WO2014053463A1 - Methods and tools for vel blood group typing - Google Patents
Methods and tools for vel blood group typing Download PDFInfo
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- WO2014053463A1 WO2014053463A1 PCT/EP2013/070403 EP2013070403W WO2014053463A1 WO 2014053463 A1 WO2014053463 A1 WO 2014053463A1 EP 2013070403 W EP2013070403 W EP 2013070403W WO 2014053463 A1 WO2014053463 A1 WO 2014053463A1
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- vel
- seq
- smim1
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- polynucleotide
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- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
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- C12Q2600/00—Oligonucleotides characterized by their use
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Definitions
- the present invention relates to the technical field of blood group determination, in particular to Vel blood group typing and specifically to tools and methods for discriminating between Vel positive and Vel negative individuals.
- erythrocyte antigens exist which do not yet meet the definition of a blood group system. Most of these antigens are either nearly universal in human blood (high-prevalence antigens) or extremely rare (low- prevalence antigens). Reagents to test for these antigens are difficult to find and many cannot be purchased commercially to date.
- PCR polymerase chain reaction
- DNA-based assays can readily determine homozygosity and heterozygosity, and in some cases, hemizygosity of a blood group allele, which is beyond the capability of conventional serology.
- Another added benefit of DNA-based blood group typing beyond the capabilities of traditional serology is the possibility to determine the foetal blood group by testing DNA derived from foetal cells in amniotic fluid or chorionic villus biopsies, or increasingly common even from cell-free foetal DNA in maternal plasma. It is a great frustration to end users and manufacturers alike that the limiting factor in providing phenotype information on patients, blood donors and reagent red blood cells (RBCs) is the availability of reliable antisera.
- RBCs red blood cells
- the Vel blood group antigen first described by Sussman and Miller (Rev. Hematol. 1952;7:368-71 ), is expressed in RBCs and is one of several so-called orphan blood group antigens for which the molecular basis is unknown.
- Vel is considered one of the most clinically relevant, as Vel negative patients, which have been immunised following transfusion or pregnancy, and subsequently transfused with red blood cells from Vel positive donors are at risk of severe side effects including acute intravascular hemolysis.
- the present inventors have uncovered the genetic basis of Vel and found a mutation (specifically, a 17-basepair deletion) in a previously uncharacterized gene (small integral membrane protein 1 ; Official Gene Symbol SMIM1, previously LOC388588; Entrez Gene ID 388588), for which all tested Vel negative individuals were
- Vel blood group antigen on RBCs is dependent on expression of the SMIM1 gene, the mRNA of which encodes a type 1 transmembrane protein.
- the Vel- negative phenotype occurs when this protein cannot be expressed.
- the present invention relates to a method of identifying the Vel phenotype of an individual, said method comprising discriminating between Vel positive and Vel negative phenotypes by analysing in a biological sample from said individual the composition of the SMIM1 gene, wherein at least one intact SMIM1 gene is indicative of a Vel positive phenotype, and wherein a SMIM1 gene comprising a mutation resulting in abolished protein expression, or in expression of a non-functional protein, is indicative of a Vel negative phenotype.
- the SMIM1 gene is typically defined by a polynucleotide sequence comprising SEQ ID NO. 1 , or a sequence variant thereof.
- SMIM1 gene may however comprise also naturally-occurring regulatory elements such as but not limited to promoters and enhancers.
- the present invention concerns a method of detection and/or quantitation of a splice variant of SMIM1 in a sample, the method comprising making
- cDNA complementary DNA
- mRNA messenger RNA
- the invention concerns a method of identifying a Vel positive individual, comprising the steps:
- a) performing amplification of a polynucleotide of the subject by contacting a polynucleotide from a cell of the subject with an oligonucleotide primer having a sequence being complementary to at least 10 consecutive nucleotides of a
- step (b) detecting an amplicon from step (a), whereby the detection of an amplicon identifies the subject as a Vel positive individual.
- the invention concerns a method of identifying a Vel negative individual, comprising the steps:
- a) performing amplification of a polynucleotide of the subject by contacting a polynucleotide from a cell of the subject with an oligonucleotide primer having a sequence being complementary to at least 10 consecutive nucleotides of a
- step (b) detecting an amplicon from step (a), whereby the detection of an amplicon identifies the subject as a Vel negative individual.
- the present invention concerns a method of detecting in a sample, a cell that expresses a Vel antigen comprising detecting in the sample a polynucleotide that encodes a Vel antigen.
- the SMIM1 gene which typically comprises SEQ ID NO. 1 or a sequence variant thereof encodes a polypeptide which is associated with the Vel antigen.
- Vel blood group antigen on RBCs is dependent on expression of the SMIM1 gene, the mRNA of which encodes a type 1 transmembrane protein.
- the Vel- negative phenotype occurs when this protein cannot be expressed, for instance due to the 17-bp deletion.
- the only way to identify Vel negative individuals has been by cell-based phenotyping assays involving antiserum from immunized patients, the supply of which is severely limited.
- the genetic testing of the present method can be performed on virtually unlimited numbers of individuals at low cost, enabling screening of large blood donor populations to identify more Vel negative donors and facilitating the procurement of Vel negative blood units to patients in need.
- the present invention concerns a method for blood transfusion comprising the steps:
- the method for blood transfusion is applicable to a method of treatment of a disease or disorder associated with erythrocytes, such as, but not limited to anemia.
- Vel negative females may carry Vel positive foetuses.
- erythrocytes from the Vel positive foetus are transferred to the Vel negative mother, this may result in an immune response of the mother, thus raising anti-Vel antibodies against the Vel positive erythrocytes.
- This is potentially lethal for the foetus, especially during a second or subsequent pregnancy of the Vel negative female.
- the present invention concerns a method of prophylactic treatment of a Vel negative pregnant female individual comprising:
- the present invention concerns an isolated polynucleotide comprising a sequence variant of SEQ ID NO. 1 , or an isolated polynucleotide comprising a sequence variant of a sequence being complementary to said SEQ ID NO. 1 , wherein the sequence variant comprises at least one mutation or polymorphism, and wherein the mutation or polymorphism results in abolished transcription and/or protein translation and/or absence of the protein on the cell surface.
- the present invention concerns an isolated polynucleotide comprising a sequence variant of SEQ ID NO. 1 , or an isolated polynucleotide comprising a sequence variant of a sequence being complementary to said SEQ ID NO. 1 , wherein the sequence variant comprises at least one mutation or polymorphism, wherein the mutation or polymorphism results in translation of a Vel antigen having an altered epitope, wherein the altered epitope is not recognised by anti-Vel antibodies.
- the present inventors have developed tools for performing the necessary analyses in order to appropriately discriminate between Vel positive and Vel negative samples.
- One such important tool includes oligonucleotide primers for the production of an amplicon which may be detected by e.g. gel electrophoresis.
- the present invention concerns an isolated oligonucleotide having a sequence selected from the group consisting of SEQ ID NO. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 and 23, as well as use of said isolated oligonucleotide in the method defined herein.
- the present invention concerns an isolated polypeptide having the sequence of SEQ ID NO. 5, or a fragment or a variant thereof, wherein said fragment comprises at least 20 consecutive amino acid residues of SEQ ID NO. 5, and wherein said variant is at least 80% identical to SEQ ID NO. 5, and wherein said variant comprises at least one Serine and/or Threonine residue, and wherein at least one Serine and/or Threonine residue of said SEQ ID NO. 5 or said fragment or variant thereof, is O-glycosylated.
- Important polypeptide fragments defining the Vel antigen are fragments of SEQ ID NO. 5.
- the present invention concerns an isolated peptide having a sequence selected from the group consisting of SEQ ID NOs. 6, 7, 8, 9 and 10.
- An isolated polynucleotide, encoding upon expression a peptide having a sequence selected from the group consisting of SEQ ID NOs. 6, 7, 8, 9 and 10 is also an aspect of the present invention.
