Test ID: AGPB
Alpha-Globin Gene Analysis

Diagnosis of alpha-thalassemia

Prenatal diagnosis of deletional alpha-thalassemia

Carrier screening for individuals from high-risk populations for alpha-thalassemia

Deletions and duplications only.

For prenatal specimens only: If amniotic fluid (non-confluent cultured cells) is received, amniotic fluid culture/genetic test will be added and charged separately. For any prenatal specimen that is received, maternal cell contamination studies will be added.

• Molecular Genetics-Congenital Inherited Diseases Patient Information Sheet
• Informed Consent for Genetic Testing

Dosage Analysis by Polymerase Chain Reaction (PCR)/Multiplex Ligation-Dependent Probe Amplification (MLPA)/Luminex Technology

(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)

Forms:

1. Molecular Genetics-Congenital Inherited Diseases Patient Information Sheet (Supply T521) in Special Instructions

2. New York Clients-Informed consent is required. Please document on the request form or electronic order that a copy is on file. An Informed Consent for Genetic Testing (Supply T576) is available in Special Instructions.

3. If not ordering electronically, submit a Molecular Genetics Request Form (Supply T245) with the specimen.

Specimen must arrive within 96 hours of collection.

Submit only 1 of the following specimens:

Specimen Type: Whole blood

Container/Tube:

Preferred: Lavender top (EDTA) or yellow top (ACD)

Acceptable: Any anticoagulant

Specimen Volume: 3 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send specimen in original tube.

Specimen Stability Information: Ambient (preferred)/Refrigerated

Due to the complexity of prenatal testing, consultation with the laboratory is required for all prenatal testing. Prenatal specimens can be sent Monday through Thursday and must be received by 5 p.m. CST on Friday in order to be processed appropriately. All prenatal specimens must be accompanied by a maternal blood specimen. Order MCC/88636 Maternal Cell Contamination, Molecular Analysis on the maternal specimen.

Specimen Type: Amniotic fluid

Container/Tube: Amniotic fluid container

Specimen Volume: 20 mL

Specimen Stability Information: Refrigerated (preferred)/Ambient

Acceptable:

Specimen Type: Confluent cultured cells

Container/Tube: T-25 flask

Specimen Volume: 2 flasks

Collection Instructions: Submit confluent cultured cells from another laboratory.

Specimen Stability Information: Ambient (preferred)/Refrigerated

The thalassemias are a group of inherited conditions characterized by decreased synthesis of 1 or more of the globin chains, resulting in an imbalance in the relative amounts of the alpha and beta chains. The excess normal chains precipitate in the cell, damaging the membrane and leading to premature red blood cell destruction. Additionally, the defect in hemoglobin synthesis produces a hypochromic, microcytic anemia. The frequency of thalassemia is due to the protective advantage against malaria that it gives carriers. Consequently, thalassemias are prevalent in populations from equatorial regions in the world where malaria is endemic.

Alpha-thalassemia is caused by decreased synthesis of alpha-globin chains. Four alpha-globin genes are normally present (2 on each chromosome 16). One, 2, 3, or 4 alpha-globin genes may be deleted or, less commonly, contain mutations. Deletions account for approximately 90% of disease-causing alleles in alpha thalassemia. Phenotypically, these deletions result in 4 categories of disease expression:

-Deletion of 1 alpha-chain: Silent carrier state, with a normal phenotype

-Deletion of 2 alpha-chains: Alpha-thalassemia trait (alpha-1 thalassemia), with mild hematologic changes but no major clinical difficulties

-Deletion of 3 alpha-chains: Hemoglobin H disease, which is extremely variable but usually includes anemia due to hemolysis, jaundice, and hepatosplenomegaly

-Deletion of all 4 alpha-chains: Hemoglobin Bart, with hydrops fetalis and almost invariably in utero demise

Less frequently alpha-thalassemia results from single point mutations. The most common nondeletion mutation is Hemoglobin Constant Spring (HbCS) (HBA2: c.427T >C). Point mutations other than HbCS and  alpha-thalassemia Saudi are not detected by this assay.

