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DDX3X Syndrome/Intellectual Disability Type 102 via DDX3X Gene Sequencing with CNV Detection

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

Sequencing and Del/Dup via NGS

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
10653 DDX3X$640.00 81479,81479 Add to Order
Pricing Comment

This test is also offered via our exome backbone with CNV detection (click here). The exome-based test may be higher priced, but permits reflex to the entire exome or to any other set of clinically relevant genes.

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 20 days.

Clinical Sensitivity

This test is predicted to detect pathogenic variants in ~1.5% of females with unexplained intellectual disability. By contrast, the diagnostic yield of this test for males with unexplained ID is predicted to be <0.1% (Wang et al. 2018. bioRxiv 283598, Snijders Blok et al. 2015. PubMed ID: 26235985, Lennox et al. 2018. bioRxiv 317974). It is important to note the high clinical and genetic heterogeneity of intellectual disability, and improved diagnostic yields that result from testing large panels of genes as well as testing parents along with the patient using a trio approach (Vissers et al. 2016. PubMed ID: 26503795). Analytical sensitivity of this test should be high because all reported pathogenic variants in DDX3X are detectable by sequencing.

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Clinical Features

Patients with DDX3X syndrome (also known as X-linked intellectual disability type 102 (MRX102)) present in infancy or early childhood with intellectual disability (ID) and developmental delay that can range from mild to severe (Snijders Blok et al. 2015. PubMed ID: 26235985). Over 90% of identified patients are female. Brain abnormalities found on magnetic resonance imaging are a consistent feature of this disorder, with corpus callosum malformations being the most frequent (>95% of patients), followed by enlargement of the lateral ventricles, diminished white matter, and polymicrogyria (Lennox et al. 2018. bioRxiv 317974). A majority of patients also have behavior problems, which are usually characterized as autism, hyperactivity, or aggression. Some children gain the ability to speak in full sentences, although about half remain completely nonverbal by age five. Most patients have hypotonia, and a significant portion develop a movement disorder, which typically includes spasticity or unusual gait. Most patients exhibit some dysmorphic features, although a recognizable facial gestalt has not been established (Snijders Blok et al. 2015. PubMed ID: 26235985).

Other major features, observed in 20-50% of patients, include seizures, microcephaly, underweight for age, joint hyperlaxity, mosaic pigmentary changes, and vision problems (including strabismus, colobomas, and cortical visual impairment). Minor features (observed in 5-20% of patients) include precocious puberty, scoliosis, variable congenital heart defects, respiratory problems, cleft lip or palate, and hearing loss (both conductive and sensorineural). Additional features are seen in a small minority of patients (Lennox et al. 2018 bioRxiv 317974). While few affected males are described in the literature, their reported phenotypic features seem largely consistent with those seen in females.

There is no available treatment for this disorder, yet advantages of testing may include prognostic information, early identification and treatment of symptoms such as seizures and autism, and ability to join the DDX3X foundation and family support groups (https://ddx3x.org/). For families with inherited causative variants, prenatal testing or pre-implantation genetic diagnosis may be implemented for future pregnancies, whereas for families with affected females, knowledge of a de novo variant and decreased recurrence risk may ease anxiety for reproductive planning.

Genetics

Pathogenic variants in the DDX3X gene cause DDX3X syndrome (Lennox et al. 2018. bioRxiv 317974). This syndrome is most commonly observed in females, who usually carry dominantly-acting de novo DDX3X variants. However, hemizygous males are also observed, with variants that are either de novo or inherited from unaffected mothers (Wang et al. 2018. bioRxiv 283598, Snijders Blok et al. 2015. PubMed ID: 26235985, Kellaris et al. 2018. PubMed ID: 29490693). None of the pathogenic variants observed in females have ever been reported in a male, suggesting these variants are embryonic lethal in the hemizygous state. Evidence suggests that pathogenic DDX3X variants account for roughly 1.5% of unexplained intellectual disability in females, making it one of the most common causes of unexplained ID in females (Wang et al. 2018. bioRxiv 283598). By contrast, DDX3X variants are estimated to account for less than 0.1% of unexplained ID in males. Unexplained intellectual disability indicates individuals whose ID is not caused by copy number variants detectable on array, fragile X triplet repeat expansion, or environmental insults. Additionally, these individuals may have one or more negative targeted gene sequencing tests (Snijders Blok et al. 2015. PubMed ID: 26235985, Lennox et al. 2018. bioRxiv 317974). DDX3X partially escapes X-inactivation, and studies suggest that X-inactivation status, including extreme skewing, is not a factor in determining the severity or type of symptoms observed in DDX3X patients (Lennox et al. 2018. bioRxiv 317974, Wang et al. 2018. bioRxiv 283598, Snijders Blok et al. 2015. PubMed ID: 26235985).

