Multiplex Ligation-Dependent Probe Amplification (MLPA)

CFH-CFHR5 Genomic Region
Genetic analyses for C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS) must include suitable technologies to detect copy number variation, hybrid genes and other complex genomic rearrangements in the CFH/CFHRs genomic region (Goodship, TH et al., 2017 PMID: 27989322).  We interrogate this region using Multiplex Ligation-Dependent Probe Amplification.

Indications for screening
Screening is offered to persons with C3G and aHUS.

MORL screening methodology
Multiplex Ligation-Dependent Probe Amplification (MLPA) is used to detect non-allelic homologous recombination events within the CFH – CFHR5. This assay is based on sequence-specific hybridization and the subsequent ligation of two directly flanking half-probes on a target nucleic acid sequence. Only when these half-probes are ligated can the resultant fragment serve as a template for PCR amplification. Multiple probes are used to cross-check validity.

Sensitivity
The standard deviation rates for each probe are 4%-10% (Schouten, J et al., 2002). MLPA has 95% confidence intervals for positive and negative predictive accuracies of 0.951-0.996 and 0.9996-1 respectively (Ahn JW et al., 2007). With the use of multiple probes, the likelihood of a spurious result is remote.

Turnaround time
Turnaround time is 3 weeks

Sample Required
8 - 10 cc. whole blood in lavender (EDTA) top tubes OR 10 μg DNA, minimum concentration: 50 ng/μl (A260/A280 1.8-2) resuspended in 0.1mM EDTA (10mM Tris HC1, 0.1mM EDTA, pH 8, Teknova Cat#T0220).

Cost & CPT Codes

The Clinical Diagnostics Service of the Molecular Otolaryngology and Renal Research Laboratories is a Joint Commission-approved CLIA-accredited diagnostic laboratory.