- the present inventors have raised antibodies capable of recognizing the above defined peptide fragments. Accordingly, in one aspect, the present invention concerns an antibody capable of recognising an epitope of a Vel antigen, wherein the epitope is defined by a peptide sequence selected from the group consisting of SEQ ID NOs. 6, 7, 8, 9 and 10, or an O-glycosylated peptide sequence selected from SEQ ID NOs. 6, 7, 8, 9 and 10.
- the invention concerns an antibody capable of recognising an epitope of a Vel antigen, wherein the epitope is defined by a peptide having the SEQ ID NO: 1
- variant is at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identical to said SEQ ID NO.
- an O-glycosylated variant of SEQ ID NO. 5 such as at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identical to a O-glycosylated variant of SEQ ID NO. 5 .
- the present invention also concerns a method of making a polyclonal antibody, the method comprising:
- the invention concerns a method of making a monoclonal antibody, the method comprising:
- step a isolating from the culture monoclonal antibodies which bind specifically to the polypeptide or polypeptide fragment of step a).
- the invention concerns a kit for detecting a Vel antigen and/or discriminating between samples from Vel negative and Vel positive individuals, said kit comprising at least two isolated oligonucleotide primers selected from the group consisting of SEQ ID NO. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 and 23.
- oligonucleotide primer is an oligonucleotide primer being complementary to a sequence selected from the group consisting of SEQ ID NO. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 and 23.
- the oligonucleotide primer is a forward primer of an oligonucleotide primer selected from the group consisting of SEQ ID NO. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 and 23.
- the oligonucleotide primer is a reverse primer of an oligonucleotide primer selected from the group consisting of SEQ ID NO. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 and 23.
- the oligonucleotide primer is a sense primer of an
- oligonucleotide primer selected from the group consisting of SEQ ID NO. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 and 23.
- the oligonucleotide primer is an antisense primer of an oligonucleotide primer selected from the group consisting of SEQ ID NO. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 and 23.
- the present invention is well suited for adaptation to high-throughput scale by applying oligonucleotides, peptides or antibodies to a surface thus forming a device for detecting Vel antigens or Vel antigen associated molecules.
- the present invention concerns a kit comprising a microchip array comprising one or more polynucleotides selected from the group consisting of SEQ ID NO. 1 , 2, 3, 4, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 32, 33, 34, 35 and 36 or one or more fragments of said polynucleotide.
- the invention concerns a kit comprising a microchip array comprising one or more peptides or polypeptides selected from the group consisting of SEQ ID NO. 5, 6, 7, 8, 9 and 10, or one or more fragments of said peptide or polypeptide wherein said fragment comprises at least 5 consecutive amino acids of said SEQ ID NO. 5, 6, 7, 8, 9 and 10.
- the invention concerns a kit comprising cells presenting a Vel antigen.
- the present invention concerns the use of an isolated
- polynucleotide which upon expression encodes a polypeptide having the amino acid sequence of SEQ ID NO: 5, or a fragment or variant thereof, as a medicament or as a diagnostic marker.
- Figure 1 Genomic organisation of SMIM1. Exons are shown as boxes while intronic sequence is depicted as a line. Dark grey represents non-coding exons, except for the alternative exon 1 (labelled exon 1 a in the figure) which is in light grey; black represents the open reading frame. Positions of the genomic DNA amplification primers are shown by the arrows.
- FIG. 1 PCR-RFLP of SMIM1 exon 3 using Siyl.
- the wild type amplicon is digested into two fragments; whilst the mutated amplicon is not digested by the enzyme. Additionally, this amplicon can be distinguished on the gel by size, based on the 17-bp difference.
- FIG. 3 Allele-specific PCR (ASP) of genomic DNA from individuals determined by sequencing to be w/w, w/m or m/m.
- the control band is generated by amplification of a region of SMIM1 encompassing the 17 bp deletion in the absence of allele-specific amplification.
- FIG. 4 Gene-specific PCR (GSP). Amplification of the region surrounding the 17-bp deletion permits discrimination of w/w, m/m and w/m samples on 3% agarose gel based solely on band size.
- Figure 5 Medium-throughput screening. An assay to detect six clinically relevant blood group polymorphisms was developed to amplify DNA directly from whole blood. These alleles are differentiated by amplicon size and can be readily identified by gel electrophoresis (shown here) or by fragment analysis using labelled probes (data not shown). Primers that amplify wild type SMIM1 have been incorporated into the assay and DNA from Vel- individuals are not amplified by the SMIM1 primers as indicated in lane 4. Figure 6. Panel A is a Western blot showing the highly specific Vel antigen
- Panel B shows the same membrane stripped and reprobed with anti-GAPDH as a loading control.
- Vel positive samples are genotypically wild type homozygotes; Vel negative samples are genotypically mutant homozygotes.
- the Vel weakly positive ( w ) sample was a genotypically determined wild type/mutant heterozygote which expresses SMIM1 weakly.
- FIG. 7a Histogram showing the natural variation in Vel antigen expression on normal RBCs homozygous for wild type SMIM1 .
- the black lines represent different examples of Vel- positive RBCs (labelled AK1 , AK8, AK10, AK1 1 ) tested with anti-Vel, followed by a FITC-labelled anti-human IgG (2o antibody).
- the shaded histograms indicate reactivity of AK1 RBCs tested with anti-human IgG only (left), and with anti-Vel (right).
- 7b Figure to show the influence of SMIM1 zygosity on Vel antigen expression.
- FIG. 8 Overview of requested high-incidence-antigen-negative blood over the period 2005 to 2010 from the American Rare Donor Program.
- Vel negative blood bar boxed for clarity.
- Vel negative blood is the most frequently requested blood type negative for an orphan blood group, i.e. where typing, selection and confirmation cannot be done by nucleic-acid-based methods.
- FIG. 9a shows the relative mRNA expression following transduction of HEL cells with a transduction controls (HEL wt and -ve), and with shRNA clone G at an MOI (multiplicity of infection) of 0.5U (G0.5-M).
- Figure 9b shows western blotting with the polyclonal rabbit anti-SMIM1 of HEL cells following transduction with a transduction control (HEL wt), and with shRNA clone G at an MOI of 0.5U, 1.0U, 2.0U, 3.0U, 4.0U. Knockdown of both mRNA transcript and protein is clearly demonstrated. Detailed description of the invention
- the Vel blood group antigen is located on red blood cells (RBCs) and is one of several so-called orphan blood group antigens for which the molecular basis is unknown. It is clinically important and thus identification of the carrier molecule would permit the development of a screening assay to identify blood donors appropriate for donation to immunized Vel negative/negative patients, and also patients at risk of producing an unwanted antibody.
- RBCs red blood cells
- the present inventors provided understanding of the molecular and genetic basis of the Vel blood group antigen. This has been achieved through a range of serological and biochemical investigations, including investigations of candidate genes. These investigations resulted in estimates of the size of the red blood cell protein carrying the Vel antigen and a likely homodimer configuration.
- the inventors determined the DNA sequence of SMIM1 in Vel negative donors and found a 17-basepair deletion that is predicted to destroy the transmembrane domain of the protein, providing sufficient explanation for a so-called null or knock-out phenotype.
- the present invention concerns an isolated polynucleotide comprising a sequence variant of SEQ ID NO. 1 , or an isolated polynucleotide comprising a sequence variant of a sequence being complementary to said SEQ ID NO. 1 , wherein the sequence variant comprises at least one non-sense mutation, and wherein the nonsense mutation results in abolished transcription and/or protein translation thus resulting in absence of the protein on the cell surface.
- the non-sense mutation is a deletion, such as a deletion of between 1 and 100 nucleotides, such as between 2 and 90 nucleotides, such as between 3 and 80 nucleotides, such as between 4 and 70 nucleotides, such as between 5 and 60 nucleotides, such as between 6 and 50 nucleotides, such as between 7 and 40 nucleotides, such as between 8 and 30 nucleotides, such as between 9 and 25 nucleotides, such as between 10 and 20 nucleotides, such as between 15 and 19 nucleotides, such as between 16 and 18 nucleotides, such as 17 nucleotides.
