Intended purpose
The SALSA MLPA Probemix P091 CFTR is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in the CFTR gene and the wild type allele of the CFTR p.Phe508del and p.Ile507del mutations in genomic DNA isolated from human peripheral whole blood specimens. P091 CFTR is intended to confirm a potential cause for and clinical diagnosis of cystic fibrosis or congenital absence of the vas deferens, and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P091 CFTR 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 CFTR gene are point mutations, which will not be detected by MLPA, with the exception of the two aforementioned 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.
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 and Coffalyser.Net analysis software.

Clinical background
Cystic fibrosis is a childhood-onset, multisystem disorder that affects epithelia of the respiratory tract, exocrine pancreas, intestine, hepatobiliary system, exocrine sweat glands and reproductive organs. The major clinical characteristics of cystic fibrosis are progressive obstructive pulmonary disease, pancreatic insufficiency, failure to thrive, meconium ileus, elevated sweat chloride levels and male infertility due to congenital absence of the vas deferens (CAVD). There is, however, a high variability in cystic fibrosis phenotypes, which range from severe forms affecting multiple organ systems (i.e. classical phenotype) to mild disease manifestations affecting a single organ (e.g. only CAVD).

Cystic fibrosis is the most common life-shortening autosomal recessive disorder in individuals of northern European (Caucasian) background, with a disease incidence of 1:3,200 live births and a carrier frequency of 1:28. The disease frequency is much lower in other ethnic populations, e.g. 1:15,000 in African Americans and 1:31,000 in Asian Americans. Cystic fibrosis and CAVD are both caused by homozygous or compound heterozygous mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which encodes a cAMP-regulated chloride channel expressed at the apical membrane of exocrine/secretory epithelial cells (Kerem et al. 1989; Riordan et al. 1989; Rommens et al. 1989). The defective protein impairs ion transport and water movement across epithelia, which among other things leads to an increased salt concentration in sweat and the formation of viscous mucus obstructing the airways of the lungs and ducts of the pancreas.

The most common CFTR variants are point mutations and small insertions/deletions, which are detected in 97-98% of the probands with cystic fibrosis and 79% of the probands with CAVD (Yu et al. 2012). The p.Phe508del (F508del; c.1521_1523del) mutation is the variant most commonly found in the general population, affecting ~66% of the CFTR alleles worldwide (Bobadilla et al. 2002). Other mutations have a much lower frequency, e.g. the p.Ile507del (I507del; c.1519_1521del) mutation constitutes ~0.5-1.3% of all CFTR mutations identified (Bobadilla et al. 2002). Large deletions and duplications are found in <3% of patients with cystic fibrosis and CAVD. The frequency and spectrum of CFTR mutations vary widely among different populations and depend upon the geographical and ethnic origin of patients (Estivill et al. 1997). Some mutations tend to be seen more frequently in specific populations. For example, a deletion of CFTR exon 2 and 3 (CFTRdele2,3 (21 kb)) is rather common among patients of German or Eastern European origin (Dörk et al. 2000). More information is available at https://www.ncbi.nlm.nih.gov/books/NBK1250/.

Probemix content
The SALSA MLPA Probemix P091-D2 CFTR contains 44 MLPA probes with amplification products between 130 and 481 nucleotides (nt). This includes 32 probes for the CFTR gene, one flanking probe for the ASZ1 gene and one flanking probe for the CTTNBP2 gene. Each exon of CFTR is covered by at least one probe. The ligation site of the exon 10 probe is located in intron 9. Exons 1, 11, 13 and 27 are covered by two probes and one probe for the upstream region of CFTR is present. One of the probes for exon 11 detects the wild type allele of the p.Phe508del and p.Ile507del mutations. A reduced signal for this probe can indicate the presence of the p.Phe508del mutation, the p.Ile507del mutation or a partial deletion of exon 11. In addition, 10 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.