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Clinical genetic testing: for professionals


Professor Graeme Black discussing about the importance of genetic testing in ophthalmology

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Purpose of genetic testing

Genetic testing is usually undertaken for the following reasons:

  • Diagnostic testing– Establishing a molecular diagnosis in patients with no previous individual/familial results
  • Confirmation testing– Confirming a molecular diagnosis in an affected patient with a known familial variant or confirming a result that was previously obtained from research participation
  • Carrier testing – Testing someone who is usually healthy but may be a carrier of a recessive or X linked genetic condition or of a non-penetrant dominant condition. It is generally carried out to guide family planning for someone who is at risk of having affected children. Depending on the specific population frequency of certain genetic alleles, and on whether there are consanguineous marriages, carriers of certain recessive conditions may be more likely to have affected children
  • Familial segregation analysis—Testing family members to evaluate intrafamilial transmission of genetic variants. For example, confirming that the mutant alleles are in trans in patients affected by autosomal recessive conditions by testing parents or children; Confirming the pathogenicity of a variant of uncertain insignificance in autosomal dominant conditions by testing other affected and unaffected family members
  • Predictive testing—Identifying asymptomatic family members of an affected patient who are at risk of developing the condition themselves  

Genetic testing is a voluntary process where patients should be counselled appropriately beforehand, either by an ophthalmic specialist in genetic eye disease, clinical geneticist or a genetic counsellor about the benefits, limitations, implications and potential ethical concerns associated with testing.

For asymptomatic family members of an affected patient, predictive testing may be helpful in planning for the future but it is a highly personal decision. Individuals wishing to undergo predictive testing need to be counselled as well given the emotional implications associated with a positive result.

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Benefits of genetic testing

  • Eligibility for approved treatment—Patients with biallelic RPE65 mutations can now be treated with a form of sub-retinal gene therapy called voretigene neparvovec (Luxturna)
  • Directing further clinical management—Some patients may harbour variants associated with systemic diseases that require input from other relevant specialties, which in turn may improve life expectancy and/or quality of life  
  • Providing a more accurate prognosis
  • Assist in family planning
  • Facilitating participation in clinical trials
  • Contributing to research if a novel gene/variant is identified

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Limitations of genetic testing

  • Limited information– Sometimes a positive result cannot predict if a person will show symptoms, how severe the symptoms will be or whether the disorder will progress over time; more research is required to establish genotype-phenotype correlations
  • Emotional implications– Stress, sadness, anxiety and guilt (parents may feel they are responsible for their child’s diagnosis)
  • Concerns about the possibility of genetic discrimination in employment or insurance 
  • Variants of unknown significance (VUS) – Variants are classified according to the American College of Medical Genetics and Genomics (ACMG) (and adopted by the Association of clinical Genomic Science [ACGS]) into 5 distinct categories: pathogenic, likely pathogenic, variant of unknown significance, likely benign and benign. A variant is termed a VUS if the criteria are not met to classify it in the other categories or if the criteria for benign and pathogenic are contradictory. Segregation analysis is often needed to help confirm if the identified variants are causal to the patient’s phenotype
Interpretation of variants of unknown significance

A proportion of patients may have an “unsolved” or “negative” genetic result. This means that a genetic mutation was not identified with the applied testing method. This does not exclude that the condition is not due to a genetic cause, but rather it may be because of the following reasons:

  • Limitations of current technology
  • Another testing method that provides more detail may be required
  • Limited knowledge about the causative genes/variants associated with that particular phenotype

Concerns on employment and insurance

Many patients are concerned about how their genetic results may affect employment, mortgage or life insurance. The genetic information that patients need to share with insurance companies are regulated by the ‘Code on Genetic Testing and Insurance’, which is an agreement between the Government and the Association of British Insurers (ABI). The purpose of this Code is to protect individuals from discrimination by insurers based on their genetic testing results.

The Code distinguishes diagnostic and predictive testing. Patients can be asked to disclose results from a diagnostic genetic test as the Code recognises that it is the same as any other diagnostic medical tests (such as a blood test). On the other hand, patients are not required to disclose results of predictive genetic testing, unless an individual has undertaken predictive testing for Huntington disease as part of his/her clinical care and is applying for life insurance over £500,000.  The insurance company will not require nor pressure any applicant to undertake a predictive or diagnostic genetic test, nor ask for, or take into account, the result of any predictive genetic test obtained through scientific research.

