Case study of Noonan-like features and Lipomatosis associated with a PIK3CA duplication investigated using Oxford Nanopore sequencing

Abstract

Copy number variants (CNVs) involving non-coding regions remain challenging to interpret due to their structural complexity and limited support from current classification frameworks. This study investigated a 229 kb duplication at 3q26.32-q26.33, classified as a variant of uncertain significance (VUS), identified in a patient with Noonan-like features and multiple lipomas. The duplicated region includes dosage-sensitive and regulatory elements, but routine testing failed to resolve its structure or clinical relevance. A stepwise molecular approach was applied, beginning with long-range PCR followed by targeted nanopore sequencing to evaluate candidate breakpoint junctions. While amplification across the proximal region was successful, off-target effects and presumed disruption near the distal breakpoint limited confirmation. Whole-genome nanopore sequencing identified two split reads spanning the junction, defining a head-to-tail tandem duplication linking the 3′ untranslated region of PIK3CA to the 3′ end of GNB4, with preserved orientation and no inversion. The resolved configuration suggests potential regulatory or dosage effects, but in the absence of segregation or expression data, the variant remains a VUS under current CNV classification guidelines. This case underscores the diagnostic ambiguity of non-coding duplications and highlights the value of long-read sequencing for structural resolution. Future work should prioritise RNA-based analysis, breakpoint-spanning PCR, and inheritance testing to support clinical re-evaluation. More broadly, this project demonstrates how targeted long-read approaches can advance the interpretation of ambiguous CNVs in rare disease diagnostics.