Position statement

Genetic and metabolic investigations for

neurodevelopmental disorders: position statement of

J Med Genet: first published as 10.1136/jmg-2022-108962 on 23 February 2023. Downloaded from [Link] on March 3, 2025 by guest.
the Canadian College of Medical Geneticists (CCMG)
Melissa T Carter ‍ ‍,1 Myriam Srour ‍ ‍,2,3 Ping-­Yee Billie Au,4 Daniela Buhas,5,6
Sarah Dyack,7,8 Alison Eaton,9,10 Michal Inbar-­Feigenberg,11 Heather Howley,12
Anne Kawamura,13,14,15,16 Suzanne M E Lewis,17 Elizabeth McCready,18,19

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Tanya N Nelson,20 Hilary Vallance,20 on behalf of the Canadian College of Medical
Geneticists

For numbered affiliations see ABSTRACT NDDs have a combined prevalence of ~17% in
end of article. Purpose and scope The aim of this position statement children aged 3–17 years, making them the most
is to provide recommendations for clinicians regarding common chronic medical conditions encountered
Correspondence to
the use of genetic and metabolic investigations for in paediatric primary care.2 Given their clinical and
Melissa T Carter, Genetics,
Children’s Hospital of Eastern patients with neurodevelopmental disorders (NDDs), aetiological heterogeneity, the cause of NDDs can
Ontario, Ottawa, ON K1H 8L1, specifically, patients with global developmental delay be challenging to diagnose. Comprehensive clinical
Canada; m​ carter@​cheo.​on.​ca (GDD), intellectual disability (ID) and/or autism spectrum care includes investigations aimed at elucidating the
disorder (ASD). This document also provides guidance underlying cause, such as brain imaging and labora-
Received 26 September 2022
Accepted 27 January 2023 for primary care and non-­genetics specialists caring for tory tests, including genetic testing.
Published Online First 23 these patients while awaiting consultation with a clinical Identifying the genetic aetiology of an NDD is of
February 2023 geneticist or metabolic specialist. clinical and personal utility.3 Increasingly, accurate
Methods of statement development A genetic diagnosis can lead to tailored therapy4–8 (and
multidisciplinary group reviewed existing literature may provide information about prognosis that in
and guidelines on the use of genetic and metabolic turn can guide medical care and therapy.8 Definitive
investigations for the diagnosis of NDDs and synthesised diagnosis may avoid unnecessary testing or treat-
the evidence to make recommendations relevant to ments,5 9 10 enable surveillance for known comor-
the Canadian context. The statement was circulated for bidities5 9–16 and help families access additional
comment to the Canadian College of Medical Geneticists disease-­specific support.9 17–19 Accurate diagnosis
(CCMG) membership-­at-­large and to the Canadian of the cause of NDDs facilitates genetic counsel-
Pediatric Society (Mental Health and Developmental ling and informs recurrence risks5 9 10 12 13 16 20 that
Disabilities Committee); following incorporation of guides reproductive decision making and enables
feedback, it was approved by the CCMG Board of prenatal diagnosis.6 When an inherited aetiology
Directors on 1 September 2022. is identified, this can provide diagnoses to other
Results and conclusions Chromosomal microarray family members with NDD.21 Finally, by ending the
is recommended as a first-­tier test for patients with diagnostic odyssey for families, identifying an aeti-
GDD, ID or ASD. Fragile X testing should also be done ology can also provide psychological benefits (eg,
as a first-­tier test when there are suggestive clinical alleviation of guilt, acceptance, closure, empower-
features or family history. Metabolic investigations ment)5 13 17 18 22 23 and improve parental quality of
should be done if there are clinical features suggestive life.24
of an inherited metabolic disease, while the patient The diagnostic process begins with a thorough
awaits consultation with a metabolic physician. clinical assessment of the patient, which includes
Exome sequencing or a comprehensive gene panel is a medical, developmental and family history with
recommended as a second-­tier test for patients with physical examination. The presence or absence of
GDD or ID. Genetic testing is not recommended for certain clinical features may provide clues as to the
patients with NDDs in the absence of GDD, ID or ASD, underlying cause and therefore direct the appro-
unless accompanied by clinical features suggestive of a priate investigations. If a known genetic condition
syndromic aetiology or inherited metabolic disease. is recognised, targeted testing should be done first.
For example, if a 2-­year-­old girl presents with clas-
sical features of Rett syndrome, it is prudent to do
INTRODUCTION MECP2 sequencing prior to other investigations.
© Author(s) (or their
employer(s)) 2023. Re-­use Neurodevelopmental disorders (NDDs) are a group However, it is more often the case that the clin-
permitted under CC BY-­NC. No of conditions first manifesting early in childhood, ical presentation is non-­ specific, and thus there
commercial re-­use. See rights characterised by impairments of personal, social, are a large number of potential genetic aetiologies.
and permissions. Published academic or occupational functioning.1 Exam- It is therefore important to broaden the testing
by BMJ.
ples of NDDs include autism spectrum disorder approach, which could result in earlier diagnosis.
To cite: Carter MT, Srour M, (ASD), global developmental delay (GDD), intel- When a known syndrome is not readily apparent,
Au P-­YB, et al. J Med Genet lectual disability (ID), learning disability (LD) and there are a variety of testing options, including chro-
2023;60:523–532. attention-­
deficit hyperactivity disorder (ADHD). mosomal microarray (CMA), fragile X syndrome
Carter MT, et al. J Med Genet 2023;60:523–532. doi:10.1136/jmg-2022-108962 523
 Position statement
(FXS) testing, testing for a range of inherited metabolic personal independence at home or in the community and
diseases (‘metabolic testing’) and tests that use next-­generation school or work functioning.
sequencing such as multigene panels and exome sequencing (ES). ► LD: also known as specific learning disorder; an NDD char-
Professional organisations from a range of disciplines recom- acterised by deficits in the ability to learn or use founda-

