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Diamond-Blackfan Anemia and Bone Marrow Failure via RPL15 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
8739 RPL15$890.00 81479,81479 Add to Order
Pricing Comment

Our 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, if 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.

For Sanger Sequencing click here.
Targeted Testing

For ordering 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

Approximately 65% of Diamond-Blackfan anemia (DBA) cases are found to have a pathogenic variant in one of the known DBA genes (Clinton and Gazda. 2016. PubMed ID: 20301769). Pathogenic variants in the RPS19 gene are found in up to 25% of patients (Gazda and Sieff. 2006. PubMed ID: 16942586). Variants in the RPL5 (6.6%), RPS26 (6.4%), RPL11 (4.8%), RPL35A (3%), RPS10 (2.6%), RPS24 (2%), and RPS17 (1%) genes are the next most frequent causes of DBA, with variants in all other associated genes accounting for a very small fraction of disease (Clinton and Gazda. 2016. PubMed ID: 20301769).

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Clinical Features

Diamond-Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome characterized by macrocytic anemia, normal leukocyte and platelet numbers, and normocellular bone marrow (Freedman. 2000. PubMed ID: 11030041; Gazda and Sieff. 2006. PubMed ID: 16942586). Physical anomalies such as craniofacial dysmorphism, thumb and neck anomalies, congenital heart defects, and genitourinary tract defects are found in ~40% of patients. Growth retardation is observed in ~30% of patients (Clinton and Gazda. 2016. PubMed ID: 20301769). Onset of hematologic complications typically occurs in the first year of life. The severity of disease varies from mild anemia with no physical anomalies to severe anemia and severe physical anomalies. DBA is also associated with bone marrow failure and increased risk for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).

Genetics

DBA is an autosomal dominant or X-linked disorder caused by inactivating variants within ribosomal protein genes RPS19 (Gazda and Sieff. 2006. PubMed ID: 16942586), RPL5 (Gazda et al. 2008. PubMed ID: 19061985), RPL11 (Gazda et al. 2008. PubMed ID: 19061985), RPL35A (Farrar et al. 2008. PubMed ID: 18535205), RPS26 (Doherty et al. 2010. PubMed ID: 20116044), RPS24 (Gazda et al. 2006. PubMed ID: 17186470), RPS17 (Gazda et al. 2008. PubMed ID: 19061985), RPS7 (Gazda et al. 2008. PubMed ID: 19061985), RPS10 (Doherty et al. 2010. PubMed ID: 20116044), RPL26 (Gazda et al. 2012. PubMed ID: 22431104), RPS27 (Wang et al. 2015. PubMed ID: 25424902), RPS29 (Mirabello et al. 2014. PubMed ID: 24829207), RPL31 (Farrar et al. 2014. PubMed ID: 25042156), RPS28 (Gripp et al. 2014. PubMed ID: 24942156), RPL15 (Landowski et al. 2013. PubMed ID: 23812780), RPL27 (Wang et al. 2015. PubMed ID: 25424902), or by variants in the GATA1 (Sankaran et al. 2012. PubMed ID: 22706301) or TSR2 (Gripp et al. 2014. PubMed ID: 24942156) genes. Pathogenic variants in the RPS19 gene are found in up to 25% of patients (Gazda and Sieff. 2006. PubMed ID: 16942586). Variants in the RPL5 (6.6%), RPS26 (6.4%), RPL11 (4.8%), RPL35A (3%), RPS10 (2.6%), RPS24 (2%), and RPS17 (1%) genes are the next most frequent causes of DBA with variants in all other associated genes accounting for a very small fraction of disease (Clinton and Gazda. 2016. PubMed ID: 20301769). Approximately 65% of DBA cases are found to have a pathogenic variant in one of the known DBA genes (Clinton and Gazda. 2016. PubMed ID: 20301769). 55-60% of DBA cases result from de novo pathogenic variants (Clinton and Gazda. 2016. PubMed ID: 20301769) with the remainder of cases resulting from inheritance of a pathogenic variant from an affected parent.

DBA results from loss of protein function and haploinsufficiency. Pathogenic variants consist primarily of missense variants and nonsense or other protein truncating variants including frameshift deletions and insertions. Large, multi-exon or full gene deletions of several ribosomal protein genes, in particular RPS19, RPL5, RPL11, RPL35A, RPS26, RPS24, RPS17, and RPL15, have been reported in patients with DBA. Dysfunctional ribosomal proteins are likely to alter the stability and/or function of the ribosomal complex causing destruction of blood-forming cells in the bone marrow and consequent anemia.

The RPL15 protein is essential for proper formation of the 60S ribosomal subunit (Farrar et al. 2014. PubMed ID: 25042156). Pathogenic variants in RPL15 have been reported in only two individuals and are a rare cause of DBA (DBA 12, Landowski et al. 2013. PubMed ID: 23812780; Farrar et al. 2014. PubMed ID: 25042156). To date, large, multi-exon or whole gene deletions are the only reported pathogenic variants found in the RPL15 gene and no obvious genotype – phenotype correlations have been reported for the RPL15 gene.

Other bone marrow failure syndromes such as Fanconi anemia, severe congenital neutropenia, dyskeratosis congenita, and Shwachman-Diamond syndrome should be considered in addition to DBA during diagnosis.

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 RPL15 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

Patients with symptoms of Diamond-Blackfan anemia or indication of bone marrow failure or MDS/AML are candidates for this test. Other candidates for this test include potential donors and patients with an indication of bone marrow failure and who have tested negative for other bone marrow failure disorders such as Fanconi anemia, Shwachman-Diamond syndrome, dyskeratoris congenita, and severe congenital neutropenia.

Gene

Official Gene Symbol OMIM ID
RPL15 604174
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Diamond-Blackfan Anemia 12 AD 615550

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Diamond-Blackfan Anemia and Bone Marrow Failure via RPS19 Gene Sequencing with CNV Detection
Diamond-Blackfan Anemia and Bone Marrow Failure via RPS26 Gene Sequencing with CNV Detection
Diamond-Blackfan Anemia and Bone Marrow Failure via the RPS17 Gene
Diamond-Blackfan Anemia Sequencing Panel with CNV Detection
Dyskeratosis Congenita (DC) and Related Disorders Sequencing Panel with CNV Detection
Neonatal Crisis Sequencing Panel with CNV Detection
Shwachman-Diamond Syndrome via the SBDS Gene

CONTACTS

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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). 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 ~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.

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.
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