Methods – Monogenic Diseases and Pharmacogenomics

In gene diagnostics, we primarily use commercially available reagents and instruments. All methods employed in our laboratory are tested and validated for diagnostic use. We continuously monitor method performance by using commercial control samples (NGS) and by regularly participating in external quality assessment programs (TaqMan®, MLPA® methods, PKD1-, PKD2-, and CYP21OH gene analyses, as well as interpretation of these results).

For diagnostic testing of monogenic disorders and pharmacogenomics, we accept EDTA whole blood or pre-isolates DNA. For MLPA analyses, we request EDTA whole blood samples. 

DNA extraction

Genomic DNA is extracted from EDTA blood using the QIAamp® DNA Blood Kit (QIAGEN). The method is usable for both fresh and frozen blood. The purity, quality and concentration of the extracted double-stranded genomic DNA – whether extracted in our laboratory or received pre-isolated – are verified fluorometrically prior to genetic analysis.

TaqMan® Assay

Common variants are analyzed using TaqMan® chemistry (Thermo Fisher Scientific). For each variant, specific primers and probes are designed. The design process accounts for repeat regions and known sequence changes to minimize their impact on analysis results. However, rare unreported variant may still affect outcomes.

The TaqMan® method is suitable for analyzing predefined genotypes, such as single nucleotide changes and short insertions/deletions. It also provides more reliable analysis of heteroplasmic samples compared to Sanger sequencing.

Sanger sequencing method

The target regions are amplified by PCR method and analyzed by capillary sequencing using the Sanger sequencing method (Thermo Fisher Scientific). Primer design considers repeat regions, known sequence changes, and other factors that may affect amplification.

Sanger sequencing detects single nucleotide changes and short insertions/deletions but does not identidy changes outside the target region. Variants within primer binding sites may lead to amplification of only one strand, causing undetected changes or misinterpretation as homozygous. GC-rich regions and repeats may prevent reliable results. 

Next Generation Sequencing (NGS)

NGS is performed on Illumina platforms using Sequencing by Synthesis (SBS) chemistry. Targeted sequencing covers exons and exon-flanking regions (at least 5 bases) of genes included in the offered panels (UCSC hg19).

NGS method is based on massive parallel sequencing in which millions of short DNA fragments are amplified simultaneously. Sequenced fragments are aligned to the human reference genome to identify bases differences. samples are prepared using commercial kits that fragment genomic DNA and enrich target regions for sequencing.

The method detects single nucleotide changes and short insertions/deletions. 

MLPA Method / Deletion Analysis

MLPA (Multiplex Ligation-dependent Probe Amplification, MRC-Holland) is used for deletion analysis by determining exon copy number or genotyping known variants. Analyses are performed on a capillary sequencing instrument using a commercial MLPA kit.

MLPA method detects deletions, other copy number changes, and predefined variants included by the kit manufacturer. MlPA is sensitive to DNA quality and extraction buffer; therefore, EDTA whole blood is recommended over pre-isolated DNA. 

MLPA does not detect:

• Changes outside probe target regions
• Variants without copy number changes
• Single nucleotide changes or short insertions/deletions unless predefined by the kit manufacturer.

Probe target regions are listed on the manufacturer’s website (https://www.mrcholland.com, accessed 06.10.2020). Detailed test-specific information is available upon request from the Gene Diagnostics Laboratory at the Genome Center of Eastern Finland.

Interpretation of Results

Variants detected by sequencing or deletion analysis are compare to databases and literature for pathogenicity assessment. Classification follows ACMG guidelines (Richards et al., 2015). 

Reports include variants classified as pathogenic, likely pathogenic, or of uncertain significance. Variant assessed as likely benign or benign are not reported. Classification may change as new information becomes available; in such cases, we inform the treating physicians.

Known Finnish founder variants previously reported as pathogenic in the literature are included. 

Reports are delivered to the referring physician.

References

Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., Grody, W., Hegde, M., Lyon, E., Spector, E., Voelkerding, K. and Rehm, H. (2015). Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine, 17(5), pp.405-423.