J Med Genet: first published as 10.1136/jmg-2022-108962 on 23 February 2023. Downloaded from [Link] on March 3, 2025 by guest.
mend CMA as a first-­tier test for children with GDD, ID and tional academic skills (reading, writing or arithmetic), which
ASD.25–30 While fragile X testing is generally recommended as is not better accounted for by ID.
a first-­tier test for these NDDs, criteria for testing are inconsis- ► NDD: a group of conditions manifesting early in devel-
tent.25 27 28 31–33 Recommendations vary considerably for meta- opment, characterised by impairments of personal, social,
bolic testing,25–28 32–35 while the use of gene panels and ES is academic or occupational functioning.
relatively new and typically not included in recommendations ► Syndromic: for an individual with an NDD, refers to the
aimed at non-­geneticists. Not surprisingly, a recent survey of presence of additional clinical features such as dysmor-

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Canadian genetic and metabolic physicians found that CMA and phisms, congenital malformations, medical comorbidities
fragile X testing are the most often used tests for patients with etc, which may suggest an underlying genetic cause (refer
NDDs, while the use of metabolic tests, gene panels and ES was to table 1).
highly variable.36 Therefore, Canadian recommendations for
genetic investigation of NDDs are urgently needed. Technical definitions
► Next-­
generation sequencing (NGS): high-­ throughput
DEFINITIONS sequencing technology used to determine nucleo-
Clinical definitions tide sequences and genome dosage at numerous loci
► ADHD: a persistent pattern of inattention and/or hyper- simultaneously.
activity impulsivity that interferes with functioning or ► Genome-­wide sequencing (GWS): sequence analysis of all
development. or a large part of the genome performed by NGS; includes
► ASD: an NDD characterised by persistent deficits in social exome and ES.
communication and social interaction and the presence of – ES: an NGS approach that determines the DNA sequence
restricted, repetitive patterns of behaviour, interests or activ- of most of the protein-­coding exons found in the genome
ities. May be diagnosed with or without accompanying intel- of an individual. As of 2022, this test is available in most
lectual or language impairment. provinces for patients meeting specific clinical criteria.
► Dysmorphic features: visible morphological findings that – Genome sequencing (GS): an NGS approach that deter-
differ from those commonly seen in the general population mines the sequence of most of the DNA content encom-
or same genetic ancestry. passing the entire genome of an individual. As of 2022,
► GDD: an NDD characterised by significant delay (at least two this test is only available on a research basis or through
SD below the mean using standardised testing) in achieving clinical pilots within Canada.
at least two developmental domains, including gross or fine ► Multigene panel: simultaneous sequencing of multiple genes
motor, speech and language, cognition, social and personal associated with a specific clinical presentation.
functioning, and activities of daily living. A diagnosis of ► First-­tier test: a laboratory test or other diagnostic investiga-
GDD applies to individuals under the age of 5 years who tion that should be pursued prior to all others.
are too young to participate in standardised testing to assess ► Second-­tier test: a laboratory test or other diagnostic inves-
intellectual functioning. tigation that should be pursued if first-­tier tests are negative
► IMDs: genetic disorders that result in metabolic defects due or inconclusive.
to deficiency of enzymes, membrane transporters or other ► Metabolic testing: blood and/or urine tests that measure
functional proteins. analytes (such as amino and organic acids) in order to iden-
► ID: an NDD characterised by deficits in intellectual and adap-
tify patterns indicating an inherited metabolic disease.
tive functioning. Intellectual functioning includes reasoning,
problem solving, planning, abstract thinking, judgement, METHODS
academic learning and experiential learning. Adaptive To develop recommendations, the Canadian College of Medical
functioning are the skills needed to live independently Geneticists (CCMG) formed an NDDs working group of physi-
and responsibly and include communication, social skills, cians and clinical scientists from medical genetics and genomics,

Table 1 Clinical features that may be suggestive of a syndromic aetiology for patients with neurodevelopmental disorders
Clinical feature Definitions and/or examples
Dysmorphic features Visible morphological findings that differ from those commonly seen in the general population or same genetic ancestry.
For example, hypertelorism and syndactyly.
Congenital malformations A non-­progressive morphological anomaly of a single organ or body part that is present at birth.
For example, cleft palate, tetralogy of Fallot and polydactyly.
Abnormal head size Occipitofrontal circumference less than or greater than 2 SD from the mean for age, sex and ethnicity.
For example, microcephaly and macrocephaly.
Unexplained growth abnormalities Growth parameters greater than or less than 2 SD from the mean for age, sex and ethnicity, particularly if parental heights are average.
For example, prenatal growth restriction, postnatal failure to thrive, short stature and overgrowth.
Family history consistent with There are similarly affected individuals related to the proband in a pattern suggestive of an autosomal recessive, autosomal dominant or X-­linked
Mendelian inheritance condition
For example, siblings with similar phenotype and consanguineous parents (may suggest autosomal recessive condition); affected males related
through maternal line with similar phenotype (may suggest X-­linked inheritance).
Additional medical comorbidities Medical conditions, particularly when multiple or uncommon, that are not expected to be present secondary to the neurodevelopmental disorder itself.
For example, sensorineural hearing loss, vision impairment, renal disease, epilepsy, ataxia and neuromotor deficits.

