Laboratory Testing for Developmental Delay, Intellectual Disability, and Autism Spectrum Disorder

Developmental delay (DD) is defined as any significant lag in a child's physical, cognitive, emotional, or social maturity. Intellectual disability (ID) is characterized by broad impairment in cognitive and adaptive functioning, typically with an intelligence quotient (IQ) <70 diagnosed before 18 years of age. A global DD diagnosis often precedes a diagnosis of ID, as cognitive skill or IQ cannot be reliably assessed before age 6. Those with severe DD diagnosed before age 6 are most likely to develop ID.  

Autism spectrum disorders (ASDs) represent a neurodevelopmental continuum with varying degrees of social impairment, communication limitations, repetitive behaviors, and/or a restricted range of interests. ASDs are typically detected by 3 years of age based on parents’ and observers’ identification of abnormal interactions and behaviors.  Routine pediatric screening may identify a child with global delay and spur investigation of the underlying etiology.

Chromosomal microarray (CMA, also referred to as cytogenomic single nucleotide polymorphism [SNP] microarray), is the recommended first-line test for DD/ID or ASD of unknown etiology.    CMA offers the highest diagnostic yield (~15-20%) in individuals with unexplained DD/ID, ASD, and multiple congenital anomalies (MCAs), and is preferred to chromosome analysis (karyotyping).    ,  Multiple groups have made recommendations regarding additional testing for fragile X syndrome (FXS), inborn errors of metabolism (IEMs), X-linked intellectual disability, MECP2-related disorders, PTEN-related disorders, and/or chromosome analysis, depending on clinical findings and family history.     Refer to the Testing for Genetic Syndromes Related to Developmental Delay, Intellectual Disability, and Autism Spectrum Disorder Algorithm for suggested tiered testing approaches.

Quick Answers for Clinicians

Which genetic test provides the highest diagnostic yield for developmental delay, intellectual disability, and autism spectrum disorders of unknown cause?

Chromosomal microarray (CMA) is the recommended first-tier diagnostic test for patients with developmental delay (DD), intellectual disability (ID), or autism spectrum disorders (ASDs) of unknown etiology. The diagnostic yield varies by patient population and the presence of comorbidities, but is estimated to be ~15-20% (~10% higher than the detection rate by karyotype analysis in the DD/ID/ASD population).  If clinical features or family history suggest a specific disorder, testing for that disorder before proceeding with CMA is recommended.    ,  Refer to the Testing for Genetic Syndromes Related to Developmental Delay, Intellectual Disability, and Autism Spectrum Disorder Testing Algorithm for more information.

What are the advantages and disadvantages of chromosomal microarray compared with G-banded chromosome analysis (karyotyping)?

The main advantage of chromosomal microarray (CMA), and the reason for the shift to CMA instead of karyotype, is its superior resolution. CMA is able to identify submicroscopic deletions and duplications (less than ~10 Mb, the size of many of the deletions and duplications that lead to developmental delay/intellectual disability/autism spectrum disorders [DD/ID/ASDs]) that cannot be detected by karyotype, which leads to a higher detection rate for patients with DD/ID, ASD, and multiple congenital anomalies (MCAs) of unknown etiology. This higher detection rate justifies the increased cost of CMA and explains why CMA is recommended as a replacement for chromosome analysis rather than as follow-up testing after a normal chromosome analysis result. Another benefit of CMA is that it will also identify regions of homozygosity, which can be scrutinized for autosomal recessive conditions and imprinting disorders. Despite these advantages, CMA (with a blood specimen) takes more time than stat chromosome analysis, does not detect balanced rearrangements, and does not characterize the location of detected copy number gains. Therefore, chromosome analysis is still preferred for some indications (eg, multiple miscarriages and suspected aneuploidy).

Should other testing be performed after, or in addition to, chromosomal microarray?

