Intended purpose
The SALSA MLPA Probemix P244-AIP-MEN1-CDKN1B is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in AIP, MEN1 and CDKN1B in order to confirm a potential cause and clinical diagnosis of familial isolated pituitary adenoma (FIPA), multiple endocrine neoplasia type 1 (MEN1) or multiple endocrine neoplasia type 4 (MEN4), respectively. This assay is for use with human DNA extracted from peripheral whole blood specimens.

Copy number variations (CNVs) detected with P244 AIP-MEN1-CDKN1B 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 AIP, MEN1 and CDKN1B genes are point mutations, none of which will be detected by MLPA. 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, population screening, pre-implantation or prenatal testing. Only in a research setting can this device be used 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
Multiple endocrine neoplasia type 1 (MEN1) is predominantly characterized by the occurrence of primary hyperparathyroidism (PHPT), which occurs in 95-100% of patients; pancreatic neuroendocrine tumours, which occur in 40-75% of patients; and pituitary adenoma, which is found in 30-50% of patients. Most tumours are non-metastasizing, but many can cause striking and serious clinical effects due to the increased secretion of hormones. It is estimated that in the general population 1 to 10 in 100.000 individuals develop MEN1 during their lifetime. Nine out of ten patients diagnosed with MEN1 have the familial form. MEN1 shows dominant autosomal inheritance and the penetrance is >95% by age 40 for confirmed pathogenic mutations. The mean age of death of MEN1 patients is between 50 and 55 years. The single gene associated with MEN1 syndrome is MEN1, which encodes the menin protein. Heterozygous MEN1 pathogenic variants are found in ~90% of familial MEN1 syndrome patients and in ~65% of sporadic cases. Loss of heterozygosity (LOH) of MEN1 is observed in >90% MEN1 tumours suggesting that MEN1 acts as a tumour suppressor gene, in line with the Knudson 2-hit hypothesis for tumorigenesis. Besides point mutations, several deletions involving one or more complete exons in the MEN1 gene have been described (Carroll 2013, Concolino et al. 2016, Lemos and Thakker 2008, Romanet et al. 2019, Thakker 2014), including a pathogenic deletion of just the 5’-UTR (Kooblall et al. 2020).

Pituitary adenomas (PAs) occur with a frequency of ~1 in 1000 in the general population. Most cases are sporadic, but approximately 5% occurs as a familial cancer. The AIP gene encodes aryl hydrocarbon receptor-interacting protein (AIP), a tumour suppressor that is involved in the control of cell proliferation and differentiation. AIP loss of function mutations are found in 15-25% of familial isolated pituitary adenoma (FIPA) cases, which are subsequently referred to as AIP-FIPA. Inheritance is autosomal dominant and the average penetrance is 15-30%, although this may vary greatly. The prevalence of AIP-FIPA is estimated at 1:100,000. Similar as for MEN1, LOH is frequently observed, suggesting that AIP also acts as a tumour suppressor gene (Cai et al. 2013). Although most known germline AIP mutations are point mutations, several exon deletions have been reported: exon 1-2, exon 2, exon 1-6 (Georgitsi et al. 2008, Igreja et al. 2010, Marques et al. 2018).

MEN1 and AIP are located in close proximity on 11q13, and somatic LOH in MEN1 and FIPA associated tumours often affects both genes. Apart from tumours in MEN1 and FIPA patients, LOH of this locus also occurs in sporadic cancers, especially in endocrine tissues. As both genes are considered tumour suppressor genes this double loss may contribute to tumorigenesis. Chromosomal losses of the 11q13 chromosomal band have also been found in other cancers, such as cervical cancer and hibernomas (Newsham 1998; Nord et al. 2010).

MEN4 is a distinct MEN type but the symptoms of MEN4 largely overlap with MEN1 (Pellegata et al. 2006). In a small number (estimated at 1-3%) of MEN1 mutation-negative patients fulfilling the diagnostic criteria for MEN1, mutations in CDKN1B have been detected. Extrapolating from this, the prevalence of MEN4 is very low: <1:300,000. Like MEN1, MEN4 is primarily characterized by PHPT and PA, but the additional tumours show some differences; tumours in the reproductive organs, and adrenal and renal tumours have been found in MEN4 patients. The only way to distinguish MEN4 from MEN1 is by identification of a pathogenic mutation in CDKN1B. Somatic mutations in CDKN1B have also been identified in sporadic tumours, but LOH of CDKN1B in MEN4-related tumours has not been found.

More information on MEN1 can be found on https://www.ncbi.nlm.nih.gov/books/NBK1538/
More information on AIP-related FIPA can be found on https://www.ncbi.nlm.nih.gov/books/NBK97965/
More information on MEN4 can be found on https://omim.org/entry/610755

Probemix content
The SALSA MLPA Probemix P244-D1 AIP-MEN1-CDKN1B contains 42 MLPA probes with amplification products between 129 and 463 nucleotides (nt). This includes 25 probes for the MEN1-AIP region, five probes for the CDKN1B region. In addition, 12 reference probes are included that target relatively copy number stable regions in all cancer types. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com).

This probemix contains nine/ten 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.