Forms

Dyskeratosis Congenita (DC) via NOP10 Gene Sequencing with CNV Detection

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

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
7965 NOP10$890.00 81479,81479 Add to Order
Pricing Comment

Our most favored testing approach is exome based NextGen sequencing with CNV analysis. This will allow cost effective reflexing to PGxome or other exome based tests. However, in the event that full gene Sanger sequencing is desired for STAT turnaround time, insurance, or other reasons, please see link below for Test Code, pricing, and turnaround time information. If the Sanger option is selected, CNV detection may be ordered through Test #600.

For Sanger Sequencing click here.
Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

Approximately 70% of individuals with dyskeratosis congenital (DC) have pathogenic variants in at least 1 of the 11 known DC genes (Savage et al. 2009. PubMed ID: 20301779. Analysis of data from The Royal London Hospital (London, UK), shows that variants in DKC1 and TERC account for approximately 36% of cases reported (30% DKC1 and 6% TERC) (Walne et al. 2007. PubMed ID: 17507419). Pathogenic variants in NOP10 are rare and probably only account for a small percentage of DC cases. No large deletions or duplications involving NOP10 have been reported to date (Human Gene Mutation Database).

See More

See Less

Clinical Features

Dyskeratosis congenita (DC) is a disease characterized by multiple anomalies including abnormal skin pigmentation, nail dystrophy, mucosal leukoplakia, bone marrow failure, and an increase in cancer predisposition (Walne et al. 2007. PubMed ID: 17507419; Kirwan et al. 2008. PubMed ID: 18005359). Individuals affected with DC can exhibit other features including liver cirrhosis, osteoporosis, pulmonary fibrosis and learning difficulties. Approximately 80-90% individuals affected with DC experience bone marrow failure by age 30 (Kirwan et al. 2008. PubMed ID: 18005359). Age of onset and progression of DC may vary. Those who have minimal physical findings with normal bone marrow function are at the mild end of the spectrum, while those at the severe end of the spectrum have physical features along with bone marrow failure (Savage. 2016. PubMed ID: 20301779).

Genetics

Dyskeratosis congenita is caused primarily from defects in telomere maintenance (Walne et al. 2007. PubMed ID: 17507419; Trahan et al. 2010. PubMed ID: 20008900). It is a disorder that is characterized by three genetic subtypes: X-linked recessive, caused by variants in DKC1, autosomal dominant DC caused by heterozygous variants in TERC or TERT, and autosomal recessive DC which involves a wide variety of genes including NOP10 (Kirwan et al. 2008. PubMed ID: 18005359). NOP10 is known to be associated with the telomerase complex, and is associated with H/ACA small nucleolar RNPs (snoRNPs) in humans (Walne et al. 2007. PubMed ID: 17507419).

To date, ACD, CTC1, DKC1, NHP2, NOP10, PARN, RTEL1, TERC, TERT, TINF2, and WRAP53 are genes in which pathogenic variants have been reported to cause DC (Savage. 2016. PubMed ID: 20301779). Currently, only one missense variant has been reported in NOP10 to be causative for DC (Human Gene Mutation Database).

The 64 amino acid NOLA3 protein is coded by exons 1 to 2 of the NOP10 gene located on chromosome 15q14.

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 NOP10 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

Candidates for this test are patients with symptoms consistent with autosomal recessive Dyskeratosis Congenita and the family members of patients who have known NOP10 variants.

Gene

Official Gene Symbol OMIM ID
NOP10 606471
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Dyskeratosis Congenita Autosomal Recessive AR 224230

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Dyskeratosis Congenita (DC) via the NHP2 Gene
Dyskeratosis Congenita (DC) and Hoyeraal-Hreidarsson Syndrome via the DKC1 Gene
Dyskeratosis Congenita (DC) and Related Disorders Sequencing Panel with CNV Detection
Dyskeratosis Congenita (DC) and Revesz Syndrome via the TINF2 Gene
Dyskeratosis Congenita (DC) via the TERT Gene
Dyskeratosis Congenita (DC) via the WRAP53 Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Human Gene Mutation Database (Bio-base).
  • Kirwan et al. 2008. PubMed ID: 18005359
  • Savage. 2016. PubMed ID: 20301779
  • Trahan et al. 2010. PubMed ID: 20008900
  • Walne et al. 2007. PubMed ID: 17507419
Order Kits
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). Common benign, likely benign, and low quality variants are filtered from analysis. 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.

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 ~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

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.

Order Kits

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.
loading Loading... ×