Alpha Thalassemia Deletion/Duplication and Constant Spring Panel
- Summary and Pricing
- Clinical Features and Genetics
|Test Code||Test Copy Genes||Individual Gene Price||CPT Code Copy CPT Codes|
|6070||HBA1 and HBA2||$540.00||81404||Add|
For ordering targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 20 days.
The -α3.7, -α4.2, -(α)20.5, --SEA, --MED, --FIL, and –THAI deletions account for ~85% of all pathogenic variants detected in alpha thalassemia patients. Other full gene deletions encompassing the HBA1 and/or HBA2 genes are found in ~<5% of cases. Pathogenic variants detectable by sequencing analysis are found in ~15% of cases (Origa and Moi. 2016. PubMed ID: 20301608; Harteveld and Higgs, 2010. PubMed ID: 20507641). The most common pathogenic variant involving one or a few nucleotides found in Asian populations is Hemoglobin Constant Spring (HbCS) which abolishes the canonical termination codon in the HBA2 gene, c.427T>C (p.*143Gln).
Hemoglobin A, the main form of hemoglobin, is a polypeptide comprised of two alpha and two beta chains. Alpha chains are encoded through the HBA1 and HBA2 genes. Defects in these genes result in alpha thalassemia, which is a common hemoglobin disorder commonly found in African and Asian populations. Carrier frequencies are often >1% due to a selective advantage of malaria resistance in these individuals. There are four clinical conditions ranging in severity from asymptomatic to hydrops fetalis: silent carrier, alpha thalassemia trait, hemoglobin H (HbH), and hemoglobin Bart hydrops fetalis (Hb Bart). Disease severity is correlated to the degree of impaired alpha chain production (Origa and Moi. 2016. PubMed ID: 20301608; Harteveld and Higgs. 2010. PubMed ID: 20507641).
The two main clinical forms of alpha thalassemia are Hb Bart and HbH. Hb Bart syndrome is a neonatal lethal disease characterized by pleural and pericardial effusions, severe hypochromic anemia, ascites, and generalized edema. HbH disease, also referred to as alpha thalassemia intermedia, is characterized by microcytic hypochromic anemia, splenomegaly, jaundice, and in some cases skeletal changes primarily affecting facial features. Onset for HbH ranges from first years of life to adulthood. HbH individuals may also develop gallstones, have iron overload, or acute hemolytic episodes during infections or in response to oxidant drugs. In more severe forms HbH individuals may require blood transfusions. Patients with alpha thalassemia trait may have mild anemia, but are largely symptomatic (Origa and moi. 2016. PubMed ID: 20301608; Harteveld and Higgs. 2010. PubMed ID: 20507641; Galanello and Cao. 2011. PubMed ID: 21381239).
Two neighboring genes with high homology, HBA1 and HBA2, comprise the 4 alpha globin alleles. Classification of the different forms of alpha thalassemia is determined by the loss or inactivation of the alpha globin alleles.
Alpha thalassemia trait
-α/-α or --/αα
Hemoglobin Bart Hydrops
Deletions in the HBA1 and HBA2 genes are found in over 90% of alpha thalassemia cases with seven founder mutations accounting for ~85% of all alpha thalassemia cases: -α3.7, -α4.2, -(α)20.5, --SEA, --MED, --FIL, and –THAI. The -α3.7 and -α4.2 deletions result in loss of a single alpha globin gene whereas the -(α)20.5, --SEA, --MED, --FIL, and -–THAI deletions encompass both HBA1 and HBA2 genes. Several other loss of function variants including splicing alterations, insertions/deletions, and nonsense changes have been reported in both the HBA1 and HBA2 genes. Patients with non-deletional forms of alpha thalassemia often present with more severe disease. The most common point variant found in Asian populations is Hemoglobin Constant Spring (HbCS) which abolishes the canonical termination codon in the HBA2 gene, c.427T>C (p.*143Gln) (Origa and Moi. 2016. PubMed ID: 20301608; Harteveld and Higgs. 2010. PubMed ID: 20507641; Galanello and Cao. 2011. PubMed ID: 21381239).
