ArrayCGH
Microarray Lead: Mrs Sarah Hewitt
Telephone: 0113 206 5419
Email:
sarah.hewitt@leedsth.nhs.uk
Introduction
Learning and developmental disability (LDD) with or without dysmorphic features or
congenital abnormalities occurs in 1–3% of the general population, with the cause unknown
in over 50% of cases. The approach to genetic investigation in our laboratory has previously
been to perform karyotyping – the gold standard test for detecting chromosome abnormalities
(with a diagnostic yield of 3.7%), and MLPA (increasing the diagnostic yield up to 8%).
Advances in technology mean that array CGH provides a one stop test with a dramatic
increase in the resolution and sensitivity of genetic investigation, negating the need for
numerous additional often uninformative and occasionally invasive tests.
What is array CGH and what are the benefits
Figure of chromosome 3 showing
a loss of chromosomal material
denoted by the red bar.
Array CGH is a technique for detecting abnormalities of genomic copy number. It is a high
resolution whole genome screen which detects genetic imbalances at a greater resolution
(60Kb) compared with karyotyping (5-10Mb), ie a ~1000 fold increase in resolution. This
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increase in resolution detects a higher number of genomic gains and losses (imbalances)
directly related to the patient’s phenotype than can be seen using karyotype + MLPA. Array
CGH also accurately identifies the type and location of the pathogenic genes involved in
chromosome imbalances. This gives more accurate information on the cause of the patient’s
phenotype and enables better patient management. We have shown that array CGH has
increased the abnormality detection rate for this patient cohort to 20-25%.
The karyogram shows chromosomal gains (green bars) and losses (red bars) within the
genome.
Who will be tested by array CGH?
Array CGH was introduced into our laboratory on the 1st April 2012. The following patients
are currently tested using array CGH:
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Patients presenting with unexplained learning difficulties / developmental delay /
behavioural problems (including autism and referrals for the fragile X phenotype).
Patients with dysmorphism / multiple congenital abnormalities suggestive of a
chromosome abnormality.
Direct FISH, karyotyping and QF-PCR will continue for the following referrals:
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Rapid aneuploidy assessment, eg newborn with ?trisomy 21
Ambiguous genitalia/indeterminate gender
Delayed puberty/inappropriate secondary sexual development
Short stature or amenorrhoea in females
Isolated clinical features, eg cleft lip, heart disease
Chromosome breakage syndromes.
Recurrent miscarriage
Infertility
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Professional judgement is used to determine which patients are tested, in line with current lab
policy.
The array CGH service that we are offering is funded for 1,800 new referrals this current
financial year. We are currently unable to routinely offer this testing to patients who have had
previous karyotyping but hope to address this situation in the 2015-2016 financial year.
Those patients who you strongly suspect a chromosomal diagnosis for who have already had
karyotyping performed should be referred to the Clinical Genetics Department at Chapel
Allerton Hospital.
Specimen Requirements
The cytogenetics laboratory will require a minimum of 1ml but preferably 3ml peripheral
blood in an EDTA tube (purple top) and a minimum of 1ml peripheral blood in a Lithium
Heparin tube (green or orange top) with an array CGH referral card completed with
comprehensive clinical details.
If an EDTA blood sample is not received for a patient an array will not be performed.
Samples suboptimal for array CGH testing
A new technique, referred to as CNVseq (Copy Number Variant detection by Sequencing),
was implemented in January 2014 to complement array CGH testing. The method uses a
Next Generation Sequencing approach to look for copy number variation in the genome in a
similar way to array CGH. The technique is used for samples which are considered to be
suboptimal for array CGH due to poor quality/low amount of DNA input. These samples are
tested by CNVseq instead of array CGH, with the same guidelines for analysis and reporting
being applied. Due to the nature of the test, the resolution obtained per case will vary; this
will be stated in the report. Please note that the test is currently performed only once every
month. As a result, the turnaround times may be slightly higher compared to array CGH.