Multiple guidelines recommend FMR1 analysis for fragile X syndrome (FXS) (see FMR1 Testing for Fragile X Syndrome) in addition to chromosomal microarray (CMA). Based on clinical and family history as well as a dysmorphology exam, recommended testing may also include metabolic testing, X-linked disorder testing, and/or targeted testing for a specific suspected condition. Multiple guidelines recommend evaluation for inborn errors of metabolism (IEMs), which have a low diagnostic yield but significant benefit for patients. Finally, testing for MECP2-related or PTEN-related disorders may be indicated in addition to CMA (see Laboratory Testing). Neither cytogenomic single nucleotide polymorphism (SNP) microarray (CMA) nor chromosome analysis will detect sequence variants, triplet repeat expansions (as in fragile X syndrome), copy number variations (CNVs) outside of probe coverage, heterodisomy uniparental disomy, or some other etiologies.     CMA will miss exon-level deletions and duplications. Therefore, additional and/or concurrent testing may be needed when a patient has DD/ID and/or ASD of unknown etiology. For more information on the limitations of CMA testing and additional guidance on testing to inform diagnosis after negative CMA, see the 2018 ACMG clinical practice resource. 

What role does chromosome analysis (standard karyotyping) play in the workup for developmental delay/intellectual disability or autism spectrum disorder?

Chromosomal microarray (CMA) has replaced karyotype analysis as the first laboratory test for developmental delay (DD), intellectual disability (ID), and autism spectrum disorders (ASDs) of unknown etiology.    ,  CMA is also now the first-line test for patients with multiple congenital anomalies (MCAs). Although chromosome analysis was historically the first-line test for patients with DD/ID, it is currently only recommended as a first test for patients with apparent aneuploidy (eg, Down syndrome, trisomy 13, trisomy 18, Klinefelter syndrome, Turner syndrome). Chromosome analysis is also indicated when there is a family history of chromosome rearrangement or multiple miscarriages because it can detect balanced chromosomal abnormalities, which CMA does not detect.    To evaluate a patient with MCAs/DD/ID/ASD who appears to have aneuploidy, chromosome analysis with reflex to CMA can be ordered.

Is there a role for FISH analysis that targets a specific microdeletion or microduplication syndrome?

The consensus is not clear or consistent regarding chromosomal microarray (CMA) versus targeted fluorescence in situ hybridization (FISH) analysis.   FISH probes assess a specific copy number variant (CNV) associated with a specific syndrome. CMA detects CNVs across the entire genome. FISH is less expensive than CMA, but only if the suspected diagnosis is confirmed; otherwise, FISH must be followed by CMA. CMA can identify CNVs that are too small to identify via FISH, and tandem submicroscopic duplications may be easier to confirm by CMA than FISH. Metaphase FISH shows whether a duplicated region is at its normal location, whereas CMA does not. Thus, FISH metaphase analysis is often used to assess the relatives of the affected patient for balanced rearrangements.

When should comprehensive metabolic testing be considered for patients with developmental delay, intellectual disability, or autism spectrum disorder?

Guidelines recommend that specific testing for inborn errors of metabolism (IEMs) should be performed after reviewing newborn screening (NBS) results and assessing for clinical indicators. This recommendation is based on the benefit of diagnosing conditions for which treatments and/or interventions exist. Although the incidence of IEMs is low, the potential to improve outcomes by intervention and treatment after diagnosis is high,    and a diagnosis may allow for dietary management or other treatment options. Not all metabolic conditions are included on NBS panels, which vary by state. Clinical features of an IEM may include developmental delays in isolation or in combination with autism, regression (neurodegeneration), failure to thrive, poor physical endurance/lethargy, episodic symptoms such as epilepsy and encephalopathy, multiple organ dysfunction, dietary selectivity, unusual odors, and hearing loss.

Where can I go for more information on the evaluation, including laboratory testing, of global developmental delay, intellectual disability, and autism spectrum disorders?