524 Carter MT, et al. J Med Genet 2023;60:523–532. doi:10.1136/jmg-2022-108962

 Position statement
developmental paediatrics, paediatric neurology, biochemical severities of impairment or for patients in different age groups
genetics, cytogenetics and molecular genetics. The CCMG is a (paediatric or adult). Evidence was also reviewed for patients
Canadian organisation responsible for certifying medical genet- with other NDDs (eg, ADHD and LD), but there are no high-­
icists and clinical laboratory geneticists and for establishing quality studies that enable distinction between patients with and

J Med Genet: first published as 10.1136/jmg-2022-108962 on 23 February 2023. Downloaded from [Link] on March 3, 2025 by guest.
professional and ethical standards for clinical genetics services without comorbid GDD or ID. Therefore, until such evidence
in Canada. emerges, we do not recommend genetic investigations for
The working group considered laboratory tests currently clin- patients without GDD, ID or ASD unless there are other clin-
ically available in Canada to investigate the aetiology of NDDs, ical features suggestive of a genetic (‘syndromic’; see table 1) or
organised into three subgroups: (1) CMA and FXS testing, (2) metabolic aetiology.
metabolic testing and (3) NGS-­based testing including ES and The recommendations herein apply to probands of all ages.
multigene panels. Each subgroup, in consultation with the The urgency of testing is typically higher for younger children,

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broader working group, identified key questions, conducted particularly if the parents are planning a pregnancy. This does
a literature search, reviewed and discussed the evidence and not preclude testing and/or genetic/metabolic specialist referral
drafted recommendations based on quality and level of evidence. for adolescents and adults, particularly if the patient has not
The working group reviewed all draft recommendations and been tested or evaluated in 5 years or more.
reached consensus. The recommendations herein also apply to probands with
The draft manuscript was reviewed by subcommittees of the ID regardless of severity, as there was insufficient evidence to
CCMG (laboratory practice, clinical practice and metabolics exclude probands from such testing based on level of disability.
committees). It was then circulated for comment to the CCMG Further studies addressing this are required.
membership-­at-­large and, following incorporation of feedback, There may be specific clinical scenarios that demand a different
approved by the CCMG Board of Directors. This position state- approach to that suggested herein, so clinicians should always
ment was also circulated for comment to the Mental Health and use their judgement as to whether the recommended investi-
Developmental Disabilities committee of the Canadian Pediatric gations are appropriate. Patient and family preferences should
Society. always be considered. This position statement is not intended to
be a comprehensive guide to the medical investigation of indi-
viduals with NDDs, and as such, we do not make recommenda-
CONSIDERATIONS tions regarding neuroimaging or health surveillance (eg, thyroid
Target audience and scope screening, vision assessment, etc).
These recommendations apply to patients with NDDs for whom
no specific aetiological diagnosis is suspected after a thorough
history and physical examination. If a specific genetic disorder is The Canadian context
suspected with a high degree of clinical certainty, targeted testing Canada’s population of nearly 39 million is concentrated in the
should be done first, and if negative, then one should proceed southern parts of Ontario, Quebec and Alberta, with relatively
with the testing recommended in this position statement. If sparse population density in the rest of the country. Twenty-­one
no diagnosis is reached after the recommended investigations, per cent of the population lives in rural or remote areas with
periodic re-­evaluation of the patient by a geneticist is recom- limited access to specialist medical care (Canadian Medical Asso-
mended, as testing technology and genomic knowledge is rapidly ciation Policy Statement, 2014). The Canada Health Act stipu-
evolving. The time interval for re-­evaluation is specific to the lates that all Canadians should have universal insurance coverage
patient’s unique situation (eg, age and testing done) and should for medically necessary healthcare service. Healthcare is a
be determined by the geneticist. provincial and territorial responsibility, and nationwide there is
We acknowledge that the demand for genetic consultation is variability regarding the types of genetic and metabolic tests that
high, and waitlists are often long for non-­urgent referrals. Thus, are publicly funded. There is also some variability in the condi-
we provide recommendations for first-­tier testing that primary tions screened for by newborn screening. The most expensive
care and/or non-­ genetic specialist clinicians can order while and resource-­intensive genetic tests, such as ES, are not always
the patient awaits a clinical genetics or metabolic assessment to accessible due to provincial healthcare insurance exclusions
ensure faster access to genetic testing. The non-­geneticist should or limited access to specialists qualified to order and interpret
this testing. The working group has taken these inequities into
have a good understanding of the benefits and limitations of
consideration, and where possible, we have offered alternatives,
first-­tier tests and should obtain informed consent for testing
such as recommending multigene panel testing when ES is not
from the patient or their guardian(s). Should first-­tier testing be
available.
abnormal, a referral to the appropriate specialist is indicated,
and many genetic/metabolic clinics will expedite consultation
for patients referred with an abnormal result. We also provide Limitations
guidance as to which patients should be referred to genetics if The working group formulated the recommendations using
first-­tier testing is not diagnostic. The role of the geneticist is the best available evidence at the time of writing (2022). The
to determine whether second-­tier testing is indicated to ensure quality of the evidence, however, generally falls at a level II–
that the patient and family have an opportunity to discuss the IV (moderate) based on the GRADE system,37 so these recom-
test results and to make recommendations for follow-­up care if mendations should be considered conditional on completion of
a diagnosis is made. higher quality studies. Expert opinion was used to fill in the gaps
The evidence reviewed for the development of this position where evidence was lacking.
statement applies specifically to patients with GDD, ID and/ It was beyond the scope of the working group to conduct
or ASD. The existing evidence was highly heterogeneous with an independent cost-­effectiveness analysis as part of the devel-
respect to the clinical phenotypes of the patients studied, so it opment of this position statement. Recognising that resources
was not possible to make separate recommendations for patients are finite, these recommendations attempt to strike a balance
with and without syndromic features, for patients with different between avoiding over-­testing and the risk of missing a diagnosis
Carter MT, et al. J Med Genet 2023;60:523–532. doi:10.1136/jmg-2022-108962 525
 Position statement