- the deletion is a deletion of 17 nucleotides.
- the sequence variant of the above defined polynucleotide is between 60% and 99.9% identical, such as between 70% and 99.8% identical, such as between 80% and 99.7% identical, such as between 90% and 99.8% identical, such as between 95% and 99.56% identical, such as between 96% and 99.55% identical, such as between 97% and 99.54% identical, such as between 98% and 99.9% identical, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 99.1 , at least 99.9%, such as 100% identical to SEQ ID NO. 1 or a sequence being complementary to SEQ ID NO. 1. or a sequence being complementary to SEQ ID NO. 1.
- sequence variant of the above isolated polynucleotide has the sequence selected from the group consisting of SEQ ID NO. 2 and 4 or a sequence being complementary to SEQ ID NO. 2 or 4.
- the isolated polynucleotide has the sequence of SEQ ID NO: 31 , and in another embodiment the isolated polynucleotide comprises the sequence of SEQ ID NO: 31 .
- the present inventors have developed tools for performing the necessary analyses in order to appropriately discriminate between Vel positive and Vel negative samples, methods which are described herein below.
- One important tool useful in said methods includes oligonucleotide primers for the production of an amplicon (an amplified polynucleotide sequence) which may be detected by conventional methods, e.g. gel electrophoresis.
- the present invention concerns an isolated oligonucleotide having a sequence selected from the group consisting of SEQ ID NO. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 and 23, as well as use of said isolated oligonucleotide in the method defined herein.
- the analysis can be performed using any suitable method known by those of skill in the art.
- suitable methods include Gel electrophoresis, Real-time PCR, Mass spectrometry (e.g. MALDI-TOF), Flurochrome-labelled oligonucleotide extention, Fluorochrome-labelled sequence-specific amplification (e.g. xMAP technology), sequencing including dye-termination sequencing, and other next generation sequencing techniques that include but are not limited to Pyrosequencing, sequencing by ligation (SOLiD) and Ion Torrent semiconductor sequencing.
- a Vel negative individual according to the present invention has a Vel negative phenotype.
- a Vel positive individual according to the present invention has a Vel positive phenotype. Accordingly the expressions Vel negative individual and Vel negative phenotype are use interchangeably herein, the meaning of which is well known by the person of skill in the art.
- the methods provided in the present invention have the aim of characterizing the Vel antigen phenotype of an individual such as a blood donor or blood recipient (patient receiving blood e.g. via transfusion).
- a blood group antigen phenotype is defined by the presence or absence of blood group antigens on the surface of the erythrocyte.
- Blood group antigens are unique, inherited, structural differences found on the proteins, glycoproteins and glycolipids that make up the external surface of the erythrocyte. All blood group antigens have been identified by specific antibodies present in the plasma of different individuals. With the exception of some "naturally-occurring" antibodies, most antibodies to blood group antigens have been made as a response to the introduction e.g.
- Antigens that do not belong in a blood group system are assigned to one of six blood group collections (201 series) or to either the 700 series (low frequency antigens) or the 901 series (high frequency antigens) as defined by the International Society for Blood Transfusion (ISBT) Working Party on Red Cell
- Phenotype can also be used to summarize the known antigens on a person's erythrocytes, e.g. Patient A's phenotype was D+C-E+c+e+, K-, Fy(a+b+). This means that Patient A's erythrocytes expressed E, c, e, Fy a and Fy b blood group antigens but did not express C or K antigens.
- the present invention thus concerns a method of identifying the Vel phenotype of an individual, said method comprising discriminating between Vel positive and Vel negative phenotypes by analysing in a biological sample from said individual the composition of the SMIM1 gene, wherein at least one intact SMIM1 gene is indicative of a Vel positive phenotype, and wherein a SMIM1 gene comprising a mutation resulting in abolished protein expression, or in expression of a non-functional protein, is indicative of a Vel negative phenotype.
- the mutation described herein above may be any mutation, however in a preferred embodiment the mutation is a deletion, such as a deletion of SEQ ID NO: 31 from the wild type SMIM1 gene.
- the mutation may also be a fragment or variant of said SEQ I D NO: 31 , wherein said fragment comprises at least 12 consecutive nucleotides of said SEQ ID NO: 31 , and wherein in said variant, no more than 5 nucleotides have been altered to 5 different nucleotides.
- the mutation is a SNP, such as a dinucleotide exchange 2907t>c and 2908g>a of SEQ ID NO:1 .
- the SMIM1 gene comprises multiple mutations such as a combination of said deletion and said SNPs.
- the method according to the present invention comprises the steps: a) providing a sample,
- the present invention relates to a method of determining the Vel blood group phenotype of an individual, said method comprising discriminating between Vel positive and Vel negative phenotypes by analysing in a biological sample the composition of:
- SMIM1 is typically defined by a polynucleotide sequence comprising SEQ ID NO. 1 or a sequence variant thereof.
- the concept SMIM1 gene may however comprise naturally occurring regulatory elements such as but not limited to promoters and enhancers.
- the present invention comprises detecting SMIM1 associated genetic markers located upstream or downstream of SEQ ID NO. 1 in the genome. This may be performed by applying any one of the methods of the present invention where the amplification is performed by:
- i) providing a biological sample comprising genomic DNA ii) contacting the sample comprising genomic DNA with a first and a second PCR oligonucleotide primer, wherein said first primer comprises at least 10 nucleotides being complementary to at least 10 consecutive nucleotides located located upstream (5') of nucleotide position 1 of SEQ ID NO. 1
- said second primer comprises at least 10 nucleotides being complementary to at least 10 consecutive nucleotides located downstream (3') of nucleotide position 3195 of SEQ ID NO. 1 , with the proviso that said first and said second primer are not both selected from a sequence being complementary to SEQ ID NO.
- step iii) obtaining an amplicon; iv) performing qualitative and/or quantitative analysis of the amplicon of step iii); comparing the length of the amplicon, to at least one Vel positive control, wherein a length differing from the Vel positive control indicates that the sample is Vel negative.
- the SMIM1 gene comprises the sequence of SEQ ID NO: 1 or a sequence variant thereof wherein the sequence variant is between at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as between 99.1 and 99.9%, such as between 99.4 and 99.6%, such as 100% identical to SEQ ID NO. 1 or a sequence being complementary to SEQ ID NO. 1
- the SMIM1 gene comprises the sequence being complementary to SEQ ID NO. 1 or a sequence variant thereof wherein the sequence variant is between at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as between 99.1 and 99.9%, such as between 99.4 and 99.6%, such as 100% identical to the sequence being complementary to SEQ ID NO. 1
- the method of the present invention can in principle be applied to any genetic material originating from any organism, however the present invention is particularly useful for detecting Vel negative blood.
- the method of the present invention is for detection of Vel negative blood.
- the method of the present invention is for detection or identification of a Vel negative subject such as a human being.
- the method for discriminating between Vel negative and Vel positive samples, or individuals, by analysing the SMIM1 composition comprises identifying subjects whose genome comprises SEQ ID NO. 1 , said subjects being Vel positive, and subjects whose genome comprises SEQ ID NO. 2,
- the method for discriminating between Vel negative and Vel positive samples, or individuals, by analysing the SMIM1 composition comprises identifying subjects whose genome comprises SEQ ID NO. 1 , 3 or 35, said subjects being Vel positive, and subjects whose genome comprises SEQ ID NO. 2, 32, 33, 34 or 36,
- the only way known for identifying Vel negative blood donors was by cell-based phenotyping assays based on serum or plasma from immunized patients, the supply of which is severely limited.
- the genetic testing of the present method can be performed on virtually unlimited numbers of individuals at low cost, enabling screening of large blood donor populations to identify more Vel negative donors and facilitating the procurement of Vel negative blood units to patients in need.