The American Academy of Pediatrics (AAP) global developmental delay (DD) and intellectual disability (ID) evaluation guideline provides guidance for clinical evaluation of DD and ID, including medical and family histories; physical, dysmorphology, and neurologic exams; imaging; patient management and communications; and laboratory testing.  The American College of Medical Genetics (ACMG) practice guidelines outline the approach to a genetic diagnostic evaluation for patients with autism spectrum disorders (ASDs). 

Indications for Testing

Laboratory evaluation of suspected DD, ID, or ASD should be considered for individuals with any of the following presentations   :

  • Failure to meet developmental milestones
  • IQ <70 and difficulty performing daily living activities
  • Comorbidities (eg, dysmorphic features, congenital anomalies) that may guide testing

Determining Diagnosis for Care Planning

Clinical evaluation of DD/ID and ASD will guide the laboratory testing strategy and should include  :

  • Medical history, including prenatal and birth histories
  • Family history with at least a three-generation pedigree
  • Physical and neurologic exam with particular attention to dysmorphology (minor anomalies)
  • Examination of behavior and neurologic symptoms

Following the clinical evaluation, the judicious use of laboratory testing, imaging, and other techniques is recommended.    If a specific condition is suspected, consider targeted testing. If the etiology of DD/ID/ASD is unknown, proceed with tiered testing based on highest diagnostic yield in the patient population.

Laboratory Testing

The American College of Medical Genetics and Genomics (ACMG) developed a tiered evaluation system to assist clinicians in the clinical genetic diagnostic evaluation of ASD.   The logic behind the tiered approach is that tests performed in higher (or earlier) tiers have a greater expected diagnostic yield, are less invasive, and provide better potential for intervention. The tiered approach also allows for customization to the clinical situation at hand.

The American Academy of Pediatrics (AAP) recommends a similar stepwise approach in the evaluation of children with ID or global DD.  The American Academy of Neurology (AAN) also supports laboratory testing in the evaluation of patients with global DD/ID. 

First-Tier Evaluation

The first-tier evaluation for DD/ID includes a detailed clinical evaluation, as described in Determining Diagnosis for Care Planning (above). If a specific disorder is suspected, targeted testing should be performed with appropriate follow-up. If no specific etiology is suggested, testing should proceed with CMA (and/or chromosome analysis in limited circumstances) and fragile X testing. Especially for global DD/ID, consider testing for IEMs, which has a low yield but high benefit for diagnosed patients.   

As with DD/ID, the first-tier evaluation for ASD includes a detailed clinical evaluation and should include an audiogram.  Certain disorders that have firmly established associations with ASD (eg, Angelman syndrome and Prader-Willi syndrome) may be identified through disorder-specific testing.  If such a diagnosis is made, no further testing to identify the etiology of ASD is required.  Otherwise, proceed with tiered testing and/or consider referral for medical genetics evaluation.

Chromosomal Microarray​

CMA is the preferred first-tier test for DD/ID, ASD, and MCAs in patients for whom the causal diagnosis is unknown. CMA offers a higher diagnostic yield (~15-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCAs than does chromosome analysis (~3%; excluding Down syndrome and other recognizable chromosomal syndromes).    CMA detects syndrome-associated microdeletions and microduplications that are below the resolution of karyotype analysis. Cryptic copy number variations (CNVs) that indicate unbalanced translocations may be identified. Compared to chromosome analysis, CMA provides far superior detail that facilitates the interpretation of results.

If CMA results are normal, review of other first-tier test results and consideration of second-tier testing and/or referral to medical genetics is recommended. If CMA results are abnormal, genetic counseling is recommended.

