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Molecular Genetics

Single gene Analysis

Single gene analysis is a targeted genetic examination method at the DNA or RNA
level, which is ideal for clarifying diseases associated with one or a few genes.

In a single gene analysis, the coding regions (exons) with flanking intronic regions of
the corresponding genes are examined using Sanger sequencing. Due to the technical
limitations of this method (e.g. due to allele dropout, mosaicism), changes might not
be detected in rare cases of Sanger sequencing.

A special requirement in the context of single gene analysis is the investigation of
repeat expansions (e.g. Huntington, SCA, Kennedy). Smaller repeats can be analysed
in-house using fragment analysis. Sequencing is carried out with the sequencers "3500 Genetic Analyzer" (Thermo Fisher Scientific) and "3500xl Genetic Analyzer" (Thermo Fisher Scientific); valuation is carried out with SeqPilot (JSI medical systems). The description and classification of the identified alterations is done according to an internationally valid nomenclature based on the HGVS and ACMG guidelines (http://www.hgvs.org/content/guidelines; Richards et al., Genet. Med. 2015, 17(5):
405-424).

During the DNA analysis, the genomic DNA is extracted from the material sent
(usually EDTA blood or tissue) and then regions of interest are amplified with specific
primers using PCR and Sanger sequencing.In addition to Sanger sequencing, DNA analysis, if appropriate. availableappropriate, also includes screening for duplications and deletions of individual exons or genes using MLPA kits (MRC Holland). For large deletions
affecting several genes, an array CGH analysis is recommended.

If it is not possible to clearly classify an already identified change in the splice area,
an RNA analysis can be used. In this case, a lymphocyte or fibroblast culture is
prepared from EDTA blood or tissue and puromycin is added to suppress the
premature degradation of aberrant transcripts. The mRNA is then transcribed into
cDNA using a reverse transcriptase and Sanger sequenced. This analysis is currently
used as standard for genes in which pathogenic intronic mutations are frequently
causal for the corresponding disease (neurofibromatosis type 1) or for genes that are
difficult to sequence due to pseudogenes (PMS2). If the classification of a potential
splice mutation is unclear or requested, we will gladly try to establish an RNA
analysis for the corresponding gene. Depending on whether the transcripts of the
corresponding gene are expressed in the blood cells, we require an EDTA blood
sample or a tissue sample (e.g. skin punch) for the examination.

Next-Generation Sequencing (NGS)

In the case of indications for which several genes with causal changes are known, or if
the symptoms cannot be narrowed down to a specific disease, a whole exome analysis (WES), using next-generation sequencing, is usually recommended for time and cost reasons. In this case, the coding regions of all known genes are sequenced and analysed. In addition, an evaluation of the CNVs (copy number variations) can be carried out. The exome analysis is performed on a NextSeq500 (Illumina) and evaluated with "VarSeq" (Golden Helix). The description and classification of the identified alterations is done according to an internationally valid nomenclature based on the HGVS and ACMG guidelines (http://www.hgvs.org/content/guidelines; Richards et al., Genet. Med. 2015, 17(5): 405-424).