In terms of employment, employers are restricted about what they can ask about in pre-employment medical checks by the Equality Act 2010. They can only ask for information that is directly relevant to the applicant’s ability to carry out the work and they are required by law to make ‘reasonable adjustments’ to the workplace to enable a particular person to work there.

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This is based on current UK regulations and might differ in other countries.

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Potential ethical concerns of genetic testing

During the consenting process, genetic counsellors need to discuss with patients the potential ethical issues associated with testing. These include:

  • Pre-symptomatic/predictive testing in children—Parents may want to know if clinically unaffected sibling(s) of the affected child are at risk of developing the same condition in the future. Ethical concerns may arise if parents decide to undertake testing on behalf of the unaffected child, specifically for conditions where there is no effective treatment/where the onset of symptoms typically occur in adulthood (removing the child’s autonomy to decide). The best interest of the child should be the primary reason for undertaking predictive genetic tests, or disclosing predictive results from existing data sets.[1] We do not advocate testing asymptomatic healthy children unless there is a treatment available to them. It is better to advise that testing can be done in adulthood if they choose to and are concerned about family planning
  • Pre-symptomatic/predictive testing in adult—Asymptomatic/mildly affected individuals with a positive result may experience unwanted anxiety
  • Familial implications—Affected relatives of a patient may be pressured by family members to undertake genetic testing instead of making a well-informed autonomous decision
  • Unexpected paternity issues/family relationships which may create tension among family members
  • Data ownership and protection—The massive data generated from genetic sequencing are highly sensitive and can be accessed by multiple professionals (scientists, technicians and clinicians). Breaches in security and/or trust may occur which can lead to malicious use of these personal data. Issues such as data security, access and usage should be discussed thoroughly with patients during the informed consent process
  • Unexpected incidental findings—Results from whole genome sequencing may reveal mutations associated with a separate disease, which can cause significant distress to patients and require assessment by other relevant specialists

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Types of clinical genetic testing

There are various types of testing methods available currently which can be broadly categorised into:

Animation by the New England Journal of Medicine on genetic testing. Please refer to the online article by Adams and Eng 2018 for the full video

Targeted gene panels (part of NGS), and now rarely Sanger sequencing, are the primary route of molecular analysis. Cytogenetic testing may be more suitable for syndromic conditions as a result of copy number variations (CNVs).  

As of 2018, all genetic testing within NHS England are co-ordinated by a single national testing network called the NHS Genomic Medicine Service, which consists of seven genomic laboratory hubs (GLHs) across the country.  Testing for rare ophthalmic conditions are conducted in the following GLHs:

  • London North GLH led by Great Ormond Street Hospital for Children NHS Foundation Trust
  • West Midlands, Oxford and Wessex GLH
  • North West GLH led by Manchester University NHS Foundation Trust

The turnaround time is highly variable. Depending on the type of sequencing technique used and the amount of data generated, it could take at least 6-9 months for results to be returned.

A National Genomic Test Directory for rare and inherited diseases is available to guide clinicians to select the most appropriate tests for eligible patients. Alongside the directory, clinicians/researchers can also use the Genomics England PanelApp to identify genes that are deemed to have adequate evidence by a panel of experts to be included in a diagnostic panel for a specific condition. In departments with limited infrastructure for clinical genetic testing, patients can be referred to tertiary centres with clinical genetics or molecular ophthalmology services for counselling, testing and further management.

Sanger sequencing

This method was first developed by the British scientist Fred Sanger in 1977, where DNA is sequenced using the chain termination technique.[2] The principles of Sanger sequencing can be summarised as follows:

  • DNA unravels into single strands
  • A complementary strand is formed by using a primer as a template and DNA polymerase enzymes to synthesise new complementary nucleotides
  • This strand continues to expand until a dideoxy-nucleotide (chain-terminating nucleotide) is added to the chain, forming a fragment
  • The dideoxy-nucleotide is marked with different coloured dyes depending on the nucleotide (adenine, guanine, cytosine and thymine) it carries
  • This process is repeated several times and fragments of different lengths are produced
  • The resultant fragments are analysed with capillary gel electrophoresis; The shortest fragment will be at the lowest lane
  • The DNA sequence is built one nucleotide at a time based on the detected coloured dyes
Sanger sequencing