Pathogenic variant types include missense, splicing, nonsense, in-frame, and frameshift alterations. To date, no causative copy number variants have been reported. Pathogenic frameshift and nonsense variants span a majority of the gene, excepting the last exon, whereas causative missense changes cluster in the two helicase domains (amino acids 211-403 and 414-575) (www.uniprot.org/uniprot/O00571). Limited evidence suggests phenotype-genotype correlations for specific missense changes in the helicase domains (T532, I415, R326) causing the most severe form of this disorder (Lennox et al. 2018. bioRxiv 317974). Overall, this gene is highly intolerant of both missense and loss of function variation, as indicated by the paucity of these changes in large datasets of normal individuals (Snijders Blok et al. 2015. PubMed ID: 26235985). Some positions of the DDX3X sequence appear prone to recurrent de novo variants (notably amino acids R376 and A488); however a majority of reported DDX3X variants are unique. Interestingly, this gene as a whole may be particularly prone to de novo alterations, as indicated by a cohort from Baylor identifying this gene as one of the most likely genes to carry de novo changes (ranked third out of 450 genes)(Wang et. al. 2018. bioRxiv 283598).

The DDX3X gene is located at Xp11.4, and encodes a 661 amino acid DEAD-box RNA helicase protein important for a wide array of basic cellular processes including transcription, splicing, translation, and transport. It is expressed highly in the developing brain where it is implicated in neuronal differentiation and migration (Lennox et al. 2018. bioRxiv 317974).

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 DDX3X gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Indications for Test

This test is suitable for females with syndromic unexplained intellectual disability and no family history of ID. DDX3X could be included as part of a larger sequencing panel or exome test for males with unexplained ID. Prenatal testing and targeted testing are also available in families with a confirmed diagnosis of DDX3X syndrome.

Gene

Official Gene Symbol OMIM ID
DDX3X 300160
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Mental Retardation, X-Linked 102 XL 300958

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Epilepsy and Seizure Plus Sequencing Panel with CNV Detection
Non-syndromic Intellectual Disability (NS-ID) Sequencing Panel with CNV Detection
X-Linked Intellectual Disability Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Kellaris et al. 2018. PubMed ID: 29490693
  • Lennox et al. 2018. bioRxiv 317974
  • Snijders Blok et al. 2015. PubMed ID: 26235985
  • Vissers et al. 2016. PubMed ID: 26503795
  • Wang et al. 2018. bioRxiv 283598
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TEST METHODS

Sequencing and Deletion/Duplication Testing via NextGen Sequencing using PG-Select Capture Probes

Test Procedure

NextGen Sequencing

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.  All pathogenic, likely pathogenic, or 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.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Deletion and Duplication Testing via NGS

Copy number variants (CNVs) such as deletions and duplications are detected from next generation sequencing 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, PCR or qPCR before they are reported.
Analytical Validity

NextGen Sequencing

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

Deletion and Duplication Testing via NGS
 
In general, sensitivity for single, double, or triple exon CNVs is ~80% and for CNVs of four exon size or larger is close to 100%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.
Analytical Limitations

NextGen Sequencing

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 Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

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 PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes 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.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

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.

Deletion and Duplication Testing via NGS
 
This CNV calling algorithm used in this test detects most deletions and duplications; 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. single vs. two or more exons), and inadequate coverage. 
 
Balanced translocations or inversions within a targeted gene, or large unbalanced translocations or inversions that extend beyond the genomic location of a targeted gene are not detected.
 
In nearly all cases, our ability to determine the exact copy number change within a targeted gene is limited. In particular, when we find copy excess within a targeted gene, we cannot be certain that the region is duplicated, triplicated etc. In many duplication cases, we are unable to determine the genomic location or the orientation of the duplicated segment with respect to the gene. In particular, we often cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we may not be able to determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.
 
Our ability to detect minor CNVs, due for example to somatic mosaicism is limited.
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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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