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Implications of an array CGH result
Following internal audit the laboratory anticipates around 25% of cases will have imbalances
detected. These will require some follow-up testing which will be undertaken on the samples
already received by the laboratory where possible. It may sometimes be necessary to request
a repeat sample for follow up studies.
Reports
Once analysed and checked a final or interim report will be issued to the referring clinician as
follows:
-Final Report: No further work required.
-Interim Report: Follow-up work required.
The interim report will then be updated when follow-up studies have been completed. If an
imbalance is detected and confirmed in a proband, parental blood samples will be required to
complete our investigations / determine inheritance. Other studies have shown that overall
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~15% of cases are shown to have de novo imbalances and therefore are almost certainly
pathogenic and explain the clinical phenotype.
Array CGH will also detect imbalances in the genome known as normal ‘copy number
variation’ (CNV’s). The laboratory uses databases and published literature to distinguish
between those deletions and duplications that are normal variation and those that may be
pathogenic in the patient. Only known or potentially pathogenic imbalances will be reported.
Complex results may be telephoned prior to the written report being sent and the
interpretation and implication discussed.
Clinical Advice
Clinical advice is available from the Department of Clinical Genetics, Chapel Allerton
Hospital, Leeds. The team are happy to receive referral letters for patients that have
had abnormal array CGH results and/or where there are clinical implications for other
family members.
Turnaround Times
The laboratory turnaround times are taken from the ACC (Association of Clinical
Cytogeneticists) professional best practice guidelines and are as follows:
Proband (no follow up studies required):
95% within 4 weeks from receipt of a suitable DNA sample.
Parental blood samples requested after detection of imbalance in proband:
95% within 4 weeks from receipt of suitable parental blood samples + follow-up test
materials (eg FISH probes)
Limits to Array CGH
-Array CGH will not detect all genetic abnormalities
-Array CGH cannot detect balanced chromosome rearrangements such as reciprocal
translocations and inversions. Karyotyping will be carried out for patients suspected of these
abnormalities.
-Array CGH cannot detect low level mosaicism (< 30% mixture of abnormal and normal
cells)
-Anomalies may occur which cannot always be recognised or properly interpreted.
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It is local policy to only confirm less well delineated imbalances detected by array CGH by a
second independent technique. Any imbalances well characterised in the literature will be
reported using only the array CGH result. The laboratory currently uses FISH, qPCR, CNV
sequencing or MLPA to confirm array CGH findings where appropriate.
The resolution of these techniques means that we cannot always confirm imbalances smaller
than 150kb. We will therefore only report on imbalances larger than 150kb within a gene
region and 200kb in non-gene regions unless imbalances appear clinically significant.
Frequently Asked Questions
What if I can’t obtain parental blood samples?
We are aware that family circumstances can be difficult and it may not be possible to obtain
parental samples in some cases. Please indicate on the array CGH referral card if in the event
of requiring parental samples it would be possible to obtain them. A report will be issued
even if parental samples are not available.
Why is it important to obtain parental blood samples?
Although we understand that it is not always possible for the clinical area treating the child to
organise parental bloods, interpretation of an imbalance detected on array CGH is less
informative without inheritance studies. If appropriate a referral to clinical genetics should be
made.
Will array CGH detect mutations?
No. The array platform we will be using will detect small genomic imbalances which may
disrupt genes and give a similar phenotype to a mutation but it is not sensitive enough to
detect mutations within genes. Requests for specific mutation testing can be made to the
Leeds DNA laboratory.
What about fragile X testing?
Currently this testing is organised by the Leeds DNA laboratory. These patients will also be
tested by array CGH. It is hoped that sequential testing may be initiated in the 2015-2016
financial year.
What about Prader Willi and Angelman’s Syndrome testing?
Currently this testing is organised by the Leeds DNA laboratory. This will continue to be the
case. Any sample received by the cytogenetics laboratory for PWS or AS testing will be
passed to the DNA laboratory and if appropriate will also have an array test.