- the methods of the present invention are based on the finding of the present inventors of the molecular basis for Vel negativity. In principle, any method which utilises the differences in the DNA code of the SMIM1 gene can be applied to discriminate between Vel negative and Vel positive individuals. This also includes pre-processing the genomic DNA (gDNA) prior to performing the analysis, as well as preparing cDNA from transcribed mRNA.
- sample of c) is from a Vel positive subject
- sample of d is from a Vel negative subject.
- the method of the present invention comprises: a) providing a biological sample comprising a SMIM1 polynucleotide, b) amplifying at least a fragment of the SMIM1 polynucleotide, wherein the SMIM1 polynucleotide has a sequence selected from the group consisting of SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 and 36 or a fragment or variant thereof wherein the variant is at least 90% identical to said sequence selected from the group consisting of SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 and 36,
- any SMIM1 or SMIM1 related polynucleotide such as a polynucleotide being at least at least 50% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. at least 55% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36 e.g. at least 60% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. at least 65% identical to said SEQ ID NO.
- any polynucleotide which is complementary to a SMIM1 or SA/ /7-related polynucleotide such as a polynucleotide which is complementary to a polynucleotide which is at least 50% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. a polynucleotide which is complementary to a polynucleotide which is at least 55% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g.
- a polynucleotide which is complementary to a polynucleotide which is at least at least 60% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36 e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 65% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 70% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least at least at least
- a polynucleotide which is complementary to a polynucleotide which is at least at least 83% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36 e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 84% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 85% identical to said SEQ ID NO.
- a polynucleotide which is complementary to a polynucleotide which is at least at least 86% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36 e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 87% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 88% identical to said SEQ ID NO.
- a polynucleotide which is complementary to a polynucleotide which is at least at least 89% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36 e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 90% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 91 % identical to said SEQ ID NO.
- a polynucleotide which is complementary to a polynucleotide which is at least at least 92% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36 e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 93% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 94% identical to said SEQ ID NO.
- a polynucleotide which is complementary to a polynucleotide which is at least at least 95% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36 e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 96% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 97% identical to said SEQ ID NO.
- a polynucleotide which is complementary to a polynucleotide which is at least at least 98% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36 e.g. a polynucleotide which is complementary to a polynucleotide which is at least at least 99% identical to said SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 or 36, e.g. a polynucleotide which is complementary to a polynucleotide which has a sequence selected from teh group consisting of SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 and 36.
- the method of discriminating between Vel negative and Vel positive individuals as outlined above can utilise any suitable tool or method known in the art which tool or method is suitable for analysing biomolecules such as DNA, RNA and proteins.
- the method used for analysing SMIM1 composition of an individual or a sample is carried out by a process which may be selected from the group consisting of: gene specific PCR, allele specific PCR, PCR- RFLP, allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, sequencing, 5' nuclease digestion, molecular beacon assay,
- oligonucleotide ligation assay size analysis, and single-stranded conformation polymorphism.
- the method for discriminating Vel positive and Vel negative samples or individuals is gene specific PCR.
- the gene specific PCR method comprises the steps of: i) providing a biological sample, ii) amplifying a SMIM1 polynucleotide by applying a first and a second oligonucleotide primer, thus obtaining an amplicon,
- oligonucleotide primer pairs capable of hybridizing with the target polynucleotide are within the scope of the present invention.
- the first oligonucleotide primer is at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97% identical, more preferably at least 98% identical, more preferably at least 99%, more preferably at least 100% identical to SEQ ID NO: 22 (LOCex3f_screen), and the second oligonucleotide primer is at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97% identical, more preferably at least 98% identical, more preferably at least 99%, more preferably at least 100% identical to SEQ ID NO: 23 (LOCex3r_screen).
- said first and said second oligonucleotide primer are capable of hybridizing to said SMIM1 polynucleotide.
- the method for discriminating Vel positive and Vel negative samples or individuals is by allele specific PCR.
- the allele specific PCR method comprises the steps of: i) providing a biological sample,
- step iii) performing qualitative and/or quantitative analysis of the SMIM1 amplicon obtained in step ii), and iv) comparing the length of the amplicon, to at least one Vel positive control, wherein a length differing from the Vel positive control indicates that the sample is from a Vel negative subject.
- the oligonucleotide primers of the above allele specific PCR method are selected from the group consisting of oligonucleotide primers being at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97% identical, more preferably at least 98% identical, more preferably at least 99%, more preferably at least 100% identical to SEQ ID NO: 15 (388588int2f), SEQ ID NO: 19 (388588int3R2), SEQ ID NO. 20
- the method for discriminating Vel negative from Vel positive samples or individuals is by allele PCR-RFLP.
- the gene specific PCR-RFLP method comprises the steps of:
- restriction enzyme is Styl.
- the oligonucleotide primers of the above PCR-RFLP method is at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97% identical, more preferably at least 98% identical, more preferably at least 99%, more preferably at least 100% identical to SEQ ID NO: 15 (388588int2f), and the second oligonucleotide primer is at least 80%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97% identical, more preferably at least 98% identical, more preferably at least 99%, more preferably at least 100% identical to SEQ ID NO: 19 (388588int3R2), wherein under high stringency hybridization conditions, said first and said second oligonucleotide primer are capable of hybridizing to said SMIM1 polynucleotide.
- the method of the present invention is based on molecular discrimination between Vel negative and Vel positive individuals or samples from such individuals.
- the method of the present invention comprises the steps:
- said first primer comprises at least 10 nucleotides being complementary to at least 10 consecutive nucleotides selected from the sequence identified as SEQ ID NO. 1 and located upstream (5') of nucleotide position 2667 of SEQ ID NO. 1
- said second primer comprises at least 10 nucleotides being complementary to at least 10 consecutive nucleotides selected from the sequence identified as SEQ ID NO: 1 and located downstream (3') of nucleotide position 2649 of SEQ ID NO. 1 , with the proviso that said first and said second primer are not both selected from a sequence being complementary to SEQ ID NO.
- the qualitative and/or quantitative analysis performed in the method of the present invention can be made by any suitable tool or method known by those of skill in the In one embodiment the qualitative and/or quantitative analysis is performed by gel electrophoresis.
- the reference control used in the method of the present invention may be any suitable molecular marker, such as a molecular marker based on Mw.
- the reference control is a polynucleotide reference composition comprising one or more polynucleotides selected from polynucleotides consisting of 161 and 178 nucleotides.
- the Vel positive control is a
- polynucleotide comprising exon 3 (SEQ ID NO. 30) of SMIM1.
- the size of the amplicon may vary depending on how the oligonucleotide primers are selected.
- the amplified genetic material is between about 5 and about 4000 nucleotides in length, such as between 10 and 100 nucleotides in length, e.g. 17 nucleotides in length.
- the present invention concerns a method of detection and/or quantitation of a splice variant of SMIM1 in a sample, the method comprising making
- cDNA complementary DNA
- mRNA messenger RNA
- the invention concerns a method of identifying a subject presenting a Vel antigen, comprising the steps:
- a) performing amplification of a polynucleotide of the subject by contacting a polynucleotide from a cell of the subject with an oligonucleotide primer having a sequence being complementary to at least 10 consecutive nucleotides of a
- step (b) detecting an amplicon from step (a), whereby the detection of an amplicon identifies the subject as a Vel antigen.
- the present invention concerns a method of detecting in a sample, a cell that expresses a Vel antigen, comprising detection in the sample of a
- the method of detection and/or quantitation of a splice variant of SMIM1 in a sample comprises making complementary DNA (cDNA) from messenger RNA(mRNA) in the sample, amplifying portions of the cDNA corresponding to the SMIM1 gene or parts thereof and detecting and quantifying the amplified cDNA in order to detect or quantify the splice variant.
- cDNA complementary DNA
- the present inventors have generated data which further characterizes the protein encoded by the SMIM1 (LOC388588) gene as well as the demographic distribution of the mutation causing the Vel negative phenotype. Thus far, no gene homologues to the human Vel protein have been found. However, the gene appears to be evolutionarily conserved as it has orthologous homologues in other species, from primates to zebrafish.
