Intended purpose
The SALSA MLPA Probemixes P351 PKD1 and P352 PKD1-PKD2 are in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assays² for the detection of deletions or duplications in the PKD1 and PKD2 genes, in order to confirm a potential cause for and clinical diagnosis of autosomal dominant polycystic kidney disease (ADPKD). P351 PKD1 can also be used to detect deletions or duplications in TSC2 exons 36, 38 and 42. Deletions disrupting both PKD1 and TSC2 can confirm a potential cause for and clinical diagnosis of TSC2/PKD1 contiguous gene deletion syndrome. Both assays are for use with genomic DNA isolated from human peripheral whole blood specimens, and are also intended for molecular genetic testing of at-risk family members.

The detection of copy number variations (CNVs) in PKD1 requires the use of both P351 PKD1 and P352 PKD1-PKD2, whereas the detection of CNVs in PKD2 only requires the use of P352 PKD1-PKD2. CNVs detected with P351 PKD1 and P352 PKD1-PKD2 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 PKD1, PKD2 and TSC2 genes are point mutations, none of which will be detected by MLPA. It is therefore recommended to use these assays 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.

These devices are 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.

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

Clinical background
Autosomal dominant polycystic kidney disease (ADPKD), the most common hereditary kidney disease, is characterised by bi-lateral development and expansion of renal cysts, hypertension and a progressive decline in renal function. In ~50% of affected individuals, ADPKD results in end-stage renal disease (ESRD), and 4-10% of ESRD worldwide is due to ADPKD. Although the kidney is the main organ involved, ADPKD is a multisystem disorder with profound extra-renal manifestations, including liver cysts and intracranial aneurysms. There is a substantial intra- and interfamilial variability in the severity of both renal and extra-renal disease manifestations. ADPKD is typically a late-onset disease diagnosed in adulthood, but about 2-5% of the ADPKD patients show a very early onset of the disease (during childhood or even prenatally) and a severe phenotype.

ADPKD is caused by heterozygous pathogenic mutations in either the PKD1, PKD2, GANAB, DNAJB11, ALG5, ALG9 or IFT140 genes (Besse et al. 2019; Cornec-Le Gall et al. 2018; Lemoine et al. 2022; Porath et al. 2016; Senum et al. 2022). Most patients carry a defect in PKD1 (~78%) or PKD2 (~15%), whereas a minority of the cases is explained by defects in GANAB (<0.5%), DNAJB11 (<0.5%), ALG5 (<0.5%), ALG9 (<0.5%) and IFT140 (1-2%). In ~5% of the cases, the underlying genetic defect is unknown. Patients with a PKD1 mutation, especially those with truncating mutations, generally have a more rapidly progressive disease with an earlier onset of ESRD than patients with a PKD2 mutation. It is estimated that ~3% of the PKD1 and PKD2 mutations are deletions or duplications (Carrera et al. 2016; Consugar et al. 2008; Schonauer et al. 2020; Xu et al. 2018). More information about ADPKD is available at https://www.ncbi.nlm.nih.gov/books/NBK1246/.

TSC2/PKD1 contiguous gene deletion syndrome is a disorder in which the phenotypes of tuberous sclerosis complex and ADPKD are combined. Tuberous sclerosis complex is a neurocutaneous disorder that involves abnormalities of the skin, brain, kidney, heart and lungs. When combined with ADPKD, it is characterised by a very early onset of severe polycystic kidney disease, that is diagnosed in utero or in infancy. The PKD1 gene lies directly adjacent to the TSC2 gene in a tail-to-tail orientation. Large PKD1 deletions that also disrupt the adjacent TSC2 gene result in TSC2/PKD1 contiguous gene deletion syndrome (Consugar et al. 2008).

Probemix content
The SALSA MLPA Probemix P351-D1 PKD1 contains 43 MLPA probes with amplification products between 135 and 481 nucleotides (nt). The SALSA MLPA Probemix P352-E1 PKD1-PKD2 contains 45 MLPA probes with amplification products between 136 and 490 nt.

The P351-D1 and P352-E1 probemixes contain 29 probes and 16 probes for the PKD1 gene, respectively. Together, these probemixes cover 41 of the 46 PKD1 exons. There are two probes upstream of PKD1 and three probes for PKD1 exon 15. The P351-D1 probemix also contains three probes for the TSC2 gene, located just downstream of PKD1. Furthermore, the P352-E1 probemix contains 18 probes for the PKD2 gene. All exons of the PKD2 gene are covered and there are two probes present for exons 1, 2 and 6. The P351-D1 and P352-E1 probemixes contain 11 reference probes each 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).

Each of the probemixes 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.