Using long-read sequencing (LRS) technology to achieve molecular diagnosis in patients with rare genetic diseases who have already been tested by state-of-the-art genetic analysis with ambiguous or negative results. This will lead to efficient and reliable identification and clinical interpretation of cryptic and complex structural genomic variants, which represent the central challenge for the coming decades in human genetics.

The application of LRS to a diagnostic setting could have an impact on detection rate and diagnostic yield, leading to a better understanding of the etiology, prognosis and recurrence risk of rare genetic diseases (RGD), but also to a targeted treatment. One of the main benefits of LRS is the detection of balanced and unbalanced structural variants (SVs), including complex rearrangements, with high sensitivity and accuracy, through reliable alignment and precise breakpoint definition.

Among the main challenges of modern genetics, are identified 4 subgroups of patients that would benefit from the application of LRS to better characterize the genetic diagnosis and disease mechanisms.

• The first subgroup of patients are those with SVs of unknown significance or uncertain disease mechanism. In particular, clinical interpretation of duplications is challenging, due to the inability of Array Comparative Genomic Hybridization (aCGH) to detect whether they occur in tandem or are duplicated and inserted elsewhere in the genome, thus possibly disrupting genes involved in the duplication or altering their regulation.
• Other complex rearrangements can be studied with LRS to gain a better understanding of the molecular mechanisms of pathogenicity. A better definition of complex SVs, together with an accurate description of the phenotype, will allow the genotype-phenotype correlation to be determined.
• A third subgroup of patients eligible for the study are those with identified monoallelic alteration in autosomal recessive (AR) genes. The aim is to conduct a targeted analysis of the specific gene in order to look for a second mutation that wasn't detected by previous analysis (i.e. intronic variants, SVs, variants in difficult genomic regions).
• Lastly, patients with a phenotype that is strongly suggestive for a genetic condition, in whom several analyses, including Whole Exome-Sequencing (WES), retrieved negative results, could benefit from LRS. In this context, the aim is to apply a whole genome LRS approach to identify missed coding variants in difficult-to-sequence regions, variants in non-coding regions or SVs.

Inclusion Criteria:

• patients/relatives of patients with Copy Number Variations (CNVs), previously detected by aCGH, with uncertain clinical significance;
• patients/relatives of patients with inconclusive WES and aCGH data (no pathogenic/likely pathogenic variant);
• patients/relatives of patients with a known single hit (a pathogenic or likely pathogenic variant) in an AR gene detected with WES or aCGH;
• patients/relatives of patients with a finding of complex structural variants whose molecular disease mechanism is to be elucidated.

Exclusion Criteria:

• none

The study population will include:

• patients previously in diagnostic routine, who have provided informed consent for storage and secondary use of their biological samples and for being recontacted; they will be informed about the current project and requested to provide a new specific informed consent;
• patients' parents, when segregation analysis is required for diagnostic purpose; they will be informed about the current project and requested to provide a new specific informed consent;
• age: from 0 years (newborns) with no upper age limit.