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Figure 1 Summary of first and second tier testing recommendations for individuals with neurodevelopmental disorders presenting for diagnostic
investigations. ADHD, attention-­deficit hyperactivity disorder; ASD, autism spectrum disorder; GDD, global developmental delay; ID, intellectual disability; LD,
learning disability.

that may have treatment or recurrence risk implications for the The diagnostic yield of fragile X testing varies between studies,
patient and their family. based on the study design and characteristics of the study popu-
Finally, input from patients and their families was not solicited lation. Taken together, the sex-­specific prevalence of FXS in
in the development of these recommendations, but for future individuals with NDD is 2.2%–2.5% for males and 1.3%–1.6%
iterations of this statement, this will be an important addition. in females.38 47–53 Retrospective reviews of laboratory databases
The sections further will briefly summarise the evidence for demonstrate a diagnostic yield of 0%–2.5% for FXS testing.54–57
each test modality and list recommendations for use of the test as The diagnostic yield of FXS testing increases (9.5%–17%) when
first or second tier for patients with NDDs. It should be under- inclusion criteria are restricted to males with NDD and char-
stood that first tier tests are, for the most part, able to be ordered acteristic physical and behavioural features or family history
by any qualified clinician and therefore should generally be done suggestive of FXS.58–60 Use of a diagnostic ‘checklist’ to deter-
prior to (or at the same time as) referral for genetics consulta- mine likelihood of FXS in males generally has high sensitivity,
tion. Figure 1 summarises the recommendations and provides but only if a low threshold is used.58 60–64 There is insufficient
guidance as to when a patient should be referred to genetics or data to determine whether this approach yields a higher diag-
metabolics for further evaluation. nostic rate in females with NDDs.60–62 No studies have evaluated
the cost-­effectiveness of selective FXS testing based on clinical
Fragile X testing (FMR1 CGG repeat analysis) criteria.
FXS) is an X-­linked condition caused by the unstable expansion
of a CGG repeat in the 5′UTR of the FMR1 gene and subsequent
Recommendations for fragile X testing
hypermethylation, preventing gene expression. It is a common
1. FXS testing is recommended as a first-­tier diagnostic test for
monogenic cause of NDDs, with a prevalence of 1.4 per 10 000
individuals presenting with:
males and 0.9 per 10 000 females.38 FXS testing is widely avail-
– GDD, ID or ASD and a clinical presentation or family
able and can be ordered by a variety of clinicians. FXS cannot, at
history suggestive of FXS (see table 2).
this time, be diagnosed by CMA or ES.
– Any NDD and a family history of FXS or other FMR1-­
The ‘classic’ FXS phenotype in males includes moderate-­to-­
related disorder.
severe ID, macro-­orchidism and distinctive facial features (long
2. FXS testing is not recommended for individuals with GDD
face, large ears and prominent jaw).39 Common comorbid condi-
or ID who do not meet the above criteria and have a com-
tions include ASD, anxiety and hyperactivity.40 Deviation from
plex clinical presentation that is not consistent with FXS
the classical phenotype is influenced by age and sex (with females
(eg, multiple congenital anomalies, profound neurological
and prepubescent children manifesting fewer physical character-
impairment).
istics) and the presence of DNA methylation or repeat size mosa-
icism.41 The phenotype in females is highly variable, with IQ
ranging from normal to moderate ID, likely due to the effects of
random X-­inactivation.42 FXS has a high recurrence risk in fami- Chromosomal microarray
lies, as children with a full mutation have mothers with a premu- CMA uses comparative genomic hybridisation or SNP array
tation that confers a risk for expansion in the oocyte,43 or with a to detect gains and losses of chromosomal material, known as
full mutation. If the diagnosis of FXS is delayed, parents may not CNVs. SNP-­based arrays are also able to detect areas of long
have the opportunity to have prenatal diagnosis in subsequent contiguous stretches of homozygosity, which may suggest
pregnancies, which may result in multiple affected children.44 parental consanguinity or uniparental disomy. While CMA can
For these reasons, a variety of professional organisations recom- detect CNVs as small as approximately 20–50 kb,29 resolution
mend FXS testing as a first-­line diagnostic test (concurrent with varies depending on the technology used and density of DNA
CMA) for GDD, ID and ASD.27 45 46 These recommendations probes. CMA is widely available and can be ordered by a variety
were largely based on expert opinion and/or published prior to of clinicians.
publication of larger studies examining the diagnostic yield of CNVs can be classified as pathogenic, likely pathogenic, uncer-
NGS-­based testing for NDDs. tain significance, likely benign or benign.65 CNVs of uncertain
526 Carter MT, et al. J Med Genet 2023;60:523–532. doi:10.1136/jmg-2022-108962
 Position statement
absence of biochemical markers that lead to a diagnosis of IMD.
Table 2 Clinical features that may be suggestive of fragile X
While individually rare, there can be clinical value in diagnosing
syndrome
IMDs: for some disorders, early identification and treatment can
Proband history and physical exam Family history improve outcomes, and most of the conditions are autosomal

