Intended purpose
The SALSA MLPA Probemix P236 CFH Region is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in the CFH, CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5 genes in genomic DNA isolated from human peripheral whole blood specimens. P236 CFH Region is intended to confirm a potential cause for and clinical diagnosis of atypical hemolytic uremic syndrome (aHUS), systemic lupus erythematosus (SLE) or C3 glomerulopathy, and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P236 CFH Region should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require conformation by another method. Most defects in the CFH gene are point mutations, none of which will be detected by MLPA. It is therefore recommended to use this assay in combination with sequence analysis. Suitable reference samples should be identified for proper data analysis. Not all exons of the aforementioned genes are covered.

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.

¹ Please note that this probemix is for in vitro diagnostic use (IVD) 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
Hemolytic uremic syndrome (HUS) is a rare disease characterised by haemolytic anaemia, thrombocytopenia and acute renal failure. Most cases result from infections with Shigatoxin-producing E. coli (STEC). Around 10% of HUS cases are not associated with STEC infections (atypical HUS, aHUS). The onset of aHUS ranges from the neonatal period to adulthood. Genetic aHUS accounts for an estimated 60% of all aHUS. Predisposition to aHUS is inherited in an autosomal recessive or autosomal dominant manner with incomplete penetrance.

aHUS is caused by hyperactivation of the alternative complement pathway. This hyperactivation can be due to mutations in complement regulators (CFH, CFI and CFHR proteins). Patients with genetic aHUS predominantly carry pathogenic variants in complement factor H (CFH), membrane cofactor protein CD46 (CD46) and complement factor I (CFI). The latter two genes are covered by probes in SALSA MLPA Probemix P296 aHUS. Genetic aHUS can also be caused by large genomic rearrangements in the CFH region (CFH, CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5) through mechanisms of gene conversion and non-homologous recombination. The CFH, CFHR3, CFHR1, CFHR4, CFHR2 and CFHR5 genes are arranged in tandem in the 1q31.3 region, approximately 195 Mb from the p-telomere. Most major rearrangements in the CFH region result in the deletion of the neighbouring CFHR1 and CFHR3 genes and, occasionally, in the generation of CFH::CFHR1 hybrid genes (Szarvas et al. 2016; Valoti et al. 2014; Venables et al. 2006; Zipfel et al. 2007). Gene rearrangements without copy number changes will not be identified by MLPA.

CFHR1-CFHR3 deletions have also been reported to occur in healthy individuals, with ethnic variations in its prevalence, including ~50% of Africans, ~25% in Europeans and less than 10% of Asians (https://www.ncbi.nlm.nih.gov/books/NBK1367/). CFHR1-CFHR3 deletions are associated with a decreased risk of developing age-related macular degeneration (ARMD). This deletion was found in nearly 20% of the chromosomes of control individuals and in only 8% of the chromosomes of individuals with ARMD (Hughes et al. 2006).

Copy number changes in the CFH region are also associated with an increased risk for the polygenic autoimmune disease systemic lupus erythematosus (SLE) (Zhao et al. 2011) and the complement-mediated renal disease C3 glomerulopathy (https://www.ncbi.nlm.nih.gov/books/NBK1425/).

Probemix content
The SALSA MLPA Probemix P236-B1 CFH Region contains 53 MLPA probes with amplification products between 130 and 500 nt. This includes 16 probes for CFH, 8 probes for CFHR3, 6 probes for CFHR1, 4 probes for CFHR4, 4 probes for CFHR2, and 5 probes for CFHR5. 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.

SALSA Reference Selection DNA SD072
This product requires the identification of suitable reference samples for proper data analysis. Suitable reference samples have two copies of all target sequences. To facilitate the selection of suitable reference DNA samples from your own sample collection, Reference Selection DNA SD072 can be used (catalogue number SD072) which needs be ordered separately from MRC Holland. Reference Selection DNA SD072 should only be used for initial experiments on DNA samples from healthy individuals with the intention to identify suitable reference samples. SD072 should not be used as a reference sample in subsequent experiments. For further details, consult the Reference Selection DNA SD072 product description, available online: www.mrcholland.com.

Sample DNA
Sample DNA developed for this product:

• SD072 Reference Selection DNA - to be ordered separately.