Pathogenic cryptic variants detectable through exome data reanalysis significantly increase the diagnostic yield in...

Area of Science:

• Genetics
• Neuroscience
• Molecular Biology

Background:

• Joubert syndrome (JS) is a complex neurodevelopmental disorder caused by mutations in various genes.
• Current diagnostic methods, including exome sequencing (ES), leave a significant portion of JS patients undiagnosed.
• Identifying the genetic basis of JS is crucial for diagnosis, prognosis, and potential therapeutic strategies.

Purpose of the Study:

• To enhance the diagnostic yield for Joubert syndrome by reanalyzing existing exome sequencing data.
• To uncover cryptic genetic variants, such as copy number variants (CNVs) and intronic splicing alterations, that were missed in initial analyses.
• To determine the frequency and impact of these cryptic variants in undiagnosed JS cases.

Main Methods:

• Targeted reanalysis of raw exome sequencing (ES) data from JS patients initially lacking a definitive diagnosis.
• Systematic search for copy number variants (CNVs) and intronic variants affecting splicing.
• Validation of identified variants using techniques like real-time PCR, chromosomal microarray, RT-PCR, and minigene assays.
• Extension of the search for cryptic variants to a cohort of exome sequencing-negative JS individuals.

Main Results:

• Cryptic "second hit" variants were identified in 54% (14/26) of patients with apparent heterozygous pathogenic coding variants.
• Biallelic cryptic variants were found in 7% (3/44) of patients initially negative by exome sequencing.
• The focused reanalysis strategy achieved an overall diagnostic gain of 24% (17/70 patients).
• Copy number variants and intronic splicing variants were confirmed as significant mutational mechanisms in JS.

Conclusions:

• Reanalysis of exome sequencing data is a highly effective strategy for increasing the diagnostic yield in Joubert syndrome.
• Cryptic variants, particularly CNVs and splicing defects, are a common cause of undiagnosed JS.
• This approach significantly improves diagnosis, especially for patients with heterozygous variants in genes like KIAA0586, CC2D2A, and CPLANE1.