Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Comprehensive Sequencing Panel with CNV Detection
|Test Type||Test Code||Total Price|
|Duo (Proband + Family Member)||5045, 5044||$3490.00|
|Trio (Proband + 2 Family Members)||5045, 5044(x2)||$4490.00|
|Genes x(1908)||81175(x1), 81200(x1), 81302(x1), 81321(x1), 81403(x5), 81404(x30), 81405(x53), 81406(x76), 81407(x18), 81408(x11), 81479(x1711)|
For a full list of genes click here.
Maximum of 6 weeks.
CMA and FMR1 CGG-repeat expansion testing have a combined diagnostic yield of 11-15% and should always be carried out as primary testing (Schaefer and Mendelsohn 2013). CMA analysis is limited in its ability to identify low-level mosaicism and balanced translocations, however, these variants are infrequently (<1%) the cause of phenotypes in ASD/ID patients (Miller et al. 2010). Genetic variants have been found responsible in 25-50% of ID cases and this percentage increases proportionally with the severity of the phenotype (McLaren and Bryson 1987). For ASD, while heritability estimates have been reported as high as 90% (Bailey et al. 1995; Lichtenstein et al. 2010), only 20% of ASD cases can be explained to date using combined genetic approaches (Devlin et al. 2012).
Trio-based studies have reported molecular diagnostic rates as high as 30-40% for developmental phenotypes (Lee et al. 2014; Fitzgerald et al. 2015; Wright et al. 2015). Therefore, all attempts should be made to utilize trio-based testing in order to maximize the clinical sensitivity of this test through clear identification of compound heterozygous and de novo variants in the probands (singleton studies cannot resolve either of these situations) (Lee et al. 2014; Wright et al. 2015; Retterer et al. 2016).
Reporting: Reports will consist of two different sections:
- Variants in genes known to be associated with phenotype
- Variants in genes possibly associated with phenotype
All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories (Pathogenic, Likely Pathogenic, Variant of Uncertain Significance, Likely Benign and Benign) per ACMG Guidelines (Richards et al. 2015). Pathogenic, Likely Pathogenic and Variants of Uncertain Significance considered to contribute to the proband's phenotype will be reported in the first and second sections (1 & 2).
Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).
Limitations and Other Test Notes: Interpretation of the test results is limited by the information that is currently available. Enhanced interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder accumulate. A negative finding does not rule out a genetic diagnosis.
When sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not capture or amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele.
For technical reasons, the ASD-ID test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants relevant to the patient's health will not be detected.
In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.
We sequence coding exons for most given transcripts, plus ~10 bp of flanking non-coding DNA for each exon. Unless specifically indicated, test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions, uncharacterized alternative exons, chromosomal rearrangements, repeat expansions, epigenetic effects, and mitochondrial genome variants.
In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative variants for recessive disorders, we cannot be certain that the variants are on different alleles, unless parental specimens are also tested.
Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient's nucleated cells may not be detected.
This test targets most, but not all, of the coding parts of genes within the panel (called exons).
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during amplification.
Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken from whole blood). Test reports contain no information about the DNA sequence in other cell-types.
We cannot be certain that the reference sequences are correct.
We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.
Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.
Autism Spectrum Disorders (ASDs) and Intellectual Disability (ID) are a heterogeneous group of neurodevelopmental disorders. ASD is characterized by varying degrees of social impairment, communication ability, propensity for repetitive behavior(s), and restricted interests (Levy et al. 2009); whereas ID refers to significant impairment of cognitive and adaptive development (intelligence quotient, IQ<70) due to abnormalities of brain structure and/or function (American Association of Intellectual and Developmental Disabilities, AAIDD). ID is not a single entity, but rather a general symptom of neurologic dysfunction that is diagnosed before age 18 in ~1%-3% of the population, irrespective of social class and culture (Kaufman et al. 2010; Vissers et al. 2016). In contrast, ASD symptoms usually present by age 3, and diagnosis is based on the degree and severity of symptoms and behaviors (McPartland et al. 2016). ASDs and ID are highly comorbid, suggesting shared etiologies in many forms. For ASD specifically, comorbidities have been observed in more than 70% of cases, and include ID, epilepsy, language deficits, and gastrointestinal problems (Sztainberg and Zoghbi 2016).
ASDs and ID are inherited in a multifactorial fashion, with heritability estimates ranging between 50-90% for ASDs and 15-50% for ID (Larsen et al. 2016; Karam et al. 2015). ASD concordance is as high as 70% in monozygotic twins. Familial recurrence rates are 7% if the first affected child is female and 4% if first affected child is male (Schaefer and Mendelsohn 2008). Incidence of ASD is approximately 1 in 68 individuals with a male-to-female ratio of 4:1 (CDC 2014). Interestingly, although ~30% more males are diagnosed with ID than females, the male-to-female ratio decreases as IQ decreases (American Psychiatric Association 2000). However, co-occurring ASD and ID has a similar male-to-female prevalence ratio of ~4:1 (Christensen et al. 2016).
