Syndromic X-linked intellectual Disability Type 34, via NONO Gene Sequencing with CNV Detection
- Summary and Pricing
- Clinical Features and Genetics
Sequencing with CNV
|Test Code||Test Copy Genes||Price||CPT Code Copy CPT Codes|
For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 26 days.
The diagnostic yield of this test for males with syndromic intellectual disability is predicted to be very low (<0.1%), however no large cohorts estimating clinical sensitivity have been published for this gene. 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. The analytical sensitivity of this test is expected to be high, as it will detect all types of NONO variants identified to date, including sequence variants as well as multiple-exon deletions or duplications in this gene.
Pathogenic variants in the NONO gene cause syndromic X-linked intellectual disability (XLID), type 34. This disorder is male-limited and presents in infancy with global developmental delay—including hypotonia, motor delays, cognitive deficits, and speech delay. Initial brain scans often appear normal; however, most patients develop dysplasia of the corpus callosum and some develop other central nervous system abnormalities, including cerebellar defects. Many people with this disorder learn to crawl or walk with ataxia, and can also develop language abilities over time. Physically, individuals with pathogenic NONO variants have a slender build, relative macrocephaly, and frontal bossing. Congenital heart defects are also common, notably left ventricular non-compaction cardiomyopathy (LVNC), as well as right ventricular hypertrophy, patent foramen ovale, patent ductus arteriosus, and atrial or ventricular septal defects. Some patients have early difficulties with feeding or breathing, requiring medical support. Behaviorally, these individuals are usually characterized as shy and anxious, though cheerfulness, autism, impulsiveness, aggressiveness, and hyperactivity have also been reported. Facial dysmorphism is variable, but can include strabismus, hypertelorism, epicanthal folds, long face, malar hypoplasia, prominent nose, short philtrum, and open mouth. Other minor features of this disorder are cryptorchidism, joint hyperreflexia, joint hyperlaxity, kyphosis, scoliosis, plagiocephaly, fifth finger clinodactyly, pes planus, hallux valgus, long narrow hands and feet, sleep apnea, myopia, café au lait macules, accessory nipples, and intention tremor (Mircsof et al. 2015. PubMed ID: 26571461, Reinstein et al. 2016. PubMed ID: 27329731, Scott et al. 2017. PubMed ID: 27550220).
While there are no treatments for NONO-related intellectual disability syndrome, patients and their families may benefit from a molecular diagnosis for prognostic information, early identification and treatment of symptoms, or for connecting with NONO-related XLID support groups. For reproductive planning, families can get maternal testing to determine if a variant arose de novo in the proband, or is present in the maternal germline.
NONO-related intellectual disability syndrome is inherited in an X-linked recessive pattern, and to date only males have been characterized. Roughly half of the pathogenic NONO variants are inherited from unaffected mothers and the others arise de novo (Mircsof et al. 2015. PubMed ID: 26571461, Reinstein et al. 2016. PubMed ID: 27329731, Scott et al. 2017. PubMed ID: 27550220). Carrier females do not express any apparent features of the disorder; however, it is important to note that the total numbers of patients identified is still very small (less than 10 documented in the literature). Therefore, the possibility of affected females (perhaps due to skewed X-inactivation) has not yet been thoroughly explored.
NONO is a non-POU domain-containing octamer-binding gene located at Xq13.1. NONO encodes a nucleic acid binding protein involved in RNA transcriptional regulation, splicing, nuclear retention, and transport (www.genecards.org, Stelzer et al. 2016. PubMed ID: 27322403). Pathogenic variants include nonsense, frameshift, canonical splice, non-canonical splice, and large deletions. Functional studies show that little or no NONO protein is produced from these altered transcripts, indicating a loss of function mechanism of disease (Mircsof et al. 2015. PubMed ID: 26571461, Scott et al. 2017. PubMed ID: 27550220). The small causative variants are all located in the latter half of the protein, including in the last exon (Reinstein et al. 2016. PubMed ID: 27329731). One large deletion includes coding exons 1-3 (Scott et al. 2017. PubMed ID: 27550220). Two pathogenic NONO variants have been observed more than once—namely a nonsense alteration at amino acid 365, and a frameshift beginning at amino acid 466. The differential diagnosis for NONO XLID is quite broad because the presenting features can be general. Pathogenic NONO variants are expected to have 100% penetrance in males.
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 numbers of sequence reads.
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 NONO 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
This test is suitable for males with syndromic intellectual disability and a family history consistent with X-linked inheritance. Due to the high clinical and genetic heterogeneity of syndromic ID, NONO could be included as part of a larger sequencing panel or exome test.
|Official Gene Symbol||OMIM ID|
|Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection|
|X-Linked Intellectual Disability Sequencing Panel with CNV Detection|
- Genetic Counselor Team - email@example.com
- Renee Bend, PhD - firstname.lastname@example.org
Exome Sequencing with CNV Detection
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).
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.
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.
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.
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.
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.