Alpha-thalassemia occurs in all ethnic groups but is especially common individuals of Southeast Asian and African ancestry. It is also frequent in individuals of Mediterranean ancestry. The carrier frequency is estimated to be 1 in 20 for Southeast Asians, 1 in 30 for African Americans, and 1 in 30 to 1 in 50 for individuals of Mediterranean ancestry. Both deletional and nondeletional (caused by point mutations) forms of alpha-thalassemia are found in individuals with Mediterranean ancestry. Deletions in cis (deletions on the same chromosome) are rare in African or Mediterranean populations, but are prevalent in Asian populations. Couples in which both partners carry deletions in cis are at risk of having a child with the fatal hemoglobin Bart hydrops fetalis syndrome.

An interpretive report will be provided.

An interpretive report will be provided.

In addition to disease-related probes, the multiplex ligation-dependent probe amplification technique utilizes probes localized to other chromosomal regions as internal controls. In certain circumstances, these control probes may detect other diseases or conditions for which this test was not specifically intended. Results of the control probes are not normally reported. However, in cases where clinically relevant information is identified, the ordering physician will be informed of the result and provided with recommendations for any appropriate follow-up testing.

Rare polymorphisms exist that could lead to false-negative or false-positive results. If results obtained do not match the clinical findings, additional testing should be considered.

A previous bone marrow transplant from an allogenic donor will interfere with testing. Call Mayo Medical Laboratories for instructions for testing patients who have received a bone marrow transplant.

Test results should be interpreted in the context of clinical findings, family history, and other laboratory data. Errors in our interpretation of results may occur if information given is inaccurate or incomplete.

This assay cannot be performed on chorionic villus specimens.

Hemoglobin Constant Spring and alpha-thalassemia Saudi are the only nondeletion types of alpha-thalassemia that will be detected by this assay. This test is not useful for diagnosis or confirmation of beta-thalassemia or hemoglobinopathies.

Hemoglobin electrophoresis should usually be done prior to this test to exclude other diagnoses or to identify non deletion types of alpha-thalassemia.

1. Harteveld CL, Voskamp A, Phylipsen M, et al: Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterized by high resolution multiplex ligation-dependent probe amplification. J Med Genet 2005; 42:922-931

2. Harteveld CL, Higgs DR: Alpha-thalassemia. Orphanet J Rare Dis 2010;5:13

This is a direct mutation analysis. Deletions within the alpha-globin locus and the Hemoglobin Constant Spring and  alpha-thalassemia Saudi point mutations are identified by a multiplex ligation-dependent probe amplification assay. Fifteen probes that hybridize throughout the alpha-globin locus from the HS40 promoter region through the 3'HVR region are utilized in order to maximize the information needed to map the approximate location of nearly all DNA deletions that occur. In addition, a PCR-based assay is used to detect the presence of the alpha-3.7 and alpha-4.2 deletions.(Bunn HF, Forget BG: Hemoglobin: Molecular, Genetic and Clinical Aspects. Philadelphia, WB Saunders Company, 1986; Weatherall DJ, Higgs DR, Clegg JB, et al: Heterogeneity and origins of the alpha-thalassemias. Birth Defects: Original Article Series 1987;23:3-14; Schouten JP, McElgunn CJ, Waaijer R, et al: Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002 Jun 15;30(12):e57)

Wednesday, Friday

Alpha-Globin Gene Analysis

81257-HBA1/HBA2 (alpha globin 1 and alpha globin 2) (eg, alpha thalassemia, Hb Bart hydrops fetalis syndrome, HbH disease), gene analysis, for common deletions or variant (eg, Southeast Asian, Thai, Filipino, Mediterranean, alpha3.7, alpha4.2, alpha20.5, and Constant Spring)

Reflex Tests

Amniotic Fluid Culture for Genetic Testing

88235-Tissue culture for amniotic fluid (if appropriate)

88240-Cryopreservation (if appropriate)

Maternal Cell Contamination, Molecular Analysis

81265-Comparative analysis using Short Tandem Repeat (STR) markers; patient and comparative specimen (eg, pre-transplant recipient and donor germline testing, post-transplant non-hematopoietic recipient germline [eg, buccal swab or other germline tissue sample] and donor testing, twin zygosity testing or maternal cell contamination of fetal cells (if appropriate)