Biochemical and genetic testing methods and workflows
CENTOGENE´s in-depth medical expertise is supported by the application of cutting-edge technologies. We utilize almost all of the existing testing methods in our workflows, processing every sample in-house. We adhere for each testing method to the highest international standards (CAP, CLIA, ISO) for establishment and validation.
Genetic and biochemical testing methods at CENTOGENE
The term DNA sequencing refers to methods for determining the order of the nucleotide bases adenine, guanine, cytosine and thymine – in a molecule of DNA using a capillary sequencer.
Investigates mutations that show an increased frequency in the population. There are different hotspot mutations based on the ethnicity/ancestry of each patient (as provided by physician).
Single gene sequencing:
Analysis of the entire protein-coding regions of a gene (associated with a specific disorder ) including exon-intron boundaries.
Fragment length analysis
Some diseases are caused by insertions/deletions which cannot be detected via classical Sanger sequencing. Moreover, in some genes, there are repeats of nucleotide motives CAG. The size of the repeat regions consisting of e.g. CAGs varies between individuals and is polymorphic in normal individuals. However, when the number of repeats exceeds a certain threshold, neurological symptoms may be caused (pathological repeat expansion). In these cases, fragment length analysis (FLA) and/or repeat primed assays are utilized to detect the extent of the expanded repeats using a capillary sequencer.
It identifies large deletions or duplications using MLPA, qPCR or ddPCR.
MLPA (multiplex ligation-dependent probe amplification) is a semiquantitative multiplex PCR method detecting abnormal copy numbers of up to 50 different genomic DNA or RNA sequences, which is able to distinguish sequences differing in only one nucleotide. Probes bind to the DNA, are ligated and amplified by PCR. Results are obtained using a capillary sequencer.
Although for most hereditary conditions (partial) gene deletions or duplications account for less than 10% of all disease-causing mutations, for many other disorders this is 10 to 30 % or even still higher. The inclusion of MLPA in clinical settings can therefore significantly increase the detection rate of many genetic disorders.
The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA template can be copied, or “amplified,” many thousand-fold to a million-fold using sequence-specific oligonucleotides, heat-stable DNA polymerase, and thermal cycling. In real-time PCR (qPCR), the amount of DNA is measured after each cycle via fluorescent dyes that yield increasing fluorescent signals in direct proportion to the number of PCR product molecules (amplicons) generated. Data collected in the exponential phase of the reaction yield quantitative information on the starting quantity of the amplification target.
In digital droplet PCR (ddPCR) is somewhat similar to a qPCR approach, however a water oil emulsion technique is used to divide the PCR solution into smaller reactions. The sample is partitioned into nanoliter-size samples and encapsulated into oil droplets. In every droplet, the amplification process (40 cycles) takes place independently. Only in droplets containing the target DNA sequence can amplification occur resulting in a fluorescent signal based on probes or DNA intercalating dye. Thus, there are only two possibilities: “positive/negative” –> digital: “on/off.” The number of positive droplets correlates to the starting amount of DNA thus allowing for absolute quantification.
Scientific articles - Deletion/duplication testing
Chromosomal rearrangements involving 17q23 have been described rarely. Deletions at 17q23.1q23.2 have been reported in individuals with developmental delay and growth retardation, whereas duplications at 17q23.1q23.2…
AMPA-type glutamate receptors (AMPARs), key elements in excitatory neurotransmission in the brain, are macromolecular complexes whose properties and cellular functions are determined by the co-assembled constituents of…
Niemann–Pick disease type C (MIM 607625; NP-C) is an autosomal recessive, lysosomal storage disorder caused by impaired cholesterol and glycolipid trafficking due to pathogenic variations in either NPC1 (accounting for…
Measurement of enzyme activities for lysosomal storage disorders (LSDs): enzymes extracted from dried blood spots (DBS) or leucocytes convert artificial substrates into products which can be quantified using tandem mass spectrometry or via fluorimetric/spectrophotometric methods.
Measurement of biomarker concentrations in dried blood spots via tandem mass spectrometry.
Scientific articles - Biomarker and biochemical analysis
The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles…
Identification of a novel deletion in the MMAA gene in two Iranian siblings with vitamin B12-responsive methylmalonic acidemia
Adenosylcobalamin (vitamin B12) is a coenzyme required for the activity of methylmalonyl-CoA mutase. Defects in this enzyme are a cause of methylmalonic acidemia (MMA). Methylmalonic acidemia, cblA type, is an inborn…
Molecular, biochemical, and structural analysis of a novel mutation in patients with methylmalonyl-CoA mutase deficiency
Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting from genetic defects in methylmalonyl-CoA mutase (MCM). We found one homozygous nucleotide change in intron 12 of the MUT gene (c.2125-3 C > G).
Next generation sequencing
Next generation sequencing (NGS) enables us to generate a large amount of sequencing data in a massively parallel manner (millions of sequencing reactions at the same time). This makes sequencing much faster and much more cost-effective, and thus allows us to sequence large panels, whole exomes (WES) and even whole human genomes (WGS).
Whole exome sequencing
Identifies the molecular basis of a genetic disorder in an affected person by analyzing the total number of exons (= exome), the complete protein-coding region of the genome. These regions make up in total approx. 1% of the genome.
Whole genome sequencing
Reveals comprehensive information about the genetic composition of an individual by exhaustively covering the whole genome, thus both protein-coding and non-coding regions.