5%, consistent with biallelic inactivation. Additional gains (fold change > 1.5) were found in AH and confirmed in plasma, suggesting a germline 13q duplication. Third-generation LR-WGS, performed with Oxford Nanopore Technology (ONT), on blood confirmed a 24.6 Mb duplication on chromosome 13, compatible with the rare 13q duplication syndrome characterized by psychomotor delay, craniofacial dysmorphism, and hemangiomas. AH-cfDNA revealed additional somatic copy-number alterations, including amplifications (i.e., MDM4 and ALK) and deletions (i.e., BRCA2), indicating progressive clonal tumor evolution. Conclusions: This experience tells us that a combined approach with TSO500 Illumina NGS on cfDNA, along with LR-WGS, is able to help solve complex cases and define the appropriate treatment and surveillance strategy. Full article
(This article belongs to the Special Issue Next-Generation Sequencing in Rare Genetic Diseases)
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