Which referral card do I use?
It's the Cytogenetics and DNA Joint referral card which you can download from
What about incidental findings?
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here.
Due to the increased resolution of the screening there may be occasional unexpected
incidental findings. It is important to explain to the parents that the test is a very detailed look
at the chromosomes and occasionally unexpected incidental findings are detected. In the
event of the laboratory detecting a clinically relevant incidental finding, clinical genetics will
be informed and will liaise with the referring clinician.
Can I have a quick result for a neonate?
Array CGH has a turnaround time (TAT) guideline of 28 days, and it will not be possible to
meet the 10 day TAT currently achieved for neonatal karyotyping . Only neonates with 2 or
more congenital abnormalities and dysmorphism will be tested and for these cases no
karyotype will be performed. All other neonates will have karyotyping which will be reported
within 10 days.
I have discussed array CGH with the carers and only karyotyping is required. Is this
possible?
We will not perform array CGH on any cases that specifically indicate array CGH is not
wanted.
VOUS - ‘Information for the clinician’ Please click here for more information
Understanding VOUS
This information is designed to aid clinicians in the interpretation of variants of
uncertain significance and detail how the laboratory deals with imbalances of this
kind. Further information can be obtained from the American College of Medical
Genetics standards and guidelines for interpretation and reporting of postnatal
constitutional CNV’s.
Following array CGH imbalances may occur which cannot always be interpreted with
regards to the clinical significance for this patient.
Copy number variation either loss or gain exists throughout the human genome.
Some of this variation is seen frequently in the general population and appears not to
have any phenotypic effect. Patients with variation of this nature will be described as
having a normal result with no clinically significant abnormalities being identified.
Other copy number variation is well documented as being pathogenic with
established phenotypic features described. Patients with imbalances of this nature
will have reports issued linking their imbalances to their clinical features.
The third category of copy number variation is harder for the laboratory to interpret
and classify. This is because the genetic material within these imbalances may not
appear to be relevant to the patients’ phenotypic features given on the referral card
or the imbalance may be rare and not reported in the general population as a
common variant. This group of imbalances are classed as variants of uncertain
clinical significance of VOUS for short.
The VOUS category is further divided within our laboratory into VOUS likely benign,
VOUS uncertain and VOUS likely pathogenic.
VOUS – Likely benign
These variants are not described in the database which holds information on variants
in the general population and have not been detected in the local population in >1%
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of tested patients. They also contain no OMIM genes. It is likely that these variants
are benign in nature.
VOUS-Uncertain
These variants have disease genes within them although there is no correlation
between the patients’ clinical features on the referral card and /or clinic letter that has
been received by the laboratory. The analysts within the laboratory endeavour to
interpret the array findings from the literature in conjunction with the clinical features
received. When this is not possible these regions will be listed in the report as they
may be of relevance to the patient with regards to other medical conditions they may
have or develop in the future.
VOUS – Likely pathogenic
Copy number variants within this category involve genes that have been tentatively
implicated as causing or contributing to a phenotype or set of features. This evidence
will be newly emerging and there could be conflicting reports. Therefore this category
states a possible link with the phenotype and will recommend frequent review of the
literature.
The information on the reports is recorded so that clinicians who are involved in
patient care can themselves review imbalances and if felt clinically relevant the
laboratory can then be contacted and further validation work can be undertaken.
Please note the laboratory only has funding to validate those imbalances that may
be clinically relevant to the patient and their family. Any copy number variants that
are classified as VOUS are not routinely validated by a second technique.
As with all array findings, should the significance of a copy number variant change,
validation studies would be undertaken and an amended report issued with the
current interpretation.
Clinical advice is always available from the Clinical Genetics Department at Chapel
Allerton Hospital should it be required.
Information for Patients and their Families
Please click
here (19 KB) for more information provided by the Yorkshire Regional
Clinical Genetics Department..
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