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SALSA MLPA Probemix P140 HBA

SALSA® MLPA® Probemix P140 HBA detects copy number variations in the alpha-globin (HBA) gene cluster and its regulatory region.

Specifications

Contents: 45 MLPA probes, including 33 probes for the alpha-globin gene cluster and its flanking regions, and 1 probe specific for the Hb Constant Spring mutation in HBA2.

Tissue: genomic DNA isolated from human peripheral whole blood and specified prenatal samples (see Intended Purpose).

Application: alpha-thalassaemia.

CE-marked and registered for in vitro diagnostic (IVD) use in selected territories.

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Key related products

SALSA MLPA Probemix P102 HBB

Contains probes for the HBB gene region.

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Intended purpose

The SALSA MLPA Probemix P140 HBA is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative assay for the detection of deletions or duplications in the alpha-globin (HBA) gene cluster and its regulatory region in genomic DNA isolated from human peripheral whole blood specimens. P140 HBA can be used to confirm parental aberrations in prenatal samples, in DNA isolated from (un)cultured amniotic fluid obtained in week 16 of pregnancy or later and free from blood contamination, or (un)cultured chorionic villi free from maternal contamination. In addition, this probemix can be used to detect the presence of the Hb Constant Spring mutation in the HBA2 gene. P140 HBA is intended to confirm a potential cause for and clinical diagnosis of alpha-thalassaemia, for molecular genetic testing of at-risk family members and for carrier screening in at-risk populations.

For the full intended purpose, see the product description.

Clinical background

Alpha-thalassaemia is the most common inherited haemoglobin disorder in the world. It is characterised by a reduced production of the alpha-globin chain, resulting in a decrease in the total amount of haemoglobin. In normal adult life, about 97% of the total haemoglobin level comprises haemoglobin A (HbA), which is composed of two alpha- and two beta-globin chains. The remaining 3% of adult haemoglobin consists of HbA2 and HbF (foetal haemoglobin), consisting of two alpha chains combined with two delta-globin chains or two gamma-globin chains, respectively.

The alpha-globin chains are encoded by the haemoglobin alpha 1 (HBA1) and alpha 2 (HBA2) genes, located in the alpha-globin gene cluster on chromosome 16p13.3. Defects in the HBA genes can lead to two clinically significant forms of alpha-thalassaemia. In the lethal Hb Bart’s hydrops foetalis syndrome, the two HBA1 and two HBA2 copies are all absent or defect. In HbH disease, only one functional HBA copy remains. In addition, two alpha-thalassaemia carrier states can be discriminated: in alpha-thalassaemia trait (heterozygous α0-thalassaemia or homozygous α +-thalassaemia), two functional HBA copies remain, whereas in “silent” alpha-thalassaemia (heterozygous α +-thalassaemia), three functional HBA copies are present. Next to defects in the HBA genes, alpha-thalassaemia can also be caused by deletions in the upstream hypersensitive (HS) sites, which constitute the regulatory elements of the alpha-globin gene cluster.

Alpha-thalassaemia patients can present with a wide variety of clinical symptoms, ranging from very mild anaemia to severe transfusion-dependent haemolytic anaemia. The phenotype depends on the gene that harbours the mutation (HBA1 or HBA2), the type of mutation, and the number of affected alpha-globin genes.

Alpha-thalassaemia is inherited in an autosomal recessive manner, with about 85% of all alpha-thalassaemia phenotypes caused by genomic deletions in the HBA1 and HBA2 genes. Most of these deletions can be detected by the MLPA technique, including commonly occurring deletions such as the 3.7 kb deletion (-α3.7), the 4.2 kb deletion (-α4.2), the South-East Asian deletion (--SEA), and the Filipinian deletion (--FIL). The remaining 15% of the alpha-thalassaemia cases result from one of at least 70 different point mutations, usually located within the HBA2 gene (Higgs and Weatherall, 2009; Harteveld and Higgs, 2010). The most common non-deletion mutation, which is frequently seen in Southeast Asia, is Hb Constant Spring, resulting from a mutation in the stop codon of the HBA2 gene. This mutation leads to the production of an elongated α-globin chain. Hb Constant Spring is produced in very small amounts because its mRNA is unstable. Heterozygotes for elongated globin chain variants such as Hb Constant Spring present with an α0-thalassemia phenotype. Presence of the Hb Constant Spring mutation can be detected by the P140 probemix.

In addition to many deletion types, several duplications have also been described in the alpha-globin gene cluster. These duplications vary in size, ranging from only a single duplicated HBA gene to large segmental duplications of the complete alpha-globin gene cluster, including the regulatory elements. Duplication of one or both HBA genes is clinically benign. However, when co-inherited with a beta-thalassaemia mutation, an HBA duplication leads to a more severe phenotype in beta-thalassaemia patients because it aggravates the balance between alpha- and beta-globin chains.

More information on alpha-thalassaemia is available on http://www.ncbi.nlm.nih.gov/books/NBK1435/.

Regulatory status

SALSA MLPA Probemix P140 HBA is CE-marked for in vitro diagnostic (IVD) use. This assay has also been registered for IVD use in Colombia and Israel.

