The SALSA MLPA probemix P102 HBB is an in vitro diagnostic (IVD)1
or a research use only (RUO) assay for the detection of deletions or duplications in the human beta-globin (HBB
) gene cluster and its regulatory region located on chromosome 11p15.4 in order to confirm a potential cause and clinical diagnosis for beta-thalassaemia or hereditary persistence of foetal haemoglobin (HPFH). In addition, this probemix can be used as confirmation of sequencing results for the presence of the mutation causing sickle cell anaemia (SCA) or sickle cell disease (SCD). This product can also be used for carrier testing of at-risk family members/individuals. This assay is optimised for use with human DNA derived from peripheral blood.
Deletions or duplications detected with the P102 HBB probemix should be verified by another technique. In particular, deletions and duplications detected by only a single probe always require validation by another method. Most defects in the HBB
gene region are point mutations, the majority of which are not detected by MLPA. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of the HBB
gene. This assay is not intended to be used as a standalone assay for clinical decisions. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.
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).
The beta-globin gene cluster, consisting of five functional genes (HBE1, HBG2, HBG1, HBD
), is located on the short arm of chromosome 11. Five nuclease hypersensitive sites are located upstream of this locus, constituting the beta-globin locus control region (LCR) which is responsible for correct transcription of the globin genes. Mutations involving one or more of the genes and/or the LCR may lead to a variety of haemoglobin disorders, together referred to as haemoglobinopathy.
Beta-thalassaemia is an autosomal recessive disorder, characterised by a reduction or absence of beta-globin chain production. Depending on the type of mutation, beta-thalassaemia patients experience a wide variety of symptoms, ranging from mild anaemia to severe transfusion-dependent haemolytic anaemia.
Beta-thalassaemia can be classified into three categories:
• Beta-thalassaemia minor: carrier of a single beta-thalassaemia mutation. Carriers present with microcytic hypochromic anaemia, however, in general they do not require any treatment.
• Beta-thalassaemia intermedia: both beta-globin genes are affected by (usually not completely inactivating) mutations. A substantial amount of functional beta-globin chains is still produced. Patients require sporadic blood transfusions, iron chelation therapy and folic acid supplementation. In some cases, a splenectomy may be necessary.
• Beta-thalassaemia major (also known as Cooley’s anaemia): function of both beta-globin genes is completely disrupted, leading to severe haemolytic anaemia, jaundice, hepatosplenomegaly and dysmorphic features due to expansion of the bone marrow. Patients are dependent on life-long regular blood transfusions and iron chelation therapy to survive. The only option for cure at this moment is bone marrow transplantation from a matching donor.
Until now, more than 600 point mutations in the HBB
gene have been described (http://globin.cse.psu.edu/hbvar/menu.html
) that lead to structural variants of the beta-globin chain and subsequently to abnormal haemoglobin molecules. A relatively small number of these abnormal haemoglobins can cause severe disease in homozygous or compound heterozygous combinations. The most frequently occurring mutation is the Haemoglobin S (HbS; HBB
:c.20A>T, p.Glu7Val) mutation. Other well-known haemoglobin variants are HbC, HbE, HbD-Punjab and HbO-Arab. Homozygosity for HbS or combined heterozygosity of HbS with a beta-thalassaemia mutation, a deletion of the HBB
gene or with another haemoglobin variant causes sickle cell disease (SCD). SCD patients suffer from infarctions in the microcirculation which lead to severe pain crises and organ damage, particularly in the bones, spleen, heart and lungs. Treatment consists of pain management and regular blood transfusion or erythrocyte exchange transfusion. Like for beta-thalassaemia major, the only option for cure at this moment is bone marrow transplantation.
In addition to beta-thalassaemia, deletions in the beta-globin gene cluster may also lead to other types of haemoglobinopathy. Depending on the gene(s) involved, they include epsilon-gamma-delta-beta-thalassaemia, gamma-delta-beta-thalassaemia, delta-beta-thalassaemia and hereditary persistence of foetal haemoglobin (HPFH). These conditions cause a variety of phenotypes, ranging from borderline-normal haematological indices with high levels of HbF (up to 40%) to microcytic hypochromic anaemia comparable to ‘regular’ beta-thalassaemia. During embryonic and foetal development, carriership of epsilon-gamma-delta-beta-thalassaemia can lead to severe haemolytic anaemia which may require intra-uterine blood transfusion (http://www.ncbi.nlm.nih.gov/books/NBK1426/
; Weatherall 2010; Steinberg et al. 2009; Galanello et al. 2010).
The SALSA MLPA Probemix P102-D1 HBB contains 49 MLPA probes with amplification products between 130 and 502 nucleotides (nt). This includes 40 probes for the beta-globin gene cluster and its flanking regions. In addition, nine 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.mlpa.com
Twenty-six probes are present that detect sequences in or very close to the globin genes. This includes two probes (136 and 180 nt) that detect sequences present in both HBG1
. Furthermore, one probe is specific for the HbS mutation in HBB
and will only generate a signal when the mutation is present. The probemix also contains seven probes for the locus control region. Finally, two probes detect a sequence centromeric of the locus control region and five probes detect a sequence telomeric of the beta-globin gene cluster. These flanking probes are included to delineate the extent of larger deletions/duplications in the beta-globin gene cluster.
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.mlpa.com
SALSA Binning DNA SD067:
The SD067 Binning DNA provided with this probemix can be used for binning of the HbS mutation (HBB
:c.20A>T, p.Glu7Val) specific probe (21234-L29609). SD067 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 SD067 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, as for this purpose true mutation positive patient samples or cell lines should be used. It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. RNase treatment is not required for SD067. For further details, please consult the SD067 Binning DNA product description, available online: www.mlpa.com