Genome Diagnostics

Re-analysis of WES/WGS data

In the event that an whole exome application has been submitted for a single gene panel analysis, it is still possible to apply for another gene panel or an exome-wide analysis retrospectively. Existing data will receive a new analysis according to the latest scientific knowledge and panels.
 
For an exome-wide (open-exome) re-analysis, involvement of a clinical geneticist is needed for the application to facilitate a written ‘informed consent’ form for the patient. The clinical geneticist will also be responsible for saving the ‘informed consent’ form.

Array/Exome CNV analysis

Within our department we annually perform 2500-3000 genome-wide array analyses for the detection of germline and acquired genomic abnormalities (Copy Number Variants (CNVs) and Regions Of Homozygosity (ROHs)).

We have two different array platforms from Thermo Fisher available:

The CytoScan HD array contains more than 2.6 million probes, including 750,000 single nucleotide polymorphism (SNP) probes, and we use this array to examine the patients’ DNA for CNVs and homozygous regions.

The CytoScan XON array contains almost 7 million probes, including 150,000 SNP probes, has excellent exon coverage and is therefore most suitable for the detection of intragenic CNVs, but this array can also be used for genome wide CNV analysis. If necessary, we will also examine the DNA of the respective parents (to perform so called patient-parent trio analysis).

Please note that as of 2022 in most cases the CNV detection will be performed by exome wide CNV analysis in Whole Exome Sequencing data instead of genome wide array analysis. This exome wide CNV in exome data analysis is a validated, good and efficient alternative with a comparable diagnostic yield compared to genome wide array analysis. Furthermore, it is possible in one test to analysis and interpret both CNVs as well as nucleotide variants either simultaneously or sequentially. Nucleotide variants are always first analysed using one or multiple gene panels, depending on the reason for referral.

The array is employed for many indications including:

• Multiple congenital disorders
• Intellectual disabilities
• ‘Homozygosity mapping’ as a screening tool
• Carrier testing in an unaffected individual
• Validation / segregation analysis for intragenic CNVs (instead of MLPA)
• Ultrasound anomalies (see also Prenatal diagnostics)
• Intra uterine foetal death (see also Prenatal diagnostics)
• Hemato-oncological disorders (ALL, CLL, MM, MPN, MDS) (see Tumour genetics)

Using our Electronic Application System  you can request for array diagnostics. The system also includes all information regarding shipping instructions and the expected turn-around times.

Please order array diagnostic requests for hemato-oncological disorders (ALL, CLL, MM, MPN, MDS) using the form of our Tumour Genetics Laboratory.

Gene panels

These following gene panels are widely used in diagnostics using single molecular Molecule Inversion Probes (smMIPs) or Ion Torrent technology, sometimes in combination with Sanger sequencing and MLPA-analysis:

• Breast- and ovarian cancer-panel (smMIPs)
• Cardiomyopathy (smMIPs, conducted by our partner MUMC+)
• Kidney cancer-panel (smMIPs)
• Paraganglioma-panel (smMIPs)
• Paraganglioma/Pheochromocytoma -panel (smMIPs) 
• Polyposis-panel (smMIPS)

By making an application through our Electronic Application System you will be able to see which genes per gene panel are tested, the expected turnaround times and shipping instructions. We also offer various gene panels/exome panels via whole exome sequencing.

Single gene analysis

Per gene, a specific test is performed, ensuring that the coded area and the intron-exon boundaries are comprehensively covered. Depending on which gene it is, a MLPA-analysis might also be performed. Using our Electronic Application System you can see if we offer a specific test for your gene of interest, if that particular gene is part of an existing panel, which biological samples need to be shipped for the test and what the expected turnaround time is. For variant(s) that are identified by whole exome sequencing and for which targeted gene analysis is unavailable, we can offer carrier screening for the relevant variant(s).

Familial mutation

When performing familial mutation screening, several different tests are used by our diagnostics department. We will often make use of Sanger sequencing as a test, but other frequently used test methods are single molecule Molecular Inversion Probes sequencing, fragment analysis, array analysis, chromosome analysis, MLPA and/or deletion PCR. The expected turnaround times for carrier screening is 4 weeks.

In prenatal carrier screening, we work with different turnaround times (see prenatal diagnostics). Ot note, prenatal carrier screening for gene defects also requires a biological sample from the mother in order to exclude the possibility of maternal cell contamination.
 
For presymptomatic carrier screening in children younger than 18 years, the involvement of a clinical geneticist is a requirement.

By using our application form you can apply for carrier screening for a specific gene mutation or chromosome abnormality.