This assay is for research use only (RUO) in all other territories.

SALSA Sample DNA for this product

SALSA Binning DNA SD031 is an artificial DNA sample with a signal for all probes in the P140 HBA probemix. Inclusion of a reaction with SD031 in initial experiments and in experiments following a change in electrophoresis conditions is recommended to aid in the creation of a bin set that links peaks to the probes that produce them. Binning DNA cannot be used as a reference sample in the MLPA data analysis, and cannot be used to quantify the signals of mutation-specific probes.

A vial of SALSA Binning DNA SD031 is included with every order of the P140 HBA probemix, but it is possible to order additional vials separately.

For more information, see the product description.

Product documentation

Version C1

Other versions

Contact us for earlier versions.

List prices

Product

Item no.
Description
Technology
Price
P140-025R
SALSA MLPA Probemix P140 HBA – 25 rxn
MLPA
€ 281.00
P140-050R
SALSA MLPA Probemix P140 HBA – 50 rxn
MLPA
€ 550.00
P140-100R
SALSA MLPA Probemix P140 HBA – 100 rxn
MLPA
€ 1075.00

Required reagents

A general SALSA MLPA Reagent Kit is required for MLPA experiments (to be ordered separately).

Item no.
Description
Technology
Price
EK1-FAM
SALSA MLPA Reagent Kit – 100 rxn – FAM (6 vials)
MLPA MS-MLPA
€ 341.00
EK1-Cy5
SALSA MLPA Reagent Kit – 100 rxn – Cy5 (6 vials)
MLPA MS-MLPA
€ 341.00
EK5-FAM
SALSA MLPA Reagent Kit – 500 rxn – FAM (5×6 vials)
MLPA MS-MLPA
€ 1571.00
EK5-Cy5
SALSA MLPA Reagent Kit – 500 rxn – Cy5 (5×6 vials)
MLPA MS-MLPA
€ 1571.00
EK20-FAM
SALSA MLPA Reagent Kit – 2000 rxn – FAM (5×6 vials)
MLPA MS-MLPA
€ 6037.00

Sample DNAs (included)

A vial is included with every order of this probemix, but additional vials can also be purchased separately.

Item no.
Description
Technology
Price
SD031
SALSA Binning DNA SD031 – 6 rxn
MLPA
€ 23.70
How to Order

Positive samples

Inclusion of a positive sample is usually not required, but can be useful for the analysis of your experiments. MRC Holland has very limited access to positive samples and cannot supply such samples. We recommend using positive samples from your own collection. Alternatively, you can use positive samples from an online biorepository, such as the Coriell Institute.

The commercially available positive samples below have been tested with the current (C1) version of this product and have been shown to produce useful results.

Resources

General resources

Learning

Publications

Selected publications using SALSA MLPA Probemix P140 HBA

  • Cao J et al. (2017). Prenatal Diagnosis and Molecular Analysis of a Large Novel Deletion (- -JS) Causing α0-Thalassemia. Hemoglobin. 41:243-7.
  • Chau JFT et al. (2022). Comprehensive analysis of recessive carrier status using exome and genome sequencing data in 1543 Southern Chinese. NPJ Genom Med. 7:23.
  • Chen DM et al. (2020). Diagnosis of the accurate genotype of HKαα carriers in patients with thalassemia using multiplex ligation-dependent probe amplification combined with nested polymerase chain reaction. Chin Med J (Engl). 133:1175-81.
  • Hashemi-Soteh SMB et al. (2020). Alpha-globin gene mutation spectrum in patients with microcytic hypochromic anemia from Mazandaran Province, Iran. J Clin Lab Anal. 34:e23018.
  • Laghmich A et al. (2019). Alpha-Thalassemia in North Morocco: Prevalence and Molecular Spectrum. Biomed Res Int. 2019:2080352.
  • Mikobi TM et al. (2018). Association between sickle cell anemia and alpha thalassemia reveals a high prevalence of the α3.7 triplication in congolese patients than in worldwide series. J Clin Lab Anal. 32:e22186.
  • Perera S et al. (2019). Genotype-phenotype association analysis identifies the role of α globin genes in modulating disease severity of β thalassaemia intermedia in Sri Lanka. Sci Rep. 9:10116.
  • Pooladi A et al. (2022). The spectrum of Alpha and Beta Thalassemia Mutations: A 10-year Population-based Study of the Premarital Health Screening Program in West of Iran. Iran J Ped Hematol Oncol. 12:190-8.
  • Rizo-de la Torre LDC et al. (2021). Molecular and Hematological Analysis of Alpha- and Beta-Thalassemia in a Cohort of Mexican Patients. Genet Test Mol Biomarkers. 25:247-52.
  • Steinberg-Shemer O et al. (2017). Whole-exome sequencing identifies an a-globin cluster triplication resulting in increased clinical severity of β-thalassemia. Cold Spring Harb Mol Case Stud. 3:a001941.

References

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CE

CE-marked products are for In Vitro Diagnostic (IVD) use only in EU (candidate) member states and members of the European Free Trade Association (EFTA), and the UK.

CO

IVD-registered in Colombia.

IL

IVD-registered in Israel.