It is able to sequence small regions of DNA (single exon/gene) of up to 500 bases in detail. The usual indications for Sanger sequencing are[3]:

  • Molecular confirmation of a clinical diagnosis (suitable for conditions mainly caused by a single gene such as aniridia due to PAX6 mutations)
  • Molecular confirmation in a patient with a known familial variant
  • Segregation analysis when samples from affected and unaffected family members are available
  • Validation of genetic variants identified from NGS techniques
  • Analysis of a mutation “hot-spot” (a region of DNA that is unusually prone to mutations)

The main advantages of this Sanger sequencing are:

  • High-quality sequencing data as only a single DNA region is captured—considered the gold standard of sequencing  
  • Quicker turnaround time (may vary depending on laboratories)
  • Lower cost if only a single gene/DNA region is sequenced  

The main limitations of Sanger sequencing are:

  • Inefficient and costly to diagnose genetically heterogeneous conditions (e.g. inherited retinal dystrophies)
  • Unable to detect large CNVs
  • Unable to identify novel disease-causing genes

Next generation sequencing (NGS)

NGS enables parallel sequencing of multiple targets simultaneously (massive-parallel sequencing technology), making it the ideal primary tool for investigating genetically heterogeneous disorders.[4] There are multiple NGS platforms available commercially, each utilising different sequencing techniques and technologies.[5]

The working principles of NGS can be briefly summarised as follows:

  • The genome is broken down into multiple short DNA fragments (called “reads”)
  • Multiple sequencing reactions of the generated fragments take place at the same time (parallel sequencing)
  • The sequenced fragments are then aligned according to a “reference genome sequence” using bioinformatics software
  • Differences between the aligned DNA sequence and the reference genome are identified (variant calling)
  • The identified variants (up to millions) are filtered for quality and annotated with evidence from multiple resources (genomic sequencing databases, publications, bioinformatic filters, prediction algorithms etc.) to isolate those that are likely to have a pathogenic effect on protein function and associated with the phenotype under investigation
  • Each isolated variant is analysed by clinical scientists to determine its likelihood of causing the investigated phenotype
  • The pathogenicity of the detected variants is reported based on a standardised classification system[6,7]—Pathogenic (Class 5), Likely-pathogenic (Class 4), Variant of unknown significance (Class 3), Likely benign (Class 2) and Benign (Class 1)

There are several NGS options available:

  • Targeted gene panels
  • Whole exome sequencing
  • Whole genome sequencing

All three options differ based on the capture method used to select and enrich the regions of interest in the DNA.

(1) Targeted gene panels

These panels are custom-designed to focus on specific regions (exons and flanking intronic regions) of genes that are associated with a specific clinical phenotype.[8] This is often the primary screening method for genetic eye diseases as large number of known causative genes can be sequenced simultaneously, maximising the chances of identifying pathogenic variants with a single test.

The main advantages of this method are:

  • Cost-effective and efficient for large scale sequencing (ideal for genetically heterogenous disorders)
  • Greater read depth (the number of times a given nucleotide in a genome is read) of the sequenced regions and thus increasing the likelihood of detecting novel and rare variants[5]
  • Less computational and bioinformatic processing, which means less storage space is required
  • The identified variants are more specific to the clinical phenotype under investigation

The main limitations of this method are:

  • Unable to identify novel disease-causing genes
  • Recently discovered novel genes/variants will not be sequenced if the panel is not updated
  • Poor detection of CNVs if breakpoints are outside of the covered regions[8]

(2) Whole exome sequencing (WES)

All the exons and flanking introns of known protein-coding genes in the human genome are sequenced using this technique. It is a relatively powerful tool for the diagnosis of genetic diseases as over 85% of disease-causing variants are located within protein-coding regions even though they only constitute about 1.5% of the genome.[9,10]

The main advantages of WES are:

  • Ability to identify novel disease-causing genes
  • Suitable for patients with previous negative panel studies/those with a complex phenotype with no certain clinical diagnosis[3]
  • Lower cost than whole genome sequencing[5]

The main limitations of WES are:

  • Deep intronic and intergenic variants are not sequenced
  • Does not comprehensively represent genomic structural variants including CNVs[11]
  • DNA regions that are not previously recognised to code proteins will not be sequenced
  • Large amount of data is generated which require ample computer processing power and storage space

(3) Whole genome sequencing (WGS)

This technique sequences the coding and non-coding regions of the whole genome (approximately 3 billion nucleotides and over 20,000 genes).