Chromosome analysis

Chromosome analysis is performed for a number of indications, including:

• Habitual abortion
• Suspicion of down syndrome
• Abnormal sexual development, including the suspicion of Turner syndrome or Klinefelter syndrome
• Breakage syndromes such as Nijmegen breakage syndrome, Bloom syndrome, Fanconi anaemia, and ataxia telangiectasia. (please note that due to the necessary preparations needed for a chromosome breakage analysis, please consult with the secretariat before sending samples (gen@radboudumc.nl)
• Carrier screening for family members in case of a known chromosome disorder.
• Echoscopical disorders (including, suspicion of  triploidy, Edwards syndrome, Patau syndrome, Down syndrome)
• Hemato-oncological disorders (please note that these applications go through The Tumour Genetics Laboratory)

Chromosome analysis includes fluorescent quantitative PCR (QF-PCR) for numerical abnormalities of the chromosomes 13/18/21/X/Y.

FISH

Facilitated by the presence of a large collection of FISH-probes we are able to conduct targeted chromosome analysis to identify chromosomal abnormalities. FISH can be performed on dividing cells (which can be extracted from heparin blood, amniotic fluid, chorionic or skin biopsy), also known as metaphase-FISH. It is also possible to conduct FISH on non-dividing cells (e.g. obtained from the mucus membrane in the cheek), also known as interphase-FISH. We do however have a preference for analysis on dividing cells.

FISH is used for several indications within genome diagnostics. FISH can be used for the following:

• A known chromosomal translocation in a family
• In a supplementary role to confirm/exclude the existence of a numerical or structural chromosomal abnormality found using array analysis or karyotyping
• If there is a suspicion of (low) mosaic (sex) chromosomal disorder (such as Turner syndrome and Klinefelter syndrome)

The expected turnaround time for FISH-analysis is 2-5 weeks. In the event that a probe is not present in our database (this is the case for particularly rare (unique) disorders), then the expected turnaround time could be longer.

Tumour genetics

The Tumour Genetics Laboratory (LTG) provides molecular diagnostics within the field of oncology. These diagnostics are aimed at detecting germline mutations that lead to a genetic predisposition for developing certain types of tumour. The LTG also specializes in the detection of tumour specific changes that have been acquired. These changes are of particular importance for differential diagnosis, predicting outcomes or therapy responses.
 
Applications in relation to hereditary tumours (germline mutations) can be submitted using the application system of the genome diagnostics department. By typing the applicable test (e.g. the name of the gene) more information will be provided regarding the expected test result, shipping instructions and whether the relevant gene is present in a gene panel.

Applications in relation to acquired tumours such as haemato-oncological diseases can be submitted to the Tumour Genetics Laboratory.

The Tumour Genetics Laboratory is headed by  prof. dr. Marjolijn Ligtenberg.

Intellectual Disabilities and Congenital Disorders

Within the team ‘Intellectual Disabilities & Congenital Disorders’ both genome wide assays as well as targeted analysis are used to identify the genetic causes explaining the clinical features of patients with Intellectual Disabilities and/or Congenital Disorders or endocrine disorders. These concerns patients of all age groups from newborns to the elderly and with a wide range of clinical indications.

With the exception of genome wide SNP-based array analysis for the detection of causal CNVs (copy number variants) the majority of analyses is whole exome based, involving the detection of de novo variants, often using ‘patient-parent trio analyses’. If necessary, additional analysis is performed to find causal recessive and X-linked chromosomal disorders. Determining the cause of a genetic disorder is of great importance to (extended) families who run a higher risk of recurrence. This is not only important for recessive and X-linked chromosomal disorders, but also for families where one parent is carrier of a balanced chromosomal rearrangement, a mosaic pathogenic variant, or a genetic variant as a result of an imprinting defect, all of which can cause varying clinical consequences. The discovery of the underlying mechanism responsible for causing the genomic disorder will enable better counselling for all parties concerned and help to more accurately determine the recurrence risk.

Our team has particular expertise in the field of genetic causes of Intellectual disabilities (Dr. R. Pfundt and Dr. N. de Leeuw), craniofacial anomalies (Dr. R. Pfundt) and endocrine conditions (Dr. T. Rinne and Dr. D. Westra).

The diagnostic team for Intellectual Disabilities & Congenital Disorders is headed by Dr. Nicole de Leeuw

Muscular-, neurological-, kidney diseases and sensory disorders

Muscular disorders

Muscular disorders consists of number of muscular diseases (e.g. Atrophy, Dystrophy, Myopathy, Myotonia and Muscle weakness) and abnormalities of the neuromuscular system (i.a. Myasthenic syndromes). Diagnostic analysis usually involves a combination of whole exome sequencing, with a primary focus on the genes implicated in Muscular disorders. Targeted gene analysis is possible when dealing with clinically recognisable Muscular disorders with little locus heterogeneity. Muscular disorders caused by repeat expansions (such as Myotonic Dystrophy) will also be subject to targeted analysis.