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Macro-­orchidism (may not be present until  In relatives on the maternal side: recessive and therefore have a high recurrence risk. According
after puberty). ► Males or females with GDD, to a systematic literature review, over 100 IMDs with ID as a
Relative or mild (+2–3 SD) macrocephaly. ID or ASD. major feature have potential treatments.80 81 Examples of IMDs
Facial features: large or prominent ears, long ► Females with premature causing NDD that have disease-­modifying treatments are urea
or narrow face, tall forehead, high-­arched menopause or ovarian
cycle disorders,82 83 maple syrup urine disease,82 homocystin-
palate and prominent jaw. insufficiency.
Connective tissue findings: soft or velvety ► Males or females with
urias, including cobalamin-­ related conditions,84–86 and some
creatine disorders including guanidinoacetate methyltransferase

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skin, redundant skin on dorsum of hands, adult-­onset tremor, ataxia or
hyperextensible joints, pes planus and mitral parkinsonism. deficiency.82 84 86 87
valve prolapse. Some authors suggest that all individuals presenting with
Behaviour: ASD or autistic features, GDD/ID/ASD should be screened for IMDs,27 28 32–35 88 while
hyperactivity, shyness, gaze avoidance, hand others recommend screening only for individuals with additional
biting, tactile defensiveness and anxiety.
clinical features suggestive of an IMD.25 26 84 There is also a lack
ASD, autism spectrum disorder; GDD, global developmental delay; ID, intellectual of consensus among experts as to which metabolic investigations
disability.
comprise a comprehensive screen for IMDs. Indeed, there is
wide variability in Canadian clinical practice.36
Both the Canadian Pediatric Society27 and the American
significance require interpretation from a clinical geneticist and Academy of Pediatrics28 reference a systematic literature
may warrant additional investigations. CMA may detect ‘suscep- review80 as the rationale to screen for treatable IMDs in all chil-
tibility CNVs’: recurrent CNVs with variable expressivity and/or dren presenting with GDD/ID; however, the diagnostic yield of
incomplete penetrance.66–68 The phenotype resulting from these this approach was unknown at the time. The protocol suggested
CNVs is variable and likely influenced by other genetic and envi- by the Treatable Intellectual Disability Endeavor (TIDE) (www.​
ronmental factors. Detection of these variants in an individual [Link]) is extensive; yet, not all the tests in this protocol are
with a neurodevelopmental disorder may not entirely explain widely available, and clinical utility and cost-­effectiveness have
the observed phenotypes, so additional investigations may be not been evaluated.
warranted; however, this is not well studied. The reported diagnostic yield of metabolic testing for patients
A variety of professional organisations recommend CMA as with NDDs can vary depending on the population studied.
a first-­tier test for individuals with GDD, ID or ASD,27 28 32 33 69 However, using the best evidence available (more recent publi-
based on its high diagnostic yield compared with standard karyo- cations in populations with expanded newborn screening, larger
type.29 70 In general, the diagnostic yield of CMA is lower in case series with clear case definitions), the overall diagnostic yield
studies in which the probands have mild ID (12%–19% vs of metabolic testing for patients with NDDs is 0.25%–0.42% for
20%–30% for moderate to severe ID71–73) or ASD without GDD/ID in non-­consanguineous populations.84 87 Retrospective
syndromic features (4%–5% for non-­syndromic ASD vs up to reviews of the medical records of patients with diagnosed IMDs
25% for those with syndromic features74 75). show that most of these patients have clinical features suggestive
There are no studies that investigate the diagnostic yield of of an IMD in addition to GDD/ID.84 87 89 90 In a prospective study
CMA for isolated speech or motor delay or LD. Although three by Campistol et al83 in 2016, an IMD diagnosis was rarely iden-
studies report a detection rate of 8%–9% for rare CNVs in indi- tified in children with non-­syndromic ASD. A review of the pilot
viduals with ADHD,76–78 they lack sufficient clinical information implementation of the TIDE protocol for patients with GDD/
about the probands to determine whether ADHD was isolated ID, with and without suggestive neurological features,34 demon-
or associated with GDD/ID or syndromic features. Furthermore, strated no significant increase in diagnoses of IMD during time
in these studies, many of the detected rare CNVs were inherited periods before and after implementation despite a greater than
and the pathogenicity and clinical impact of these CNVs were fourfold increase in test volumes.84 Metabolic testing early in the
unclear. Another study found that children with isolated ADHD diagnostic process should theoretically improve ‘time to diag-
had similar detection of rare CNVs compared with controls.79 nosis’ and clinical outcomes for IMDs not detected by newborn
There is strong evidence for the utility of CMA as a first-­tier screening; however, this has not been rigorously studied.
test for individuals with GDD, ID or ASD. There is insufficient Reviews, commentaries and guidelines that quote diagnostic
evidence for the utility of CMA as a first-­tier test for other NDDs yields of metabolic investigations for NDDs as high as 5%
such as ADHD without GDD, ID or ASD unless there are other should be interpreted with caution. Literature examining the
clinical features suggestive of a syndromic aetiology (see table 1). diagnostic yield of metabolic testing for patients with NDDs is
generally of low quality due to lack of peer review, small sample
Recommendations for CMA testing sizes, variability in tests performed and lack of robust case defi-
1. CMA is recommended as a first-­tier diagnostic test for indi- nitions. Furthermore, some publications pre-­date the expansion
viduals presenting with: of newborn screening to detect more treatable IMDs.87 Although
– GDD, ID or ASD. most children with treatable IMDs are now diagnosed by routine
– Other NDDs when syndromic features are present (see provincial newborn screening programmes in Canada, newborn
table 1). screening cannot detect all treatable IMDs, neonatal screening
programmes vary among provinces/territories and false-­negative
Metabolic testing screens are possible.27 84
Inherited metabolic disorders (IMDs) comprise a group of For children with NDDs, most IMD diagnoses are made in
genetic conditions that can present with NDDs as part of the those with clinical features such as regression or plateauing
clinical spectrum. Metabolic testing can detect the presence or of development, acute encephalopathy or altered level of
Carter MT, et al. J Med Genet 2023;60:523–532. doi:10.1136/jmg-2022-108962 527
 Position statement