- the SMIM1 gene which typically comprises SEQ ID NO. 1 or a fragment or sequence variant thereof, encodes a polypeptide which is associated with the Vel antigen.
- the Vel blood group antigen on red blood cells is dependent on expression of the SMIM1 gene, the mRNA of which encodes a type 1 transmembrane protein.
- the Vel-negative phenotype occurs when this protein cannot be expressed, for instance due to the 17-bp deletion.
- a main aspect of the present invention is to avoid laborious and expensive traditional serological methods for identifying Vel negative samples or individuals
- the findings of the present inventors of the connection between SMIM1 and the Vel antigen provides a number of antibody and cell based applications which are useful for e.g. in vitro testing purposes.
- the method according to the present invention can in principle be applied to any biological sample comprising genetic material.
- the sample may be from cell scrapings, a biopsy tissue or a body fluid such as, but not limited to blood, bone marrow, plasma, serum, cerebrospinal fluid, saliva, sperm, sputum, urine and stool.
- the present inventors have provided the molecular nexus between the SMIM1 gene and the Vel antigen.
- the inventors have also designed and tested peptides corresponding to the putative protein and tested Vel positive and Vel negative RBCs with rabbit antibodies raised to these peptides.
- One such antibody demonstrates extraordinar specificity for Vel positive RBCs by Western blotting technique.
- the antibody being detected by said method is anti-Vel
- the antigen is a peptide selected from the group consisting of SEQ ID NO. 5, 6, 7, 8, 9 and 10.
- the Vel antigen identified by the present inventors is represented by the polypeptide having SEQ ID NO. 5, or a sequence variant or fragment thereof.
- the antigen is a useful tool in the methods of the present invention.
- the present invention concerns an isolated polypeptide having the sequence of SEQ ID NO. 5, or a fragment or a variant thereof, wherein said fragment comprises at least 20, such as at least 25, such as at least 30, such as at least 35, such as at least 40, such as at least 45, such as at least 50, such as at least 55, such as at least 60, such as at least 65, such as at least 70, such as at least 75 consecutive amino acid residues of SEQ ID NO. 5, and wherein said variant is at least 80% identical to SEQ ID NO.
- the variant comprises at least one Serine and/or Threonine residue, and wherein at least one Serine and/or Threonine residue of said SEQ ID NO. 5 or said fragment or variant thereof, is O-glycosylated.
- Expression of the Vel blood group antigen on red blood cells is dependent on expression of the SMIM1 gene, the mRNA of which encodes a type 1 transmembrane protein. The Vel-negative phenotype occurs when this protein cannot be expressed, for instance due to the 17-bp deletion as discussed herein above.
- Important polypeptide fragments associated with the Vel antigen include fragments of SEQ ID NO. 5.
- the present invention concerns an isolated peptide having a sequence selected from the group consisting of SEQ ID NOs.
- the variant is at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 81 %, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91 %, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identical to
- the present invention concerns a homodimer comprising two identical polypeptide chains, each having the sequence of SEQ ID NO. 5, or a fragment or a variant thereof, wherein said fragment comprises at least 20 consecutive amino acid residues of SEQ ID NO. 5, and wherein said variant is at least 80%, preferably at least 81 %, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91 %, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identical to identical to SEQ ID NO. 5.
- the polypeptide fragment defining the Vel antigen is an isolated polynucleotide, encoding upon expression a peptide having a sequence selected from the group consisting of SEQ ID NOs. 6, 7, 8, 9 and 10 is also an aspect of the present invention.
- the antigen may under certain circumstances be glycosylated, typically O-glycosylated on a Serine or Threonine residue.
- the present invention concern an isolated polypeptide having the sequence of SEQ ID NO. 5, or a fragment or a variant thereof, wherein said fragment comprises at least 20 consecutive amino acid residues of SEQ ID NO.
- said variant is at least 80% identical, preferably at least 81 %, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91 %, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identical to SEQ ID NO. 5, and wherein said variant comprises at least one Serine and/or Threonine residue, and wherein at least one Serine and/or Threonine residue of said SEQ ID NO. 5 or said fragment or variant thereof, is O-glycosylated.
- At least one Serine and/or Threonine residue of the above polypeptide is O-glycosylated.
- Threonine residue is individually and independently selected from the group consisting of:
- Tn antigen (GalNAcaSer/Thr)
- Sialyl-Tn antigen Siaa2-6GalNAcaSer/Thr
- the present invention concerns a method of detecting in a sample an antibody directed to a Vel antigen, comprising the steps:
- the present invention concerns a method of detecting in a sample an antibody directed to a Vel antigen, comprising the steps:
- the interaction is detected by studying agglutination. In another embodiment the interaction is detected by BIACORE. In another embodiment the interaction is detected by ELISA based methods. All of the above methods of detecting interaction are well known by those of skill in the art.
- the present invention concerns a method of detecting a Vel antigen on red blood cells, comprising the steps:
- the present invention concerns a method of detecting in a sample a Vel antigen, comprising the steps:
- the present invention concerns a method of identifying a subject presenting a Vel antigen, comprising the steps:
- step (b) detecting an amplicon from step (a), whereby the detection of an amplicon identifies the subject as a Vel antigen.
- the present invention concerns a method of detecting in a sample, a cell that expresses a Vel antigen comprising detecting in the sample a polynucleotide that encodes a Vel antigen.
- the present invention concerns an antibody capable of recognising an epitope of a Vel antigen, wherein the epitope is defined by a peptide sequence selected from the group consisting of SEQ ID NOs. 6, 7, 8, 9 and 10, or an O-glycosylated peptide sequence selected from SEQ ID NOs. 6, 7, 8, 9 and 10.
- the antibody is capable of recognising an O-glycosylated peptide sequence selected from SEQ ID NOs. 6, 7, 8, 9 and 10 which comprises at least one
- Serine and/or Threonine residue wherein the at least one Serine and/or Threonine residue individually and independently is O-glycosylated with a glycan selected from the group consisting of:
- Tn antigen (GalNAcaSer/Thr)
- Sialyl-Tn antigen Siaa2-6GalNAcaSer/Thr
- the antibody is either monoclonal or polyclonal.
- the present invention also concerns a method of making a polyclonal antibody, the method comprising:
- step a) screening the isolated antibodies with the polypeptide or polypeptide fragment thereby identifying a polyclonal antibody that binds specifically to the polypeptide or polypeptide fragment of step a).
- the present invention also concerns a method of making a monoclonal antibody, the method comprising:
- step a isolating from the culture monoclonal antibodies which bind specifically to the polypeptide or polypeptide fragment of step a).
- the method of the present invention is particularly useful in a clinical setting involving blood transfusions.
- the above methods further comprises the steps of:
- the present invention concerns a blood transfusion method comprising the steps:
- Blood transfusions are useful in the treatment of blood disorders.
- the present invention is particularly useful during therapy in relation to treatment of a disease or disorder associated with erythrocytes wherein the invention concerns a method comprising the steps:
- Pregnant Vel negative females may carry Vel positive foetuses. If erythrocytes from the Vel positive foetus is transferred to the Vel negative mother, this may result in an immune response of the mother thus raising anti-Vel antibodies against the Vel positive erythrocytes. This is potentially lethal for the foetus, especially during a second or subsequent pregnancy of the Vel negative female.
- the present invention concerns a method of prophylactic treatment of a Vel negative pregnant female individual comprising:
- the present invention also concerns an anti-Vel antibody for use in a method of prophylactic treatment of a Vel negative pregnant female individual comprising: a) identifying a Vel negative individual by applying the method as defined herein above, and
- the primary use of the present invention is in the field of transfusion medicine where described nucleotide deletion will be used as a basis for screening of blood donors in order to identify compatible blood for patients who have developed antibodies to the Vel antigen.
- High throughput genotyping platforms for donor testing exist in the routine laboratory today and thus in one embodiment the present invention is incorporated into these platforms.
- the present invention concerns a kit for detecting a Vel antigen, or discriminating between Vel negative and Vel positive samples, wherein said kit comprises the antibody as defined herein above.
