18.03.21 Use of microarray for LD Flashcards
Give a brief introduction to developmental delay.
Developmental delay (DD) or intellectual disability (ID) is characterized by significant impairment of cognitive and adaptive functions, and affects 1-3% of the population worldwide. Chromosomal and genetic disorders account for 30-40% of moderate to severe DD cases, environmental factors account for a further 10-30% but in the remaining 40% the cause is unknown.
Why is it important to provide a diagnosis for developmental delay patients?
Providing a diagnosis in such cases is important: although these patients are essentially incurable, a diagnosis can help provide prognostic information. An accurate diagnosis can help direct clinical care and educational needs, as well as identify appropriate support groups for patients and their families. A diagnosis may also allow for future prenatal diagnosis.
What is the estimated increase in pick-up rate of using aCGH over karyotyping as a front-line test?
Use of arrays increased the detection rate in this cohort was increased from approx 3% with karyotyping to approx 15-20% and represent a 100 fold higher resolution approach.
When might a targeted test for DD patients be requested e.g. FISH. What might cause an issue for this approach?
Whenever genotype-phenotype correlation is high and the patient fits the clinical picture
However, unless phenotype strongly points to a specific condition, a targeted approach can result in sequential additional testing following each negative result – expensive and unhelpful for patients.
Give a brief overview of aCGH.
Each array consists of thousands of immobilized DNA fragments adhered to a slide (array) surface, to allow the hybridization of complimentary sequences between these probes and target DNAs (patient and reference).
Test and reference DNA are labeled with different colors (Cy3 and Cy5) and hybridize competitively with the probes in the array. Differences in Cy3 and Cy5 fluorescence for each spot will indicate loss or gain of material in the respective chromosomal region. The log2 ratios of the test DNA (for example, Cy5) divided by the reference DNA (Cy3) are then plotted against the chromosome position.
What are the disadvantages of aCGH?
Disadvantages of array CGH are that they cannot detect:
- balanced rearrangements
- triploidy
- low level mosaicism
- marker chromosomes are sometimes missed, depending on the size, marker composition, and array coverage of the specific chromosomal region present on the marker.
What are some other applications of microarray technology?
DNA microarrays or ‘chips’ are commonly used to unmask copy number changes, for single nucleotide polymorphism (SNP) genotyping, but can also be used to study DNA methylation, alternative splicing miRNAs and protein-DNA interactions.
What is DGV?
The Database of genomic variants (DGV) provides a catalogue of structural and copy number variation in the human genome
What is DECIPHER?
The Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER): is an interactive web-based database that incorporates a suite of a suite of tools designed to aid the interpretation of submicroscopic chromosomal imbalance
What methods of array follow up are there?
FISH MLPA q-PCR karyotype Parental analysis can be by array (e.g. PvP)
What are the advantages and disadvantages of using FISH as follow up?
Adv - Provides positional information
Disadv - can mismap, difficult to detect enhancements/duplications, aberrations too small for the probe.
What are some of the advantages of using aCGH over karyotyping for diagnosis of LD?
- Identification of new syndromes.
- Higher resolution detection, i.e., unbalanced abnormalities of a few kb in size can be detected using arrays compared to approx. 5Mb using G-band karyotype analysis
* Both CGH and SNP arrays detect amplifications and deletions, but SNP arrays have the ability to also detect mosaicism, extended regions of loss of heterozygosity, uniparental disomy, provide more accurate calculation of copy numbers and determine parental origin of de novo CNVs. - DNA is extracted from uncultured cells therefore can be processed quicker than karyotype analysis in some cases. (This would depend on the workflow of the lab and on which day the sample was received etc.)
- Both array platforms and array analysis can be targeted, i.e., custom arrays or targeted arrays can be purchased depending on needs of the laboratory. Analysis of arrays can be targeted using software applications to consider only specific chromosomes or genomic regions. This would be increasingly important if using array analysis in parents of an affected proband as this approach would limit the chance of unexpected findings.
- Array files can be stored for quick retrieval and analysis in the future for consideration of newly identified genes.
- Arrays may confirm small duplications or deletions at the breakpoints of what looks like an apparently balanced translocation using G-band analysis
How many new syndromes have been estimated to have been made through the introduction of aCGH?
Chromosomal copy number changes are known to be a significant cause of ID/DD, congenital malformation and ASD and the impact of array CGH have allowed the rapid identification and delineation of many new syndromic conditions. Many new microdeletion and microduplication syndromes have been identified in patients with DD; 211 microdeletion syndromes and 79 microduplication syndromes (Weise, A. 2012)
Give a brief overview of the aims of the DDD project.
- Collaboration between the Wellcome Sanger institute and NHS clinical genetic services.
- Provide a unique, online catalogue of genetic changes linked to clinical features that will enable clinicians to diagnose developmental disorders.
- Enable the design of more efficient and cheaper diagnostic assays for relevant genetic testing to be offered to all such patients in the UK and so transform clinical practice for children with developmental disorders.
- In the long term this study will also help improve the understanding of the genetic etiology of developmental disorders.
What methodology was used in the DDD project?
The Sanger Institute perform genetic analysis using both arrays and NGS to analyse all genes (approx 19,000) in order to identify mutations in patients with DD. Focus is mainly on the 1000 genes already identified as causing disorders in children, however the remaining 18,000 genes are also scanned. 12 new disorders have been identified as a result.
The DDD initiative grew out of the groundbreaking DECIPHER database and the study aims to extend the reach of DECIPHER across a broader spectrum of disorders than is currently possible.
DDD project update:
As of October 2014, the Wellcome Trust received over 30,000 samples from 10,000 families. Approx 350 likely diagnoses have been made in the first 1000 families.