Table 3 Clinical features suggestive of inherited metabolic disorders (IMDs) and suggested metabolic testing that could be done while awaiting
metabolics (or other specialist) consultation
Targeted metabolic testing
If URGENT, clearly indicate on the requisitions when

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Clinical feature Major groups of IMDs to consider ordering tests
Developmental plateau or regression in the context of an LSD, peroxisomal disorders (X-­ALD), UCD, HCYS Ammonia, blood gas, lactate, PAA, UOA, TPH, urine MPS,
abnormal neurological exam VLCFA†
Altered level of consciousness, especially if episodic; stroke-­like UCD, MSUD, HCYS, organic acidurias, mitochondrial disorders Ammonia, blood gas, glucose, lactate, electrolytes, anion gap,
episodes PAA, TPH, ACP, UOA
Movement disorder (ataxia, dystonia, choreoathetosis, Organic acidurias, HCYS, creatine disorders, LSD, Wilson Blood gas, lactate, glucose, ACP,
myoclonus, tremor) disease PAA, UOA, TPH, urine MPS, creatine panel†, copper,

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ceruloplasmin
MRI/MRS brain abnormality (eg, white matter change, Peroxisomal disorders, organic acidurias, LSD Metabolic testing tailored to MRI findings by metabolic/
abnormal cerebellum / basal ganglia) genetic specialist
Hepatomegaly, splenomegaly* LSD, peroxisomal disorders Urine MPS (if other systemic abnormalities noted*), VLCFA†
Specific food aversions: avoiding high protein foods UCD, MSUD and organic acidurias PAA, ammonia, UOA, ACP
Ophthalmological findings (most common: cataracts, Galactosaemia, Lowe disease, sulfite oxidase deficiency, HCYS, Metabolic testing tailored to ophthalmological findings by
dislocated lens, corneal depositions, retinopathy, cherry red LSD, peroxisomal disorders metabolic/genetic specialist
spot)
Seizures: drug resistant, myoclonic or neonatal UCD, organic acidurias, HCYS, creatine disorders, vitamin-­ Ammonia, blood gas, glucose, lactate, electrolytes, anion gap,
dependent epilepsies, peroxisomal disorders, Menkes disorder PAA, UOA, TPH, creatine panel†,
VLCFA†
Abnormal tone (hypotonia or spasticity) Organic acidurias, HCYS, creatine disorders, UCD (eg, arginase Ammonia, blood gas, glucose, lactate, electrolytes, anion gap,
deficiency and HHH), biotinidase deficiency, peroxisomal PAA, UOA, TPH, VLCFA†, creatine panel†
disorders, LSD
Coarse facial features and/or skeletal abnormalities on X-­ray* MPS and other storage disorders Urine MPS
Multisystemic involvement LSD, mitochondrial disorders, peroxisomal disorders, CDG, Metabolic testing tailored to specific findings by metabolic/
SLOS genetic specialist
X-­linked ALD (adrenoleukodystrophy).135–137
Highly specialised tests that are not recommended for initial workup are not included.
*For mucopolysaccharidoses (MPS), look for noisy breathing, short stature, macrocephaly and sensorineural hearing impairment. For MPS III (Sanfilippo syndrome), aggressive behaviour, sleep
disturbance and hirsutism may be early features.
†Test ordering may be restricted to certain specialists.
ACP, acylcarnitine profile; CDG, congenital disorders of glycosylation; HCYS, homocystinuria and remethylation/cobalamin disorders; HHH, hyperornithinaemia hyperammonaemia homocitrullinuria
syndrome; LSD, lysosomal storage disorder; MRS, Magnetic resonance spectroscopy; MSUD, maple syrup urine disease; PAA, plasma amino acids; SLOS, Smith-­Lemli-­Opitz syndrome; TPH, total
plasma homocysteine; UCD, urea cycle disorder; UOA, urine organic acids; VLCFA, very long chain fatty acids.