- the kit typically comprises a microchip array wherein the antibodies defined above are conjugated to the microchip array.
- the present invention concerns a kit for detecting a Vel antigen and/or discriminating between samples from Vel negative and Vel positive individuals, said kit comprising at least two isolated oligonucleotide primers selected from the group consisting of SEQ ID NO. 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 and 23.
- the invention concerns a kit comprising a microchip array comprising one or more polynucleotides selected from the group consisting of SEQ ID NO. 1 , 2, 3, 4, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 32, 33, 34, 35 and 36 or one or more fragments of said polynucleotide.
- the kit including or excluding the microchip array may include a combination of one or more of polynucleotides, oligonucleotides, peptides and polypeptides defined herein.
- the present invention concerns a kit comprising a microchip array comprising one or more peptides or polypeptides selected from the group consisting of SEQ ID NO. 5, 6, 7, 8, 9 and 10, or one or more fragments of said peptide or polypeptide wherein said fragment comprises at least 5 consecutive amino acids of said SEQ ID NO. 5, 6, 7, 8, 9 and 10.
- a kit comprising a microchip array comprising one or more peptides or polypeptides selected from the group consisting of SEQ ID NO. 5, 6, 7, 8, 9 and 10, or one or more fragments of said peptide or polypeptide wherein said fragment comprises at least 5 consecutive amino acids of said SEQ ID NO. 5, 6, 7, 8, 9 and 10.
- Threonine residue of said peptide in said kit is O-glycosylated, such wherein at least one of said Serine and/or Threonine residue is independently and optionally O- glycosylated by a glycan selected from the group consisting of:
- Tn antigen (GalNAcaSer/Thr)
- Sialyl-Tn antigen Siaa2-6GalNAcaSer/Thr
- the kit described herein above comprises red blood cells presenting a Vel antigen.
- the invention concerns the following embodiments.
- Embodiment 1 A method of determining the Vel phenotype of an individual, said method comprising discriminating between Vel positive and Vel negative phenotypes by analysing in a biological sample the composition of:
- Embodiment 2 The method according to embodiment 1 , wherein the SMIM1 gene comprises the sequence of SEQ ID NO: 1 or a sequence variant thereof wherein the sequence variant is between at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as between 99.1 and 99.9%, between 99.4 and 99.6%, such as 100% identical to i) SEQ ID NO. 1 or ii) a sequence being complementary to SEQ ID NO. 1.
- Embodiment 3 The method according to any one of the preceding embodiments, wherein the discrimination by analysing the SMIM1 composition comprises identifying: subjects whose genome comprises SEQ ID NO. 1 , said subjects being Vel positive, and subjects whose genome comprises SEQ ID NO. 2,
- Embodiment 4 The method according to embodiment 1 , wherein the method comprises: a) providing a biological sample comprising a SMIM1 polynucleotide, b) amplifying at least a fragment of the SMIM1 polynucleotide, wherein the SMIM1 polynucleotide has a sequence selected from the group consisting of SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 and 36 or a fragment or variant thereof wherein the variant is at least 90% identical to said sequence selected from the group consisting of SEQ ID NO. 1 , 2, 3, 4, 32, 33, 34, 35 and 36;
- polynucleotide fragments based on polynucleotide length.
- Embodiment 5 The method according to embodiment 1 comprising the steps: providing a biological sample comprising genomic DNA,
- first primer comprises at least 10 nucleotides being complementary to at least 10 consecutive nucleotides selected from the sequence identified as SEQ ID NO. 1 and located upstream (5') of nucleotide position 2667 of SEQ ID NO. 1
- second primer comprises at least 10 nucleotides being complementary to at least 10 consecutive nucleotides selected from the sequence identified as SEQ ID NO: 1 and located downstream (3') of nucleotide position 2649 of SEQ ID NO. 1 , with the proviso that said first and said second primer are not both selected from a sequence being complementary to SEQ ID NO. 31 ; iii) obtaining an amplicon; iv) performing qualitative and/or quantitative analysis of the amplicon of step iii).
- Embodiment 6 A method of detection and/or quantitation of a splice variant of SMIM1 in a sample, the method comprising making complementary DNA (cDNA) from messenger RNA (mRNA) in the sample, amplifying portions of the cDNA corresponding to the SMIM1 gene or parts thereof and detecting and quantifying the amplified cDNA in order to detect or quantify the splice variant.
- cDNA complementary DNA
- mRNA messenger RNA
- Embodiment 7 An isolated polynucleotide comprising a sequence variant of SEQ ID NO. 1 , or an isolated polynucleotide comprising a sequence variant of a sequence being complementary to said SEQ ID NO. 1 , wherein the sequence variant comprises at least one mutation, wherein mutation results in abolished transcription and/or protein translation and/or absence of a polypeptide encoded by SEQ ID NO. 1 on the surface of an erythrocyte.
- Embodiment 8 The isolated polynucleotide according to embodiment 7, wherein the sequence variant has the sequence selected from the group consisting of SEQ ID NO. 2 and 4.
- Embodiment 9 An isolated polypeptide having the sequence of SEQ ID NO. 5, or a fragment or a variant thereof, wherein said fragment comprises at least 20 consecutive amino acid residues of SEQ ID NO. 5, and wherein said variant is at least 80% identical to SEQ ID NO. 5, and wherein said variant comprises at least one Serine and/or Threonine residue, and wherein at least one Serine and/or Threonine residue of said SEQ ID NO. 5 or said fragment or variant thereof, is O- glycosylated.
- Embodiment 10 An isolated peptide having a sequence selected from the group consisting of SEQ ID NOs. 6, 7, 8, 9 and 10. Examples
- SEQ ID NO. 1 SMIM1 gene
- Grey indicates exons; underline indicates the start and stop codons; bold italics indicates the 17bp deletion identified in the mutant phenotype in exon 3 and also the polymorphic nucleotides in intron 3 (c.1 10+193t/c and c.1 10+194g/a; nucleotides 2907 & 2908 in the sequence above) both of which are invariantly mutated in the mutant phenotype.
- SEQ ID NO. 2 SMIM1- mutant phenotype (17bp deletion)
- Grey indicates exons based on the wild type structure however the deletion extends the open reading frame and the sequence in blue indicates the potential new translated sequence until the next in-frame stop codon. Underline indicates the start and stop codons.
- Bold italics indicate the polymorphic nucleotides in intron 3 that are invariantly mutated (c.1 10+193t>c and c.1 10+194g>a) in the mutant phenotype.
- Nucleotides (SNPs) 2872 and 2873 are invariably associated with Vel negative but individually are not restricted to Vel negative.
- SEQ ID NO. 3 cDNA based on SEQ ID NO. 1
- SEQ ID NO. 4 cDNA based on SEQ ID NO. 2 (17-bp deletion not present)
- SEQ ID NO. 10 Peptide 5 (scrambled peptide 1 ) CPESWHSYMRVQHEQD
- SEQ ID NO. 1 1 cDNA primer 388588cDNAf CGCACCTCAGCCCACGAC
- SEQ ID NO. 12 cDNA primer 388588cDNAr TCCAGGCCTGTGCTCTCAC
- SEQ ID NO. 22 LOCex3f_screen PCR primer
- SEQ ID NO. 23 LOCex3r_screen PCR primer
- SEQ ID NO. 24 RACE primer Vel 59F
- SEQ ID NO. 30 Exon 3 of SMIM1
- SEQ ID NO. 32 SA/ /7-rmut.ant phenotype extended to subsequent stop codon
- SEQ ID NO. 33 SMIM1 var 1 (chrl :3691980 A/G)
- SEQ ID NO. 34 SMIM1 var 2 (chi :3691980 A/G)
- SEQ ID NO. 36 cDNA of SMIM1 variant (transcript according to 5' and 3' RACE in blood; 17bp deletion)
- the Vel blood group antigen is located on red blood cells and is one of a few so-called orphan blood group antigens for which the molecular basis is unknown. It is clinically important and thus identification of the carrier molecule would permit the development of a screening assay to identify blood donors appropriate for immunized Vel negative/negative patients, and also patients at risk of producing an unwanted antibody.
