Intended purpose
The SALSA MLPA Probemix P140 HBA is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² 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.

Copy number variations (CNVs) detected with P140 HBA should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Although most defects in the alpha-globin gene cluster are copy number changes, about 15% of the defects are due to point mutations in the HBA1 and HBA2 genes, most of which will not be detected by MLPA. It is therefore recommended to use this assay in combination with sequence analysis.

This device requires in depth knowledge of the complicated human alpha-globin gene cluster and assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, parental evaluation, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.

This device is not intended to be used for standalone diagnostic purposes, or for the detection of, or screening for, acquired or somatic genetic aberrations.

¹ Please note that this probemix is for in vitro diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the product is for research use only (RUO).
² To be used in combination with a SALSA MLPA Reagent Kit, SALSA Binning DNA SD031 and Coffalyser.Net analysis software.

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 is dependent on which of the genes harbours the mutation (HBA1 or HBA2), type of mutation and number of affected alpha-globin genes. More information on alpha-thalassaemia is available on http://www.ncbi.nlm.nih.gov/books/NBK1435/.

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 the commonly occurring deletions such as the 3.7 kb deletion (-α^3.7), the 4.2 kb deletion (-α^4.2), the South-East Asian deletion (--^SEA) and the Filipinian deletion (--^FIL). The remaining 15% of the alpha-thalassaemia cases results 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, alpha-globin gene duplication leads to a more severe phenotype in beta-thalassaemia patients because it aggravates the balance between alpha- and beta-globin chains.

Probemix content
The SALSA MLPA Probemix P140-C1 HBA contains 45 MLPA probes with amplification products between 130 and 481 nucleotides (nt). This includes 34 probes for the alpha-globin gene cluster and its flanking regions, including one probe specific for the Hb Constant Spring mutation in HBA2 which will generate a clear signal (>10% of the mean peak height of all reference probes in the sample) when the mutation is present. In addition, 11 reference probes are included that detect autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com).

Eight probes are present that detect sequences in the HBA genes. This includes two probes for HBA2 intron 2, two probes for HBA1 intron 2 and three probes (172, 214 and 220 nt) that detect sequences present in both HBA1 and HBA2. Next to these HBA-specific probes, the probemix contains 18 probes for sequences elsewhere in the alpha-globin gene cluster and two probes for the HS-40 regulatory element. Finally, one probe detects a sequence telomeric of the HS-40 regulatory element and five probes detect a sequence centromeric of the alpha-globin gene cluster. These flanking probes are included to delineate the extent of larger deletions/duplications in the alpha-globin gene cluster.

This probemix contains five probe pairs targeting locations with a very small sequence difference between HBA1 and HBA2: one probe detecting the HBA1 sequence and the other probe detecting the HBA2 sequence. Due to the close proximity of these genes, it is possible that in some healthy individuals the HBA2 sequence at one or more of these five locations is changed by gene conversion into the HBA1 sequence (or vice versa), without any clinical consequences. Probe pairs that can be affected in this way are the 160 & 165 nt intron 2 probes, the 244 & 250 nt intron 2 probes, the 391 & 190 nt probes, the 328 & 226 nt probes, and the 373 & 202 nt probes.

This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mrcholland.com.

SALSA Binning DNA SD031
The SD031 Binning DNA provided with this probemix can be used for binning of all probes including the Hb Constant Spring mutation (HBA2: 427T>C, p.*143Glnext*31) specific probe (S0585-SP0043-L09493). SD031 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD031 Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signal. It is not needed to perform an RNAse treatment on SD031 Binning DNA. For further details, please consult the SD031 Binning DNA product description, available online: www.mrcholland.com.

Sample DNA
Sample DNA developed for this product:

• SD031 Binning DNA - included with this probemix.