According to a large number of reports, chromosomal abnormalities (Fragile X syndrome, translocations, recurrent autosomal microdeletions/duplications) and pathogenic copy number variants (CNVs) familial and de novo, can explain ~10-15% of ASD-ID cases. The fragile X mental retardation 1 (FMR1) gene remains the most frequent (~0.5-5%) candidate (Ropers and Hamel 2005; Rauch et al. 2006; Zahir and Friedman 2007; Schaefer and Mendelsohn 2013; Vissers et al. 2016). Therefore, chromosomal microarray (CMA) is recommended as the first-tier diagnostic test in these disorders, followed by screening for Fragile X syndrome (Mefford et al. 2008; Weiss et al. 2008; Miller et al. 2010; Schaefer and Mendelsohn 2013; Vissers et al. 2016). For ASD, de novo missense and likely gene disrupting variants are 15% and 75% more frequent in patients than unaffected controls, respectively (Iossifov et al. 2014). Hence, trio testing (whenever possible) is considered the most powerful approach for genetic diagnosis of ASD-ID (Lee et al. 2014; Wright et al. 2015).
This test includes over ~1,900 genes that through literature, OMIM, and HGMD searches have at least a potential association with ASD-ID phenotypes.
For the ASD-ID comprehensive panel, Next Generation Sequencing (NGS) technologies are used to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. Genomic DNA is extracted from patient specimens, as required. Patient DNA corresponding to the targeted genes is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is then sequenced using Illumina’s Reversible Dye Terminator (RDT) platform NextSeq 500 using 150 by 100 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NextSeq 500 is converted to fastqs by Illumina Bcl2Fastq 1.8.4, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com). Since de novo variants are recorded at elevated frequencies in individuals with ASDs and ID, de novo variants in all clinically-relevant genes included in our PGxome test (Test #5000) are filtered, annotated, and interpreted. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed by Sanger sequencing.
For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.
The report will not include all the observed variants in the ASD-ID comprehensive panel due to the large number of genes included. However, the list of variants is available along with our interpretations, upon request. Since this test is performed using exome capture probes, a reflex to exome sequencing may be ordered. Please see the PGxome page (Test #5000) for limitations and reporting criteria for this test.
Indications for Test
This test is primarily implicated for the patients with ASDs and/or ID, who are negative for any kind of cytogenetic abnormalities and Fragile-X syndrome (particularly males). Whole genome chromosomal microarray (CMA) via aCGH and SNP (Test #2000) and the FMR1 CGG-repeat expansion (Test #558) tests are available to individuals who have not been previously tested.
- Genetic Counselor Team - firstname.lastname@example.org
- Greg Fischer, PhD - email@example.com
- American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, 2000. Text Revision. 4.
- Bailey A. et al. 1995. Psychol. Med. 25: 63–77. PubMed ID: 7792363
- Center for Disease Control and Prevention 2014. Morbidity and Mortality Weekly Report 63:1-21. PubMed ID: 24670961
- Christensen D.L. et al. 2016. Morbidity and Mortality Weekly Report. 65: 1-23. PubMed ID: 27031587
- Devlin B., Scherer S.W. 2012. Current Opinion in Genetics & Development. 22: 229-37. PubMed ID: 22463983
- Fitzgerald T.W. et al. 2015. Nature. 519:223-8. PubMed ID: 25533962
- Iossifov I. et al. 2014. Nature. 515: 216-21. PubMed ID: 25363768
- Karam S.M. et al. 2015. American Journal of Medical Genetics. Part A. 167: 1204-14. PubMed ID: 25728503
- Kaufman L. et al. 2010. Journal of Neurodevelopmental Disorders. 2: 182-209. PubMed ID: 21124998
- Larsen E. et al. 2016. Molecular Autism. 7: 44 PubMed ID: 27790361
- Lee H. et al. 2014. JAMA. 312: 1880-7 PubMed ID: 25326637
- Levy S.E. et al. 2009. Lancet. 374: 1627-38. PubMed ID: 19819542
- Lichtenstein P. et al. 2010. The American Journal of Psychiatry. 167: 1357-63. PubMed ID: 20686188
- McLaren J., Bryson S.E. 1987. American Journal of Mental Retardation. 92: 243-54. PubMed ID: 3322329
- McPartland J.C. et al. 2016. Encyclopedia of Mental Health. 2: 124-130
- Mefford H.C. et al. 2008. The New England Journal of Medicine. 359: 1685-99. PubMed ID: 18784092
- Miller D.T. et al. 2010. American Journal of Human Genetics. 86: 749-64. PubMed ID: 20466091
- Rauch A. et al. 2006. American Journal of Medical Genetics Part A. 140: 2063-74 PubMed ID: 16917849
- Retterer K. et al. 2016. Genetics in Medicine. 18: 696-704 PubMed ID: 26633542
- Ropers H.H., Hamel B.C. 2005. Nature Reviews. Genetics. 6: 46-57. PubMed ID: 15630421
- Schaefer G.B., Mendelsohn N.J. 2008. Genetics in Medicine. 10: 4-12. PubMed ID: 18197051
- Schaefer G.B., Mendelsohn N.J. 2013. Genetics in Medicine. 15: 399-407. PubMed ID: 23519317
- Sztainberg Y., Zoghbi H.Y. 2016. Nature Neuroscience. 19: 1408-17. PubMed ID: 27786181
- Vissers L.E. et al. 2016. Nature Reviews. Genetics. 17: 9-18. PubMed ID: 26503795
- Weiss L.A. et al. 2008. The New England Journal of Medicine. 358: 667-75. PubMed ID: 18184952
- Wright C.F. et al. 2015. Lancet. 385: 1305-14. PubMed ID: 25529582
- Zahir F., Friedman J.M. 2007. Clinical Genetics. 72: 271-87. PubMed ID: 17850622
myPrevent - Online Ordering
- The test can be added to your online orders in the Summary and Pricing section.
- Once the test has been added log in to myPrevent to fill out an online requisition form.
- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
- We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.
(Delivery preferred Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.