The main advantages of WGS are:

  • Ability to detect deep intronic, intergenic and structural variants including CNVs
  • Ability to identify novel disease-causing genes
  • Suitable for patients where a molecular diagnosis could not be made on panel and/or WES testing  

The main limitations of WGS are:

  • Higher cost than WES and targeted gene panels[5]
  • Data interpretation is more complex
  • Large amount of data is generated which require ample computer processing power and storage space (more than WES)

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Cytogenetic testing

Cytogenetic testing is used to detect chromosomal abnormalities/CNVs or to validate CNVs identified from NGS techniques.[12] Common cytogenetic testing methods are:

(1) Array-CGH

Array-CGH detects CNVs by comparing the test sample with a reference genome, which is labelled with a different colour. It can detect abnormalities between 100,000 base pairs (100 kilobases [kb]) to 5,000,000 base pairs (5 Mb) and has a high detection rate in patients with syndromic-related ocular conditions.[12,13] The main limitation of array-CGH is its inability to detect balanced chromosomal rearrangements (resulting in no net loss/gain of chromosomal material) such as inversions or balanced translocations.[12]

(2) Karyotyping

It is the process of pairing and ordering all the chromosomes of an organism, giving a gross snapshot of an individual’s chromosomal structure.[14] It is one of the most conventional ways of detecting chromosomal abnormalities. Unlike array-CGH, it can detect balanced rearrangements including inversions and balanced translocations in addition to CNVs. However, it can only detect large chromosomal anomalies with a minimum size of 5-10 Mb.[15]

All 46 human chromosomes are arranged in pairs based on sizes and the genetic materials contained. For each chromosome pairing, we inherit one copy from each parent.

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Further information

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  1.  Royal College of Physicians, Royal College of Pathologists, Medicine BSfG. Consent and confidentiality in genomic medicine: Guidance on the use of genetic and genomic information in the clinic. Report of the Joint Committee on Genomics in Medicine. . July 2019 2019
  2.  Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America. 1977;74(12):5463-5467
  3.  Adams DR, Eng CM. Next-Generation Sequencing to Diagnose Suspected Genetic Disorders. N Engl J Med. 2018;379(14):1353-1362
  4.  Heather JM, Chain B. The sequence of sequencers: The history of sequencing DNA. Genomics. 2016;107(1):1-8
  5.  Salmaninejad A, Motaee J, Farjami M, Alimardani M, Esmaeilie A, Pasdar A. Next-generation sequencing and its application in diagnosis of retinitis pigmentosa. Ophthalmic Genet. 2019;40(5):393-402
  6.  Richards S, Aziz N, Bale S, et al. 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. Genet Med. 2015;17(5):405-424
  7.  Biesecker LG, Harrison SM. The ACMG/AMP reputable source criteria for the interpretation of sequence variants. Genet Med. 2018;20(12):1687-1688
  8.  Patel A, Hayward JD, Tailor V, et al. The Oculome Panel Test: Next-Generation Sequencing to Diagnose a Diverse Range of Genetic Developmental Eye Disorders. Ophthalmology. 2019;126(6):888-907
  9.  Broadgate S, Yu J, Downes SM, Halford S. Unravelling the genetics of inherited retinal dystrophies: Past, present and future. Prog Retin Eye Res. 2017;59:53-96
  10.  Rabbani B, Tekin M, Mahdieh N. The promise of whole-exome sequencing in medical genetics. J Hum Genet. 2014;59(1):5-15
  11.  Biesecker LG, Shianna KV, Mullikin JC. Exome sequencing: the expert view. Genome Biol. 2011;12(9):128-128
  12.  Speicher MR, Carter NP. The new cytogenetics: blurring the boundaries with molecular biology. Nature Reviews Genetics. 2005;6(10):782-792
  13.  Shaw-Smith C, Redon R, Rickman L, et al. Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features. Journal of medical genetics. 2004;41(4):241-248
  14.  O’Connor C. Karyotyping for Chromosomal Abnormalities. Scitable Web site. https://www.nature.com/scitable/topicpage/karyotyping-for-chromosomal-abnormalities-298/. Published 2008. Accessed 7 July 2020
  15.  Smeets DF. Historical prospective of human cytogenetics: from microscope to microarray. Clinical biochemistry. 2004;37(6):439-446

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Updated on November 30, 2020

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