Intended use: The SALSA MLPA probemix P226 SDH is an in vitro diagnostic (IVD)¹ or a research use only (RUO) assay for the detection of deletions or duplications in the human SDHB, SDHC, SDHD, SDHAF1, and SDHAF2 genes, in order to confirm a potential cause and clinical diagnosis for Hereditary Paraganglioma/Pheochromocytoma Syndrome. This product can also be used for molecular genetic testing of at-risk family members. This assay is for use with human DNA extracted from peripheral blood.

Deletions or duplications detected with the P226 SDH probemix must be confirmed by another technique. In particular, deletions or duplications detected by only a single probe always require validation by another method. Most defects in the SDH genes are point mutations and small deletions, none of which will be detected by MLPA. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of the SDH genes. This assay is not intended to be used as a standalone assay for clinical decisions. The results of this test must be interpreted by a clinical molecular geneticist or equivalent.

¹Please note that this probemix is for In Vitro Diagnostic use (IVD) in the countries specified at the end of this product description. In all other countries, the product is for Research Use Only (RUO).

Clinical background: Paragangliomas (PGLs) are neuroendocrine tumours that originate from neural crest-derived cells. They arise from sympathetic or parasympathetic paraganglia tissues and can be situated in the head and neck region, thorax, abdomen, and pelvis. Tumours that arise from the adrenal medulla are called pheochromocytomas (PCCs). Symptoms of PGL/PCC result either from mass effects (for example carotid body enlargement, visible in the neck) or catecholamine hypersecretion. Both parasympathetic and sympathetic PGLs are rare. Estimates of the overall incidence of parasympathetic PGLs range from 1 in 30.000 to 1 in 100.000.
The hereditary PGL/PCC syndromes are inherited in an autosomal dominant manner. Pathogenic variants in the succinate dehydrogenase (SDH) genes, including SDHA, SDHB, SDHC, SDHD, and SDHAF2, cause PGL/PCC and occur in up to 40% of cases. Probes for SDHA are not included in this P226 SDH probemix, but are included in probemix P429 SDHA-MAX. SDH genes are tumour suppressor genes and loss of heterozygosity is a second hit in tumours. SDHD and SDHAF2 demonstrate parent-of-origin effects and generally cause disease only when the pathogenic variant is inherited from the father (Hao et al. 2009, Hensen et al. 2004), with a penetrance of 90% or higher by the age of 70. Mutations in SDHA, SDHB and SDHC are inherited in an autosomal dominant manner with no parent-of-origin effect and show a low penetrance (Benn et al. 2006). Mutations in the SDHAF1 gene are a cause of SDH defective infantile leukoencephalopathy (Ghezzi D et al. 2009) and might cause PGL due to the function of SDAF1, but this has not been reported.

Approximately 30% of hereditary PGL/PCC syndrome is caused by pathogenic variants in the SDHD gene, 22-38% in the SDHB gene, 4-8% in the SDHC gene, while for SDHAF2 it is unknown. The majority of mutations in the SDH genes are point mutations and small deletions. It is estimated that around 5-17% of pathogenic mutations in the SDHB, SDHC and SDHD genes is attributed to large deletions/duplications, including the founder mutations: SDHB Dutch founder deletion in exon 3 and the SDHB Spanish founder deletion in exon 1 (Bayley et al. 2005, 2009, Buffet et al. 2012).

More information is available at https://www.ncbi.nlm.nih.gov/books/NBK1548/.

Probemix content: The P226-D1 SDH probemix contains 45 MLPA probes with amplification products between 130 and 494 nt. The P226-D1 contains probes for all exons of the SDHB, SDHC, SDHD, SDHAF1 and SDHAF2 genes. In addition, 13 reference probes are included in this probemix, detecting several different autosomal chromosomal locations. The identity of the genes detected by the reference probes is available online (www.mlpa.com). This Probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity Fragments (Q-fragments), two DNA Denaturation Fragments (D-fragments), one benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mlpa.com.