FMR1 Testing for Fragile X Syndrome

FXS is the most common form of heritable ID. This X-linked condition presents as severe DD in males and as mild ID in females. Apparently spontaneous cases with negative family history arise due to the expansion of premutations in female carriers. The AAP recommends that all children who present with global DD/ID of unknown etiology be tested for FXS.  The AAN evidence report also supports testing patients of both sexes with unexplained DD/ID.  The ACMG recommends FMR1 analysis for boys with ASD and for girls with a consistent family history and phenotype.  The AAN finds that FMR1 testing has a combined yield of at least 2% in male and female patients who have mild DD/ID.  Timely diagnosis supports access to early intervention and timely reproductive risk counseling. For test specific information, see the Fragile X Syndrome Test Fact Sheet.

Chromosome Analysis (Karyotyping)

G-banded chromosome analysis is no longer recommended unless aneuploidy is suspected. Chromosome analysis has a lower diagnostic yield than CMA because it typically only detects genomic imbalances greater than 5-10 Mb. It is only cost-effective in patients whose symptoms are consistent with a specific chromosome abnormality such as Down syndrome, trisomy 13, or trisomy 18, or in certain other special cases (eg, family history of multiple miscarriages).    

Metabolic and/or Mitochondrial Disorders Testing

Metabolic and/or mitochondrial disorder testing may be considered after reviewing newborn screening (NBS) results and assessing for clinical indicators that may suggest a metabolic disorder (eg, failure to thrive, unusual odors, hearing loss, and episodic symptoms). The AAP, AAN, and ACMG support the consideration of screening for metabolic conditions in children who present with DD/ID/ASD. Many metabolic tests are available at a relatively low cost, and, despite the low prevalence of inherited metabolic conditions, the potential for improved outcomes after diagnosis and treatment is high.   

Second-Tier Evaluation

MECP2

If no etiology is found for ASD, second-tier testing includes MECP2 sequencing (for Rett syndrome) in all females and MECP2 duplication testing in males with suggestive phenotypes. 

Second-tier testing for DD/ID includes MECP2 full gene analysis in females. The AAN suggests MECP2 testing in girls with severe global DD/ID, and the AAP emphasizes complete MECP2 testing for females with global DD/ID.   Both groups emphasize that MECP2 testing in these patients is indicated even in the absence of classic Rett syndrome features.

PTEN

Patients of both sexes who have head circumferences >2.5 standard deviations above the mean should have PTEN gene analysis if ASD etiology is still unknown after initial evaluations. 

Other Testing

The AAP, ACMG, and AAN recommend considering testing for X-linked ID in males with a suggestive family history.

Brain magnetic resonance imaging (MRI) is recommended for patients with microcephaly, macrocephaly, or abnormal neurologic exams.

Development of a plan for follow-up, including potential reevaluation, is recommended. 

Refer to the Testing for Genetic Syndromes Related to Developmental Delay, Intellectual Disability, and Autism Spectrum Disorder Algorithm for suggested testing strategies.

Diagnostic Yields of Genetic Testing

The following approximate diagnostic yields are expected in the genetic evaluation of ASDs and global DD/ID:

Testing Approximate Diagnostic Yield (Global DD/ID) Approximate Diagnostic Yield (ASD)a
CMA 15-20%b 10%
FXS >2% (mild-moderate DD/ID)c 1-5%
MECP2 1.5% (females with moderate-severe DD/ID)c 4% (females)
PTEN   5% (of those tested with head circumferences >2.5 SDs)
Karyotype 3-4%b,c (excluding Down syndrome) 3%
Other (eg, IEM, X-linked disorders) Up to 10%c 10%

aSchaefer, 2013 

bMiller, 2010 ; average of 33 studies

cMichelson, 2011 

SD, standard deviations

ARUP Lab Tests

First-Tier Testing

Method

Tandem Mass Spectrometry, Electrophoresis/Spectrophotometry, Gas Chromatography/Mass Spectrometry, Liquid Chromatography/Tandem Mass Spectrometry, and Quantitative Liquid Chromatography/Tandem Mass Spectrometry, Genomic Microarray (Oligo-SNP Array), Polymerase Chain Reaction/Capillary Electrophoresis

