The SALSA MLPA Probemix P244-AIP-MEN1-CDKN1B is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative assay 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.

For the full intended purpose, see the product description.

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

SALSA MLPA Probemix P244 AIP-MEN1-CDKN1B is CE-marked for in vitro diagnostic (IVD) use.

This assay is for research use only (RUO) in all other territories.

Inclusion of a positive sample is usually not required, but can be useful for the analysis of your experiments. MRC Holland has very limited access to positive samples and cannot supply such samples. We recommend using positive samples from your own collection. Alternatively, you can use positive samples from an online biorepository, such as the Coriell Institute.

The commercially available positive samples below have been tested with the current (D1) version of this product and have been shown to produce useful results.

• Coriell NA07981: Four copies of CDKN1B affecting all probes for this gene, including flanking probes.

Various related products

• SALSA MLPA Probemix P017 MEN1: Contains the same MEN1 probes as this P244 probemix, but does not contain probes for the AIP gene, CDKN1B gene and several other genes in the 11q13 region.
• SALSA MLPA Probemix P169 Hirschsprung-1: Contains probes for the RET gene, associated with MEN2A and MEN2B.
• SALSA MLPA Probemix P177 CASR: Contains probes for the CASR gene, associated with familial hypocalciuric hypercalcaemia, a disease with certain similar symptoms as PHPT.
• SALSA MLPA Probemix P466 CDC73: Contains probes for the CDC73 gene that is associated with hyperparathyroidism-jaw tumour syndrome, a disease with certain similar symptoms as PHPT.

General MLPA resources

• MLPA technique overview
• Coffalyser.Net overview

MLPA education

• Getting started with MLPA
• Using Coffalyser.Net to analyse MLPA data
• Help centre with other learning resources

Selected publications using P244 AIP-MEN1-CDKN1B

• Belar O et al. (2012). Novel mutations in MEN1, CDKN1B and AIP genes in patients with multiple endocrine neoplasia type 1 syndrome in Spain. Clin Endocrinol (Oxf). 76:719-24.
• Carvalho RA et al. (2018). Germline mutation landscape of multiple endocrine neoplasia type 1 using full gene next-generation sequencing. Eur J Endocrinol. 179:391-407.
• Georgitsi M et al. (2008). Large genomic deletions in AIP in pituitary adenoma predisposition. J Clin Endocrinol Metab. 93:4146-51.
• Igreja S et al. (2010). Characterization of aryl hydrocarbon receptor interacting protein (AIP) mutations in familial isolated pituitary adenoma families. Hum Mutat. 31:950-60.
• Kooblall KG et al. (2021). Multiple Endocrine Neoplasia Type 1 (MEN1) 5'UTR Deletion, in MEN1 Family, Decreases Menin Expression. J Bone Miner Res. 36:100-9.
• Morotti A et al. (2020). The Oncosuppressors MEN1 and CDC73 Are Involved in lncRNA Deregulation in Human Parathyroid Tumors. J Bone Miner Res. 35:2423-31.
• Nord KH et al. (2010). Concomitant deletions of tumor suppressor genes MEN1 and AIP are essential for the pathogenesis of the brown fat tumor hibernoma. Proc Natl Acad Sci U S A. 107:21122-7.
• Pardi E et al. (2017). Mutational and large deletion study of genes implicated in hereditary forms of primary hyperparathyroidism and correlation with clinical features. PLoS One. 12:e0186485.
• Romanet P et al. (2019). UMD-MEN1 Database: An Overview of the 370 MEN1 Variants Present in 1676 Patients From the French Population. J Clin Endocrinol Metab. 104:753-64.
• Tuncer FN et al. (2018). Screening of AIP Gene Variations in a Cohort of Turkish Patients with Young-Onset Sporadic Hormone-Secreting Pituitary Adenomas. Genet Test Mol Biomarkers. 22:702-8.

References

• Cai F et al. (2013). Screening for AIP gene mutations in a Han Chinese pituitary adenoma cohort followed by LOH analysis. Eur J Endocrinol. 169:867-84.
• Carroll RW (2013). Multiple endocrine neoplasia type 1 (MEN1). Asia Pac J Clin Oncol. 9:297-309.
• Concolino P et al. (2016). Multiple endocrine neoplasia type 1 (MEN1): An update of 208 new germline variants reported in the last nine years. Cancer Genet. 209:36-41.
• Georgitsi M et al. (2008). Large genomic deletions in AIP in pituitary adenoma predisposition. J Clin Endocrinol Metab. 93:4146-51.
• Igreja S et al. (2010). Characterization of aryl hydrocarbon receptor interacting protein (AIP) mutations in familial isolated pituitary adenoma families. Hum Mutat. 31:950-60.
• Kooblall KG et al. (2021). Multiple Endocrine Neoplasia Type 1 (MEN1) 5'UTR Deletion, in MEN1 Family, Decreases Menin Expression. J Bone Miner Res. 36:100-9.
• Lemos MC et al. (2008). Multiple endocrine neoplasia type 1 (MEN1): analysis of 1336 mutations reported in the first decade following identification of the gene. Hum Mutat. 29:22-32.
• Marques P et al. (2018). Emergence of Pituitary Adenoma in a Child during Surveillance: Clinical Challenges and the Family Members' View in an AIP Mutation-Positive Family. Int J Endocrinol. 2018:8581626.
• Newsham IF (1998). The long and short of chromosome 11 in breast cancer. Am J Pathol. 153:5-9.
• Nord KH et al. (2010). Concomitant deletions of tumor suppressor genes MEN1 and AIP are essential for the pathogenesis of the brown fat tumor hibernoma. Proc Natl Acad Sci U S A. 107:21122-7.
• Pellegata NS et al. (2006). Germ-line mutations in p27Kip1 cause a multiple endocrine neoplasia syndrome in rats and humans. Proc Natl Acad Sci U S A. 103:15558-63.
• Romanet P et al. (2019). UMD-MEN1 Database: An Overview of the 370 MEN1 Variants Present in 1676 Patients From the French Population. J Clin Endocrinol Metab. 104:753-64.
• Thakker RV (2014). Multiple endocrine neoplasia type 1 (MEN1) and type 4 (MEN4). Mol Cell Endocrinol. 386:2-15.