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Autism Spectrum Disorders via KATNAL2 Gene Sequencing with CNV Detection

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
9817 KATNAL2$890.00 81479,81479 Add to Order
Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 26 days.

Clinical Sensitivity

Currently, the contribution of de novo and inherited factors to Autism Spectrum Disorders (ASD) risk is estimated to be approximately 50-60% (Krumm et al. 2015. PubMed ID: 25961944). KATNAL2 is categorized as a gene with ‘high confidence’ regarding its association with ASD in the Simons Foundation Autism Research Initiative (SFARI) Database (https://gene.sfari.org/database/human-gene/KATNAL2). However, more than 700 genes have been associated with ASD features (Bourgeron. 2016. PubMed ID: 27289453), suggesting that the clinical sensitivity of any single gene sequencing or copy number variant test in the context of autism spectrum disorder phenotypes is small. For KATNAL2, the true clinical sensitivity of sequence or copy number variant analyses is unknown.

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Clinical Features

Autism Spectrum Disorders (ASD) include several neurodevelopmental disorders characterized by varying degrees of social impairment, communication ability, and propensity for restricted interests and repetitive behavior(s) (Levy et al. 2009. PubMed ID: 19819542). ASD usually presents by age 3. Diagnosis is based on the degree and severity of symptoms and behaviors (Diagnostic and Statistical Manual of Mental Disorders (DSM-5); McPartland et al. 2016). Comorbidities occur in more than 70% of cases and include intellectual disability (ID), epilepsy, language deficits, and gastrointestinal problems (Sztainberg and Zoghbi. 2016. PubMed ID: 27786181). Recent studies using whole exome trios have identified novel gene candidates, with familial and de novo variants from several hundred genes now implicated in the development of ASD (Bourgeron. 2016. PubMed ID: 27289453).

Genetics

Genetic aberrations are reported to be responsible for 50%-90% and 15%-50% of ASD and ID cases, respectively, and inheritance overall is multifactorial (Larsen et al. 2016. PubMed ID: 27790361; Karam et al. 2015. PubMed ID: 25728503). Incidence of ASD is approximately 1 in 68 individuals with a male-to-female ratio of 4:1 (Center for Disease Control 2014). De novo missense and likely gene disrupting variants are 15% and 75% more frequent in ASD patients than unaffected controls, respectively (Iossifov et al. 2014. PubMed ID: 25363768). Multiple independent de novo sequence variants in the same gene among unrelated probands has been reported as a potential means to identify ASD risk alleles (Sanders et al. 2012. PubMed ID: 22495306).

KATNAL2 (Katanin P60 subunit A-like 2) is a putative microtubule-severing ATPase that, in mice, has been shown to impact dendritic branch formation in the developing brain (Williams et al. 2016. PubMed ID: 27161796). KATNAL2 may also play a role in ciliogenesis and centriole activity (Dunleavy et al. 2017. PubMed ID: 29136647).

De novo variants in KATNAL2 have been reported in individuals with ASD features from simplex families (Sanders et al. 2012. PubMed ID: 22495306; O'Roak et al. 2012. PubMed ID: 22495309; Stessman et al. 2017. PubMed ID: 28191889), supporting an autosomal dominant mode of inheritance. Maternally-inherited KATNAL2 likely gene disrupting variants (nonsense and splice site) have also been reported in individuals with ASD phenotypes (Yuen et al. 2015. PubMed ID: 25621899; Stessman et al. 2017. PubMed ID: 28191889). Of note, females are reported to have more variants in ASD risk genes than males, but require a greater mutational burden than males before clinical manifestation of ASD features (Jacquemont et al. 2014. PubMed ID: 24581740). Furthermore, variants predicted to disrupt KATNAL2 protein function (splice site, nonsense, frameshift) have also been reported in population databases composed of presumably unaffected individuals (Supplementary Table 18, Stessman et al. 2017. PubMed ID: 28191889) which may be related to sex-specific difference in ASD manifestations or incomplete penetrance.

Testing Strategy

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. 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.

This test provides full coverage of all coding exons of the KATNAL2 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Since this test is performed using exome capture probes, a reflex to any of our exome based tests is available (PGxome, PGxome Custom Panels).

Indications for Test

De novo and maternally-inherited KATNAL2 variants have been reported to date. A panel (or exome/genome) sequencing test would nearly always be more appropriate for patients with ASD, unless previous clinical knowledge implicates the KATNAL2 gene. For example, an affected individual with a family member having a known KATNAL2 variants and similar clinical features may consider KATNAL2 testing (targeted Sanger sequencing for the known variant may be most appropriate).

Gene

Official Gene Symbol OMIM ID
KATNAL2 614697
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Autism Susceptibility 1 AD 209850

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Autism Spectrum Disorders Sequencing Panel with CNV Detection
Autism Spectrum Disorders via CACNA2D3 Gene Sequencing with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Bourgeron. 2016. PubMed ID: 27289453
  • Center for Disease Control and Prevention. 2014. Morbidity and Mortality Weekly Report. Surveillance Summaries (Washington, D.C.). 63: 1-21. PubMed ID: 24670961
  • Dunleavy et al. 2017. PubMed ID: 29136647
  • Iossifov et al. 2014. PubMed ID: 25363768
  • Jacquemont et al. 2014. PubMed ID: 24581740
  • Karam et al. 2015. PubMed ID: 25728503
  • Krumm et al. 2015. PubMed ID: 25961944
  • Larsen et al. 2016. PubMed ID: 27790361
  • Levy et al. 2009. PubMed ID: 19819542
  • McPartland et al. 2016. Encyclopedia of Mental Health. 2: 124-130.
  • O'Roak et al. 2012. PubMed ID: 22495309
  • Sanders et al. 2012. PubMed ID: 22495306
  • Stessman et al. 2017. PubMed ID: 28191889
  • Sztainberg and Zoghbi. 2016. PubMed ID: 27786181
  • Williams et al. 2016. PubMed ID: 27161796
  • Yuen et al. 2015. PubMed ID: 25621899
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TEST METHODS

Exome Sequencing with CNV Detection

Test Procedure

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 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 NovaSeq 6000 is converted to fastqs by Illumina Bcl2Fastq, 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).

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.

Analytical Validity

Copy Number Variant Analysis: The PGxome test detects most larger deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. 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.

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

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.

For technical reasons, the PGxome 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 will not be detected.

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 mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

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.

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.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.  

In nearly all cases, our ability to determine the exact copy number change within a targeted region is limited.

Our ability to detect CNVs due to somatic mosaicism is limited.

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.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.

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Ordering Options


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.
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.

SPECIMEN TYPES
WHOLE BLOOD

(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.

DNA

(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.

CELL CULTURE

(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.
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