- the present inventors provided understanding of the molecular and genetic basis of the Vel blood group antigen. This has been achieved through a range of serological and biochemical investigations, including investigations of candidate genes. These investigations resulted in estimates of the size of the red blood cell protein carrying the Vel antigen and a likely homodimer configuration.
- the Vel negative phenotype is inherited in an autosomal-recessive manner, and (c) the gene encoding the Vel antigen, like several other blood group genes, could be preferentially expressed on erythrocytes.
- the inventors determined the DNA sequence of SMIM1 (LOC388588) in Vel negative donors and found a 17-basepair deletion that destroys the protein, causing a so-called null or knock-out phenotype. Subsequent genetic studies have identified the exact same 17-base-pair deletion in all Vel negative donors from Sweden, UK, Switzerland, Israel and the USA tested to date, suggesting that it is indeed the predominating cause of the Vel negative phenotype. Additional sequence analysis revealed homozygosity for the 17-base-pair deletion in a further five Vel-samples of Swiss origin and 2 samples of Israeli origin. This has set the stage for identification of Vel negative blood donors by genetic testing.
- DNA sample panels used for screening to establish mutation prevalence were anonymized samples from normal Swedish blood donors.
- Table 1 Discovery cohort of Vel negative and Vel positive samples analysed on the lllumina HumanOmni 2.5M BeadChip microarray.
- Genomic DNA was prepared either using a modified salting-out procedure and diluted to 100 ng/uL in water; or using the QIAamp DNA Blood Mini Kit (Qiagen AB,
- Genomic DNA from 20 Vel negative and 8 Vel positive samples were subjected to genome-wide genotyping with respect to 2,443,179 single-nucleotide polymorphisms (SNPs) using lllumina HumanOmni 2.5M BeadChip microarrays (lllumina Inc, San Diego, CA, USA; genotyping and genotype calling performed at the SCIBLU facility, Lund University, Sweden). Genotypes were called in GenomeStudio (lllumina Inc). Call rate was at least 99.5% for all samples. Four samples were genotyped twice on separate arrays with a concordance of at least 99.3%. Only the result in one of the arrays was subsequently analyzed.
- SNPs single-nucleotide polymorphisms
- Breakpoints of the detected haplotype block were estimated from inspection of unfiltered genotype call data.
- Amplified products were run on 3% agarose gel, excised, eluted, and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Life).
- RNA isolated from whole blood or cell lines was converted to cDNA using the High Capacity Kit (Applied Biosystems) according to the protocol. Amplification of mRNA was performed with the Expand High Fidelity PCR Kit (Roche Diagnostics Corporation, Indianapolis, IN, USA) using primers 388588cDNAf (SEQ ID NO. 1 1 ) and 388588cDNAr (SEQ ID NO. 12). Products were sequenced as described above.
- PCR-RFLP genomic DNA was amplified with primers 388588int2f (SEQ ID NO:
- the amplified bands are 459 and 442 bp respectively however, the size of the amplicon is arbitrary and other gene-specific primers surrounding the deletion could be designed by the person of skill in the art.
- the products were digested with Sty ⁇ at 37°C for two hours. Products were analysed on a 3% agarose gel. (see figure 2). The restriction site was abolished in the mutated sequence.
- the amplified bands are 459 and 442 bp respectively however, the size of the amplicon is arbitrary and other gene-specific primers surrounding the deletion could be used.
- Allele-specific PCR (ASP) genomic DNA was amplified with primers
- LOCex3f_screen SEQ ID NO. 22
- LOCex3r_screen SEQ ID NO. 23
- Allele-specific PCR products of 178 bp (wild type) or 161 bp (mutant) were discriminated based on size by
- RNA was isolated from total RNA extracted from bone marrow cells cultured towards erythropoietic maturation as described previously by an mRNA Isolation Kit (Roche Diagnostics Corporation). RACE was performed with the
- SMIM1 was stably knocked-down in Human Erythroleukemic (HEL) and JK-1 cell lines by lentivirus-based shRNA vectors specific for SMIM1.
- HEL Human Erythroleukemic
- JK-1 cell lines by lentivirus-based shRNA vectors specific for SMIM1.
- the following libraries were purchased from SIGMA: TRCN0000365551 , TRCN0000365607, TRCN0000365620, TRCN0000376652, TRCN0000376653, TRCN0000376654, TRCN0000422700 at a titer of 10 6 in pLKO.1 vector.
- the transduction was performed according to the manufacturers' instructions. Briefly, 1 .6 x 10 4 of HEL and JK-1 cells stably expressing SMIM1 were seeded in 96-well plate in 100 ⁇ growth media per well at the day of transduction.
- Hexadimethyrine bromide was added to each well at a final concentration of 8 ⁇ g ml.
- Transduction with shRNA lentiviral clones were carried out at a MOI (multiplicity of infection) ranging between 0.5 to 5.
- Lentiviral particles of a non-human transduction control (SH202V, SIGMA), a transduction efficiency control (SHC203V,SIGMA) and shRNA Lenti-viral particles for SMIM1 were added to the wells containing cells. The plate was centrifuged at 2300 rpm for 30 min at 37°C. Post 4-5 h transduction, wells were replenished with RPMI-1640 with 10 and 20% FCS for HEL and JK-1 cells, respectively.
- the transduced cells were incubated at 37°C in a humidified incubator in an atmosphere of 5% C0 2 for 48 h.
- the media containing lentiviral particles was removed and fresh media was added.
- Puromycin was added at a concentration of 1 .75 ⁇ g/ml and 0.25 ⁇ g/ml for HEL and JK-1 cells, respectively.
- the media containing drug was replaced after every two days for a period of 8 days. Thereafter, clones were allowed to proliferate till confluent for further analysis.
- the cell lines were analysed by real time PCR, Western blot and flow cytometry (Figure 9).
- Peptides 1 and 3 were used to immunize rabbits to produce a polyclonal antibody to the predicted protein. Peptide synthesis and antibody production was purchased as a service from Innovagen AB (Lund, Sweden). Post-immunization sera were tested by hemagglutination and by Western blotting. Neither antibody was specifically reactive by routine serological hemagglutination techniques (described below) however, anti- peptide 1 was highly specific as indicated in the section describing the Western blotting experiments below. ELISA
- ELISA was performed to determine whether polyclonal anti-Vel specifically recognized the linear peptides 1 -4.
- Peptide 5 was a scrambled sequence of peptide 1 .
- a concentration of 0.01 ug/ml_ of each peptide was used to coat microplates overnight. Following coating, the plates were blocked in 1 % PBS/BSA, washed and incubated with anti-Vel.
- An unrelated antibody, anti-K and inert plasma (AB serum) were used as controls. Unbound antibody was washed off, and the plate incubated with HRP-labelled goat anti-human IgG (BioRad Laboratories). Following washing, the reaction were developed with TMB Single Solution chromogen for ELISA ( Invitrogen) and stopped with 1 M HCI. Reactions were read at 450 nm by an ELISA reader.
- Erythrocyte membranes were prepared as described previously : and solubilised with 1 % Nonidet P-40. The solubilizate was mixed with equal volumes of Laemmli sample buffer with or without the addition of 5% mercaptoethanol depending on the conditions required. Prior to loading, samples were heated to 95 °C for 5 minutes. Solubilised RBC membranes (15 ⁇ ) were run on NuPAGE® 4-12% Bis-Tris gels (Novex, Life Technologies.) for -75 minutes at 150 v until the dye front reached the bottom of the gel. The gel was either stained with SimplyBlueTM SafeStain (Life Technologies), or the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes.