Panel includes cytogenomic SNP microarray; fragile X (FMR1) with reflex to methylation analysis; acylcarnitine quantitative profile (plasma), mucopolysaccharides screen (electrophoresis and quantitation, urine), organic acids (urine), creatine disorders panel (serum/plasma and urine), and amino acids quantitative by LC-MS/MS (plasma)

AAP and ACMG guidelines suggest testing for fragile X disorder in males only

Other Testing (Useful Only in Certain Situations)

Metabolic Testing

Panel includes acylcarnitine quantitative profile (plasma), mucopolysaccharides screen (electrophoresis and quantitation, urine), organic acids (urine), creatine disorders panel (serum/plasma and urine), and amino acids quantitative by LC-MS/MS (plasma)

Medical Experts

Contributor
Contributor
Contributor

LaGrave

Danielle LaGrave, MS, LCGC

Faculty, Genetic Counseling Graduate Program, Human Genetics, University of Utah

Licensed Genetic Counselor, Maternal Serum Screening and Cytogenetics at ARUP Laboratories

Contributor

Longo

Nicola Longo, MD, PhD

Professor, Pediatrics, and Adjunct Professor of Clinical Pathology, University of Utah

Medical Director, Biochemical Genetics at ARUP Laboratories

Contributor

Mao

Rong Mao, MD, FACMG

Professor of Clinical Pathology, Adjunct Associate Professor, Pediatrics, and Co-Director, Clinical Molecular Genetics Fellowship Program, University of Utah

Laboratory Section Chief, Molecular Genetics and Genomics, at ARUP Laboratories

Contributor

Pasquali

Marzia Pasquali, PhD

Professor of Clinical Pathology and Co-Director, Fellowship Training Program in Biochemical Genetics, University of Utah

Laboratory Section Chief, Biochemical Genetics, and Medical Director, Biochemical Genetics and Newborn Screening, at ARUP Laboratories

Contributor

Toydemir

Reha Toydemir, MD, PhD, FACMG

Assistant Professor of Clinical Pathology, and Adjunct Assistant Professor of Pediatrics, University of Utah

Medical Director, Cytogenetics and Genomics, at ARUP Laboratories

References

  1. 20231187

    Shen Y

    Dies KA

    Holm IA

    Bridgemohan C

    Sobeih MM

    Caronna EB

    Miller KJ

    Frazier JA

    Silverstein I

    Picker J

    Weissman L

    Raffalli P

    Jeste S

    Demmer LA

    Peters HK

    Brewster SJ

    Kowalczyk SJ

    Rosen-Sheidley B

    McGowan C

    Duda AW

    Lincoln SA

    Lowe KR

    Schonwald A

    Robbins M

    Hisama F

    Wolff R

    Becker R

    Nasir R

    Urion DK

    Milunsky JM

    Rappaport L

    Gusella JF

    Walsh CA

    Wu BL

    Miller DT

    Autism Consortium Clinical Genetics/DNA Diagnostics Collaboration

    Pediatrics

    2010
    125
    4
    e727-35
    PubMed
  2. 20466091

    Miller DT

    Adam MP

    Aradhya S

    Biesecker LG

    Brothman AR

    Carter NP

    Church DM

    Crolla JA

    Eichler EE

    Epstein CJ

    Faucett A

    Feuk L

    Friedman JM

    Hamosh A

    Jackson L

    Kaminsky EB

    Kok K

    Krantz ID

    Kuhn RM

    Lee C

    Ostell JM

    Rosenberg C

    Scherer SW

    Spinner NB

    Stavropoulos DJ

    Tepperberg JH

    Thorland EC

    Vermeesch JR

    Waggoner DJ

    Watson MS

    Martin CLese

    Ledbetter DH

    Am J Hum Genet

    2010
    86
    5
    749-64
    PubMed
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