Development and validation of a comprehensive mutation and deletion detection assay for SDHB, SDHC, and SDHD

Abstract

Background: Lack of sequencing validation and complexity of deletion testing hinder genetic diagnosis of SDH-associated paraganglioma/pheochromocytoma.

Methods: We developed sequencing assays and multiplex ligation-dependent probe amplification (MLPA) deletion detection for SDHB, SDHC and SDHD. Clinical performance was validated on 141 blinded samples, previously tested at NIH.

Results: Sequencing and deletion detection were highly reproducible and agreed with previous NIH results in 99.3% and 100%, respectively.

Conclusions: DNA sequencing combined with MLPA allows reliable and simplified genotyping of SDHB, SDHC and SDHD.

Figure 1. MLPA deletion detection

The top half of the figure depicts the signals of a patient sample (blue peaks) compared to multi-run averaged female control signals (red peaks). The bottom half of the figure shows the peak ratios of averaged control signal to the signal generated in a run by control samples (male, left upper and female, right upper) and the response ratios observed in the sample of a male patient with a complete deletion of the SDHB gene (left lower) and a male patient with a deletion of exon 1 of SDHB. The horizontal green lines and the green shaded area on each graph indicate the expected normal range for the signal ratios. Of note is the difference between both normal control and patient sample with regards to the signal ratio of the DMD control gene. The DMD gene is located on the X-chromosome and is therefore only present in a single copy in male patients, thus serving as a convenient control gene for the expected deviation of a heterozygous deletion from the established normal signal ratio range.

Figure 2. Decision tree and workflow for SDH genotyping

The flow chart depicts a suggested decision tree and workflow for SDH genotyping in patients with suspected or known familial paraganglioma (PGL) or pheochromocytoma (PC). This includes most patients with PGL. With regards to PC, activating mutations in RET, and inactivating mutations or deletions in VHL and NF1 also have to be considered in suspected familial cases, and this is indicated at the top of the decision tree. However, an in-depth discussion and depiction of workflow for all familial variants of chromaffin tumors is beyond the scope of this article and the figure concentrates on the SDH genotyping.