Commercially available reagents and devices are widely used in gene diagnostics. All laboratory methods we use have also been tested and validated for diagnostic use. The quality of the methods is regularly monitored by making use of commercial control samples (NGS) and by participating in the regular external quality monitoring rounds (TaqMan®, MLPA®). The analyses we use are based on the protein coding regions of the RefSeq database ( and cover either LRG ( or RefSeq- gene transcripts in human genome. Changes in transcripts have been reported in the research descriptions of our website.

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 is measured fluorometrically prior to genetic analysis.


The disease-causing variants are analyzed using TaqMan® chemistry (Thermo Fisher Scientific) and specific primers and probes have been designed for each variant analyzed. Repeat sequences and changes in known sequences are noticed by primer designs preventing their effect on analysis results. However, this cannot eliminate the effects of rare unreported variant changes. TaqMan® method is usable for analyzing known single nucleotide changes, as well as short insertions and deletions.

Sanger sequencing method

The target regions are amplified by PCR method and analyzed by capillary sequencing using the Sanger sequencing method (Thermo Fisher Scientific). Repeat sequences and changes in known sequences are noticed by primer designs for PCR amplification as well as other factors influencing PCR efficiency. Sanger method is usable for analyzing single nucleotide changes, as well as short insertions and deletions.


Next Generation Sequencing

Illumina devices are used in Next Generation Sequencing (NGS) analysis with Sequencing by Synthesis (SBS) chemistry. Exons with at least 5 bp these flanking regions of the genes are sequenced using targeted custom gene sequencing panels (UCSC hg19) and NGS method. NGS method is based on massive parallel sequencing in which millions of short DNA fragments are amplified simultaneously.

The sequenced fragments are aligned to the appropriate human reference sequence so that altered bases can be identified. The sequencing gene panels are prepared by random fragmentation of the genomic DNA and an enrichment of DNA fragments from target regions are done using streptavidin beads and PCR method for NGS. This method is used for analyzing single nucleotide changes, short insertions and deletions. The average read depth as well as the coverage at least 15x read depth (%> 15x) of the sequencing are reported in study statement.

MLPA-method/deletion analysis

The use of MLPA in deletion analysis is based on determination of exon copy number or genotypes in known variants. The target regions of deletion analysis are amplified by multiplex-PCR method kit (Multiplex Ligation-dependent Probe Amplification, MLPA®, MRC-Holland) and analyzed by capillary sequencing using the Sanger fragment analysis (Thermo Fisher Scientific). MLPA method is usable for analyzing deletions as well as other copy number changes and known variants.

Interpretation of Results

Databases and literature are used to evaluate variant alterations found in sequence and deletion analysis. These alterations are compared with previously reported variants to evaluate their causality. Variants are evaluated according to ACMG recommendations (Richards et al., 2015). Neutral sequence changes are not reported.

Increasing information of variants can change classification. Pathogenic variants of our analysis list have previously specified to be causal according to literature.

Known variants are previously found to be causal. Written study statement is sent to his/her attending physician.


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.