Intended purpose
The SALSA MLPA Probemix P256 FLCN is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in the FLCN gene, as well as the presence of the two most common point mutations, c.1285delC and c.1285dupC, in genomic DNA isolated from human peripheral whole blood specimens. P256 FLCN is intended to confirm a potential cause for and clinical diagnosis of Birt-Hogg-Dubé syndrome (BHDS) and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P256 FLCN should be confirmed with a different technique. In particular, CNVs detected by only a single probe as well as the two common FLCN point mutations always require confirmation by another method. Most defects in the FLCN gene are point mutations, which will not be detected by MLPA, with exception of the two aforementioned FLCN point mutations. 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.

¹Please 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).
²To be used in combination with a SALSA MLPA Reagent Kit, Coffalyser.Net analysis software, and SALSA Binning DNA SD032.

Clinical background
Birt-Hogg-Dubé syndrome (BHDS) is a rare inherited genodermatosis, characterised by hair follicle hamartomas, kidney tumours, pulmonary cysts, and spontaneous pneumothorax. BHDS is inherited in an autosomal dominant fashion and is associated with mutations in the FLCN gene that encodes a protein called folliculin. Folliculin acts as a tumour suppressor in the mTOR pathway and inactivation of FLCN leads to increased mitochondrial oxidative metabolism (Hartman et al. 2009).

In >95% of BHDS patients a germline mutation in FLCN is identified. In <8% of BHDS patients the causative mutation is a large CNV. Penetrance is high and there is extensive clinical variability. The prevalence is roughly estimated at ~1:200,000. A second (somatic) FLCN mutation or loss of heterozygosity is found in the majority (~70%) of BHDS-associated renal tumours, in line with the Knudsen two-hit model of tumorigenesis.

Notably, there is a hypermutable C8-tract in exon 11 that spawns the two most common FLCN mutations c.1285dupC and c.1285delC; ~20-24% of patients show a germline deletion or insertion of a cytosine at this site (Nickerson et al. 2002; Schmidt et al. 2005; Toro et al. 2007; Toro et al. 2008; Liu et al. 2017). A slippage-mediated mechanism during DNA replication is thought to be responsible for these frameshift mutations leading to protein truncation.

More information is available at: https://www.ncbi.nlm.nih.gov/books/NBK1522/

Probemix content
The SALSA MLPA Probemix P256-C1 FLCN contains 27 MLPA probes with amplification products between 129 and 380 nucleotides (nt). This includes 15 copy number probes for the FLCN gene and two probes specific for the c.1285delC and c.1285dupC FLCN mutations, which will only generate a signal when the mutation is present. In addition, ten 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.

SALSA Binning DNA SD032
The SD032 Binning DNA provided with this probemix can be used for binning of all probes including the two mutation-specific probes: the188 nt probe 08598-L31913 detecting the c.1285delC mutation, and the 195 nt probe 08598 L31789 detecting the c.1285dupC mutation. SD032 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequences detected by the above mentioned probes. Inclusion of one reaction with 5 μl SD032 Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signal(s). It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD032 Binning DNA product description, available online: www.mrcholland.com.

Sample DNA
Sample DNA developed for this product:

• SD032 Binning DNA - included with this probemix.