Multiplex Ligation-Dependent Probe Amplification (MLPA) enables the detection of deletion and duplications of single and multiple exons within a given gene (Eijk-Van Os and Schouten 2011). This test involves analysis only of the specific gene(s) of interest for each patient. The MLPA test is designed to have coverage for all exons for each targeted gene. It is a semi-quantitative technique to determine relative copy number using a multiplex PCR-based reaction. Only hybridized and ligated adjacent probe oligonucleotides of approximately 60 nucleotides in length are amplified using PCR and thus are specific for the sequence of interest. A stuffer sequence attached to the probe ensures a particular length for deciphering the probe target. Therefore, MLPA enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. For additional information please see www.mlpa.com.
This MLPA test is also capable of detecting Hemoglobin Constant Spring (HbCS), a common structural variant found in Asian populations is which abolishes the canonical termination codon in the HBA2 gene, c.427T>C (p.*143Gln) (Origa and Moi. 2016. PubMed ID: 20301608; Harteveld and Higgs, 2010. PubMed ID: 20507641; Galanello and Cao. 2011. PubMed ID: 21381239).
Indications for Test
Patients with HbH present with anemia, splenomegaly, and mild jaundice. Laboratory findings include hypochromia (low mean corpuscular hemoglobin), microcytosis (low mean corpuscular volume) with detectable inclusion bodies, moderate reticulocytosis, decreased hemoglobin levels. Ideal candidates have HPLC or capillary electrophoresis indicating Hb patterns consistent with HbH or Hb Bart (Origa and Moi. 2016. PubMed ID: 20301608; Harteveld and Higgs. 2010. PubMed ID: 20507641).
|Official Gene Symbol||OMIM ID|
|Alpha Thalassemia Sequencing Panel with CNV Detection|
|Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection|
|Neonatal Crisis Sequencing Panel with CNV Detection|
- Genetic Counselor Team - firstname.lastname@example.org
- Luke Drury, PhD - email@example.com
Multiplex Ligation-Dependent Probe Amplification Assay
As required, genomic DNA (gDNA) is extracted from the patient specimen. gDNA extracted from blood samples/submitted DNA from the patient is denatured and hybridized to MLPA probes specific to exonic or intronic regions of a particular gene(s). Each probe consists of two adjacent sequences that once hybridized to patient/reference DNA are ligated into a single probe. Fluorescently labeled PCR is then used to amplify each ligated probe using a common PCR primer set. The amplicon is then visualized during fragment separation using a capillary electrophoresis instrument. The relative peak height of each amplified probe from the patient’s sample is compared to four internal negative control results to determine relative copy number. For each patient sample the data for only the gene(s) of interest is analyzed and reported.
MLPA enables the detection of relatively small deletion and amplification mutations within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene. PreventionGenetics has established and verified this test’s accuracy and precision.
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.
Only the indicated gene or genes were analyzed. Test reports contain no information about other regions of the genome, including genes that are not requested, and genes that are not targeted. This test does not provide any information about deletions or duplications within repetitive elements.
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.
We cannot determine if the duplicated segment is inserted in tandem within the gene or inserted elsewhere in the genome. Similarly, we cannot determine the orientation of the duplicated segment (direct or inverted), and whether it will disrupt the open reading frame of the given gene.
For a single probe deletion or duplication we will compare MLPA results to sequencing results to ensure that no single nucleotide polymorphisms are underlying the specific probe, which may affect probe hybridization.
Any partial exonic deletions and duplications outside the probe binding sequence cannot be detected.
Impurities in the test and reference DNA samples can increase the chance of false positive or negative results. Where possible similar DNA extraction methods between test and reference samples are ideal for relative copy number analysis.
Our ability to detect minor copy number change, due for example to somatic mosaicism may be limited.
Unless otherwise indicated, MLPA results are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about copy number changes in other tissues.
We cannot be certain that the reference sequence(s) are correct. Exons, for example, may be misidentified.
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.
Normal findings within a targeted gene do not rule out the clinical diagnosis of a genetic disease.
Genetic counseling to help explain test results to the patients and to discuss reproductive or medical 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.