consciousness, movement disorders, intractable seizures, multi- – GDD, ID, or ASD and clinical features suggestive of an
systemic involvement, specific ophthalmological findings, IMD. Metabolic testing should be tailored to the presen-
organomegaly and/or features suggestive of a storage disease. tation (see table 3) and a referral made to a metabolic
Patients with such ‘red flags’ should be referred for specialist specialist promptly.
consultation without delay. Table 3 provides guidance on addi- 2. Metabolic testing is not recommended for individuals pre-
tional investigations that should be ordered, where possible, for senting with ASD/GDD/ID without suggestive features unless
patients with these features while they await consultation. newborn screening was not performed or the test panel was
Given its very low yield, routine metabolic testing is not not comparable with standard provincial programmes (eg,
recommended for patients with GDD/ID/ASD without sugges- newcomers to Canada).
tive clinical features listed in table 3. Rarely, a treatable IMD
may be missed by not performing metabolic testing for such ES and multigene panels
patients; for example, patients with homocystinurias, including Massively parallel ‘next-­generation’ sequencing technology has
cobalamin-­ related conditions, can occasionally present with allowed for the simultaneous analysis of hundreds to thousands
isolated NDDs.85 However, these conditions are very rare, and of genes from a single DNA sample. Currently, ES is widely
those with time-­sensitive treatments are detected by newborn available as a clinical diagnostic test. An alternative to ES is the
screening. Creatine transporter disorder, which is X-­ linked, use of comprehensive multigene panels, which are targeted to
may rarely present with isolated NDD.84 90 91 Treatment does a specific phenotype. For NDDs, commercially available panels
not alter clinical outcomes for this condition; however, diagnosis vary widely in the number of genes analysed; the largest ones
might be helpful for future family planning. include over 2500 genes.92 In future, GS is poised to replace
Clinicians should be aware of the conditions tested for via both ES and CMA, but as it is not yet publicly funded in Canada,
newborn screening in their jurisdiction. Children who have not these recommendations consider the use of panels and ES only.
had newborn screening may be at increased risk for having an
The main advantage of ES and large panels is the ability
undiagnosed IMD, so clinicians should determine what newborn
to simultaneously interrogate large numbers of genes, which
screening was done and, if limited, consider referring to a meta-
means that the clinician need not have a particular genetic
bolic specialist.
condition in mind to direct the diagnosis. However, they have
important limitations that means that a negative test does
Recommendations for metabolic testing not rule out a genetic cause. Depending on the methodology
1. Metabolic testing is recommended as a first-­tier diagnostic used, all coding exons may not be completely sequenced and,
test for individuals presenting with: thus, some variants may be missed.93 ES is less able to detect
528 Carter MT, et al. J Med Genet 2023;60:523–532. doi:10.1136/jmg-2022-108962
 Position statement
mosaicism or exon-­level deletions compared with GS or panels Clinical utility of ES for individuals with NDDs
that include deletion/duplication analysis.94 ES and panels do The clinical utility of ES for the diagnosis of patients with
not reliably assess repetitive DNA sequences (such as trinucle- suspected monogenic disorders has been well established. A
otide repeats), intronic or non-­ coding variants, methylation, 2020 Ontario Health Technology Assessment examined the

J Med Genet: first published as 10.1136/jmg-2022-108962 on 23 February 2023. Downloaded from [Link] on March 3, 2025 by guest.
epigenetic or mitochondrial DNA variants or balanced chromo- clinical utility of ES in unexplained developmental disabilities
somal rearrangements.94 and multiple congenital anomalies, concluding that ES allows
These tests can identify multiple rare variants in a patient’s for changes in clinical management, provides insight into the
sample, the clinical relevance of which can be challenging to natural history of the patient’s condition, informs reproductive
decipher. They can identify misattributed parentage (when done planning and optimises the patient’s ability to access disease-­
as a ‘trio’ with both declared parents),95 96 as well as incidental specific supports.99 Similar findings were reported in a 2020
and secondary findings (pathogenic variants in medically action- ACMG systematic evidence review that examined the health,

Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies.
able genes unrelated to the indication for testing).97 Further- clinical, reproductive and psychosocial outcomes resulting from
more, they can identify misattributed parentage (when done, ES for the investigation of congenital anomalies, developmental
as they often are, as a ‘trio’ with both declared parents) and delay or ID.125 It also considered possible negative impacts such
secondary findings (pathogenic variants in medically actionable as insurance discrimination, financial burden and psychological
genes unrelated to the indication for testing).95 96 Testing of the impact on patients and found that return of ES test results incur
proband only (vs proband-­parent trio) results in a lower diag- no clinically significant psychological harms with low levels of
nostic yield98 99 and more labour-­intensive downstream analysis test-­related distress and positive psychological effects. Genetic
counselling and an appropriate consenting process can help miti-
and/or additional testing to resolve variants, so a ‘trio’ approach
gate any associated risks.103
is the most useful, time-­efficient and informative approach.98 100
Given these considerations, these tests require robust pretest and
post-­test genetic counselling and input from a clinical geneti- Cost-effectiveness of ES
cist.97 101 The significant cost of ES has historically been a barrier to its
ES and comprehensive multigene panels were not widely implementation within the Canadian healthcare system as
available as routine clinical tests when the practice guidelines standard-­of-­care testing for NDDs. Recent evidence shows that
for genetic testing for patients with NDDs were published by the performing ES early in the diagnostic testing pathway is cost-­
American Association of Neurology in 201126 and the American effective,99 113 126–131 although other studies have been inconclu-
Association of Pediatrics in 2014.28 However, a 2015 CCMG sive.132–134 In Ontario, a 2020 cost analysis of ES testing before
position statement recommended clinical GWS (either exome or concurrently with CMA for patients with developmental
or genome sequencing) for patients with moderate-­ to-­
severe disabilities supported ES as a second-­tier test (when CMA is non-­
or syndromic ID, when appropriate first-­ tier genetic testing diagnostic) as the most cost-­effective approach99; this study is
(such as CMA) is non-­diagnostic.102 The 2021 ACMG practice based on ES analysis that does not include duplication/deletion
guidelines103 strongly recommend that GWS be considered as a analysis.
first-­tier or second-­tier test for paediatric patients with develop- In summary, the evidence strongly supports using ES as a diag-
mental delays and/or ID. nostic test for patients with ID/GDD due to its high diagnostic
yield, cost-­effectiveness and demonstrable clinical utility. The
diagnostic yield for patients with GDD/ID is significant even
Diagnostic yield of ES for patients with NDDs in the absence of syndromic features. For patients with ASD
The reported diagnostic yield of ES for patients with GDD/ID without GDD/ID, there is insufficient data to make evidence-­
demonstrates wide variation (8%–68%),9 16 74 98 103–118 likely based recommendations, although evidence suggests a higher
owing to differences in cohort size and composition, whether diagnostic yield for patients with syndromic features (table 1). A
ES was performed clinically or under a research protocol and trio-­based testing approach is recommended. While ES provides
whether a proband-­parent trio or proband-­only approach was a higher diagnostic yield, a comprehensive multigene panel for
used. When considering the largest studies from clinical labora- NDDs is a suitable substitute when ES is not available, especially
tories,105 106 the diagnostic yield of ES is 26%–31%, consistent when done as a trio-­based test. When GS becomes a widely avail-
with results from a meta-­analysis that showed pooled estimates able clinical test, it is anticipated to replace fragile X, CMA and
of 29%.104 It is difficult to tease out the diagnostic yield in ES as a single test of choice for NDDs.
specific patient subgroups (such as ASD without GDD/ID, or In most cases ES is recommended as a second-­tier test after
GDD/ID without syndromic features). A few studies dedicated CMA, as the latter has a high diagnostic yield, is more widely
available, less costly and can be ordered by non-­ geneticists.
to patients with ASD, most of whom had ID (with or without
However, it may be appropriate to do GWS before or concur-
other features), reported that ES had a diagnostic yield of
rently with CMA; for example, when the proband is highly likely
8%–25.8%.74 106 109–111 119 The few studies that have compared
to have an autosomal recessive condition (based on parental
the diagnostic yield of ES for GDD/ID patients with and without
consanguinity and/or affected siblings), or when a diagnosis
syndromic features have not found statistically significant differ-
must be rapidly obtained, such as to inform the management
ences.107 120 121
of an ongoing pregnancy (for a sibling of the proband), or to
guide medical care in a patient who is acutely ill or whose clinical
Diagnostic yield of NGS panels for patients with NDDs status is deteriorating (eg, neurological regression).
A few studies have examined comprehensive panels for patients
with NDDs, reporting a diagnostic yield of 11%–39%.118 122–124 Recommendations for ES or multigene panels
Two studies directly compared a targeted NDD gene panel to 1. Recommended as a second-­tier diagnostic test for individuals
ES, and both demonstrated slightly lower diagnostic yields for presenting with:
the panel117 120 – GDD or ID (with or without ASD).
Carter MT, et al. J Med Genet 2023;60:523–532. doi:10.1136/jmg-2022-108962 529
 Position statement
– ASD and/or other NDD and clinical features suggestive Patient consent for publication Not applicable.
of a syndrome (see table 1). Ethics approval Not applicable.
Provenance and peer review Not commissioned; externally peer reviewed.
CONCLUSIONS Open access This is an open access article distributed in accordance with the

J Med Genet: first published as 10.1136/jmg-2022-108962 on 23 February 2023. Downloaded from [Link] on March 3, 2025 by guest.
Patients with NDDs in Canada require a thorough evaluation for Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which
the underlying cause of their condition. The CCMG Neurode- permits others to distribute, remix, adapt, build upon this work non-­commercially,
velopmental Disorders working group recommends an approach and license their derivative works on different terms, provided the original work is
properly cited, appropriate credit is given, any changes made indicated, and the use
to the diagnostic evaluation that is tailored to the patient and
is non-­commercial. See: [Link]
based on current evidence. These recommendations are based
on best available evidence as of 2022 and may evolve as new ORCID iDs
evidence emerges. Therefore, they should be reviewed every Melissa T Carter [Link]

Protected by copyright, including for uses related to text and data mining, AI training, and similar technologies.
Myriam Srour [Link]
2–3 years.
An overview of these recommendations are found in figure 1.
We recommend a tiered testing strategy, such that first-­tier testing
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