Intended purpose
The SALSA MLPA Probemix P226 SDH is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in SDHB, SDHC, SDHD, SDHAF1, and SDHAF2 in genomic DNA isolated from human peripheral whole blood specimens. P226 SDH is intended to confirm a potential cause for and clinical diagnosis of Hereditary Paraganglioma/Pheochromocytoma (PGL/PCC) and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P226 SDH should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Most defects in the SDHB, SDHC, SDHD, SDHAF1, and SDHAF2 genes are point mutations, none of which will be detected by P226 SDH. It is therefore recommended to use this assay in combination with sequence analysis.

Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.

This device is not intended to be used for standalone diagnostic purposes, pre-implantation or prenatal testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations, e.g. from DNA extracted from formalin-fixed paraffin embedded (FFPE) or fresh tumour materials.

¹ Please note that this probemix is for in vitro diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the product is for research use only (RUO).
² To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.

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. PGLs are rare (incidence of ca. 2 in 1.000.000 individuals), and are most frequently found in individuals between the age of 30 and 50 years. For PCCs, the true incidence is unknown, as many affected individuals remain undiagnosed.

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 (collectively referred to as SDHx) cause PGL/PCC and occur in up to 40% of cases. Probes for SDHA are not included in this P226 SDH probemix, but SALSA MLPA Probemix P429 SDHA-MAX-TMEM127 can be used to detect CNVs in the SDHA gene. SDHx 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 the recessive disorder SDH defective infantile leukoencephalopathy (Ghezzi D et al. 2009), SDHAF1 loss-of-function might also cause PGL due to the function of SDHAF1 in the SDH complex. This association, however, has not yet been established.

It is estimated that 10-25% of hereditary PGL/PCC syndrome cases are caused by pathogenic variants in the SDHB gene, 2-8% in the SDHC gene, 8-9% in the SDHD gene. SDHAF2 mutations are very rare (<0.1%). The majority of mutations in the SDHx genes are point mutations and small deletions. It is estimated that 1.2-5.5% of patients with hereditary PGL/PCC have pathogenic CNVs in SDHB, SDHC, or SDHD, which include the known 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), all of which can be detected with this probemix.

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

Probemix content
The SALSA MLPA Probemix P226-D1 SDH contains 45 MLPA probes with amplification products between 130 and 494 nt. This includes nine probes for the SDHB gene, ten probes for SDHC, seven probes for SDHD, two probes for SDHAF1 and four probes for SDHAF2. In addition, 13 reference probes are included that detect autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.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.mrcholland.com.