Development and validation of a TTR-specific copy number screening tool, and application to potentially relevant patient cohorts.

TTR amyloidosis (ATTR) is a fatal condition caused by extracellular deposits of misfolded transthyretin. Patients often present with cardiac disease, but manifestations may also involve other organs including the peripheral nervous system. ATTR is considered familial when heterozygous mutations in the TTR gene are present (ATTRmutant or ATTRm), or acquired when no TTR aberrations are detected (ATTRwildtype or ATTRwt). We hypothesized that TTR copy number variants (CNVs), which would escape the standard diagnostic approaches, contribute to ATTR-related phenotypes, and developed a multiplex ligation-dependent probe amplification-based (MLPA-based), TTR-specific copy number screening tool. High inter-sample and intra-sample homogeneity of MLPA signals and the expected drop in signal intensity for restriction digest-based positive controls validated this tool. Subsequent application to 13 patients diagnosed with ATTRwt, and to 93 patients presenting with late onset and presumably inherited polyneuropathy did not identify TTR CNVs. We discuss insufficient sensitivity of the assay as well as non-existence and non-pathogenicity of TTR CNVs as potentially underlying our negative finding, but suggest size and composition of our cohorts as more likely explanations. Our CNV-screening tool will be made available to initiatives interested in screening additional and potentially more appropriate patient samples.