Neurological disorders

The majority of diagnostic analysis is concentrated on Cerebellar Ataxia, Spastic Paraplegia, Dystonia, and Epilepsy. The analysis itself usually involves a combination of whole exome sequencing with a primary analysis of the genes implicated in Neurological disorders. Targeted gene analysis is possible when dealing with clinically recognisable Neurological disorders with little locus heterogeneity. Syndromes caused by repeat expansions (such as the dominant SCAs) will also be subject to targeted analysis. Scientific research into Cerebellar Ataxia, Spastic Paraplegia and Dystonia will be performed at the centre of expertise for movement disorders. Research into Intellectual disabilities is classified as a separate field of research.

Hearing Loss

Hereditary deafness, or hearing impairment, can be congenital or develop at an older age. There is also a clear distinction between perceptive (sensorineural) deafness (caused by a problem in the inner ear, the vestibulocochlear nerve and/or parts of the brain) and conductive (middle ear) deafness (caused by a problem with the outer ear and/or middle ear). A combination of perceptive and conductive deafness is also possible. In addition, syndromes can occur in which hearing loss is accompanied by other health problems, with Usher syndrome being perhaps the most well-known example.
Diagnostic analysis usually involves a combination of whole exome sequencing, possibly preceded by a single gene  analysis if a single gene is known to harbour many mutations for the disease or syndrome in question (e.g. GJB2 for congenital deafness). Scientific research into hereditary deafness is conducted as part of a collaboration with a centre of expertise “Hearing and Genes”.

Hereditary Metabolic and Mitochondrial diseases

We offer a large variety of genetic tests for the diagnostics of Metabolic and Mitochondrial diseases. Additionally, we also have a comprehensive set of functional assays that can not only be requested prior to genetic diagnostics, but also for further functional follow-up for variants of unknown clinical significance.

Pharmacogenetics

What is pharmacogenetics?
Not all drug therapies have the same effect on every individual. Sometimes a medication will deliver the desired result, on other occasions there might be side-effects or no results at all, meaning that an alternative therapy has to be found. The differences in reaction to drug therapies have a varied set of underlying causes.
Known causes include environmental factors, such as smoking and diet, co-medication or an underlying medical condition. There is however a large percentage of causes that are the result of a genetic predisposition. Only a limited number of genes is involved in drug metabolisms. The primary aim of Pharmacogenetics is to identify which variants in those genes modulate drug response.

Medicines made to measure
Pharmacogenetic diagnostics play a large role in providing therapy perfectly suited to the needs of the individual patient. Testing for known variants that have an effect on the metabolic pathways of medication before starting therapy, is helpful in predicting the reaction to therapy. A genetic variant can cause a medication to be metabolized at a slower rate, increasing the chance of  side-effects. A variant can also cause an increased rate of metabolism which means that the medicine does not remain in the body long enough to have an effect, or alternatively, severely decreases the effect of the medicine. By having tests performed beforehand, you know what you can expect from your therapy. The prevention of side-effects is better than curing them!

Translational genomics

We now have a research group specialized in putting new insights into clinical and diagnostic practice, called “translational genomics”. Within this group, there is a lot of attention given to the clinical utility technology and diagnostic strategy, but there is also a lot of consideration for if the aforementioned can be optimised for more routine applications. There is also careful consideration for the advantages that can be found, compared with more traditional methods when looking at diagnostic success, the costs of health care and patient satisfaction.

The research group for Translational Genomics is headed by Prof. dr. Lisenka Vissers.

Bioinformatics

Within the Bioinformatics group, there are a number of different fields of expertise such as: data management, software-development and -design, automation, server management en statistical methodology.

The group conducts a number of routine analyses including:

• Analysis of whole exome sequencing (WES) data for diagnostic purposes (alignment, variant calling, de novo mutation detection, CNV detection, annotation and quality control)
• Analysis of Molecular Inversion Probe (MIP) data (alignment, variant calling, CNV detection, annotation and quality control)
• Analysis of whole genome sequencing (WGS) data (alignment, variant calling, de novo mutation detection)
• Support for microarray diagnostics  

The Bioinformatics group is also currently developing new analyses such as:

• Analysis of long read technologies (Pacific Biosciences Sequel technology)
• Analysis of RNA-sequencing (transcriptome) data
• Analysis of data from metabolomic experiments  

The Bioinformatics research group also conducts research for the benefit of  patient diagnostics. See also Lelieveld et al. Nature Neurosci (2016).
 
The group for Bioinformatics is headed by Dr. Christian Gilissen.