- PVDF polyvinylidene difluoride
- the membranes were blocked in 5% non-fat milk/phosphate-buffered saline (PBS) for 1 hour at room temperature, then rinsed in PBS before incubation with anti-peptide 1 (bleed #2, diluted 1 :20,000) for 2 hours at room temperature. Following incubation, the membranes were washed 3 x 10 minutes in PBS/Tween (PBST), then incubated for 1 hour at room temperature with horseradish peroxidise (HRP)-labelled goat anti-rabbit IgG (Agrisera, Vannas, Sweden) diluted 1 :10000 in PBS-T. The PVDF membranes were washed as before and then developed with ECL reagent (Life Technologies) according to the manufacturer's instructions.
- PBS non-fat milk/phosphate-buffered saline
- the labelled membranes were sealed in plastic and then exposed to film (Hyperfilm ECL, Life Technologies) for an appropriate time, as determined by an initial 60 second exposure, and developed manually.
- the membranes were reprobed with a murine anti-GAPDH (clone 6C5, Millipore, Solna, Sweden), diluted 1 :5000, washed and incubated with HRP-labelled goat anti-mouse IgG diluted 1 :10000 in PBS-T; and visualised as above ( Figure 6).
- the results show that the antibody generated to the N-terminal sequence of the SMIM1 proteins specifically recognised a protein that was present in Vel positive individuals but absent in Vel negative individuals.
- Flow cytometry was performed on RBCs, cell lines and transfected cells.
- a suspension of approximately 0.5 x 10 6 RBCs or 10 6 cultured cells in PBS containing 1 % bovine serum albumin (PBS/BSA) were tested with polyclonal human anti-Vel, diluted 1 :4 in PBS/BSA.
- AB serum was included as a control for unspecific binding. After washing unbound primary antibody, cells were incubated with FITC-conjugated F(ab') 2
- Vel antigen expression is well-known in the field to vary between the RBCs of one Vel positive individual and another.
- Figure 7a shows the variation between RBCs from normal blood donors known to be homozygous for wild type SMIM1.
- Figure 7b shows the mean antigen expression obtained by flow cytometric analysis from 37 blood donor samples (16 w/m; 21 w/w genotype).
- Figure 7c shows the Vel antigen expression following over-expression of full-length SMIM1.
- the full cDNA sequence (exons 1 -4) of the SMIM1 gene were amplified from Vel positive and Vel negative blood donors and inserted into the green fluorescent protein(GFP)-positive plasmid vector plRES2-ZsGreen1 by restriction enzyme digestion using £coR1 and Sam/-/1. Folllowing re-ligation, One Shot® TOP10 chemically competent E. coli (Invitrogen) cells were transformed with the new constructs and incubated overnight on agar plates containing Kanamycin. Colonies were PCR-amplified and sequenced to confirm the presence and orientation of the insert.
- Colonies containing the wild type sequence (pEFI oLOCwt), the mutant sequence (pEFI oLOCmut) or empty vector (pEF1 a-IRES-ZsGreen1 ) were cultured overnight in multiple 15ml tubes containing 5ml LB medium and 5 ⁇ Kanamycin.
- Minipreps were prepared using GeneJET Plasmid Miniprep Kit (Thermo Scientific, #K0502). Plasmid DNA concentration and purity measurements were assessed using the Quant-iTTM dsDNA Broad-Range Assay Kit (Invitrogen, Life Technologies), and were re-sequenced to confirm the presence of the correct insert.
- K562 cells were cultured in RPMI 1640 medium (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C and humidified atmosphere containing 5% C0 2 .
- Cultured K562 cells were mixed with 10 ⁇ g plasmid containing wild type, mutant or control vectors and electroporated using the Gene Pulser II electroporation system (BioRad Inc. Carlsbad, CA) and incubated for 48h before being analysed by flow cytometry.
- Example 6 Method for screening for Vel negative/negative blood
- GSP Gene-specific PCR
- LOCex3f_screen SEQ ID NO. 22
- LOCex3r_screen SEQ ID NO. 23
- 161 bp (mutant) are readily discriminated based on size by electrophoresis for 75 minutes at 165V on a 3% agarose gel (see figure 4).
- genomic DNA can be amplified with primers 388588int2f (SEQ ID NO. 15) and 388588int3R2 (SEQ ID NO. 19).
- the amplified bands are 459 and 442 bp respectively however, the size of the amplicon is arbitrary and other gene-specific primers surrounding the deletion could be used.
- 388588int2f (SEQ ID NO. 15) (1 pM), 388588int3R2 (SEQ ID NO. 19) and either 388588wtex3f (SEQ ID NO. 20) or 388588mutex3f (SEQ ID NO. 21 ).
- An internal control band was provided by the amplification of a 460bp (wild type) or 443bp (mutant) product. Allele-specific bands of 266bp (wild type) or 249 bp (mutant) were specifically amplified by their respective primers (see figure 3).
- primers amplifying the 266 bp wild type SMIM1 fragment (388588int3R2 (SEQ ID NO. 19) and 388588wtex3f (SEQ ID NO. 20) have been incorporated into a medium-thoughput, albeit manual, screening method for high-prevalence blood group alleles (see figure 5).
- Vel- individuals i.e. homozygous SMIM1 mutants are identified by the absence of the SMIM1- specific band in the presence of the remaining five bands specific for other blood group loci. Further methods useful include semi-automated (medium- and high-throughput) genotyping-based assays which readily incorporate screening for Vel into their platforms.
- Probe sequences that are useful are primer sequences 388588wtex3f (SEQ ID NO. 20) and 388588mutex3f (SEQ ID NO. 21 ).
- BLOODChip is a microarray platform that incorporates allele-specific probes (usually based on single nucleotide polymorphisms) that capture labelled PCR products containing the allele of interest.
- allele-specific probes usually based on single nucleotide polymorphisms
- a multiplex PCR reaction amplifies from genomic DNA selected regions of different blood group genes that contain antigen-defining polymorphisms.
- the PCR products are labelled and annealed to the microarray that is printed with both permutations of an allele.
- the PCR product will anneal to the probe containing the allele-specific SNP at a specific position. Positive reactions are determined by a laser reader. Probe sequences that could be incorporated into such a platform would include SEQ ID NO.
- the probes could be elongated at either end with the consensus sequence without affecting specificity.
- HEA BeadChipTM Kit (Immucor, Inc.) is a technology which employs beads to which allele-specific oligonucleotide probes are attached to coloured beads. The colour determines the signature of each bead, and post production, they are sorted to a silicon wafer which is the test platform.
- a multiplex PCR reaction amplifies from genomic DNA selected regions of different blood group genes that contain antigen- defining polymorphisms. These products are mixed with the beads, and in contrast to the above, the technology relies on allele-specific extension of the DNA strand and incorporation of the labelled PCR product. Again, the 17-bp deletion in SMIM1 could be exploited by this technology using probes that incorporated SEQ ID NO.
- MassARRAY® iPLEX® technology (Sequenom® GmbH) have developed a matrix- assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) platform for the detection of specific blood group alleles.
- MALDI-TOF matrix- assisted laser desorption/ionisation time-of-flight mass spectrometry
- This technology bases detection of a specific allele on the mass difference between one allele and its counterpart and has been shown to be very specific [5]. It is a flexible technology that allows rapid incorporation of desirable alleles.
- Sequenom® GmbH have a platform in place for the detection of common blood group alleles and are looking to expand into the detection of alleles encoding other clinically significant blood group antigens.
- the present invention could be integrated into and make use of any solid or fluid phase matrix for genotyping that relies on the allele-specific annealing, extension or recognition could incorporate probes that include SEQ ID NO. 20 or SEQ ID NO. 21 for the specific determination of the Vel positive and Vel negative phenotype respectively.
Abstract
Description
Claims
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AU2013326613A AU2013326613A1 (en) | 2012-10-01 | 2013-10-01 | Methods and tools for Vel blood group typing |
CA2885967A CA2885967A1 (en) | 2012-10-01 | 2013-10-01 | Methods and tools for vel blood group typing |
EP13773677.3A EP2904107B1 (en) | 2012-10-01 | 2013-10-01 | Methods for vel blood group typing |
US14/431,424 US9989537B2 (en) | 2012-10-01 | 2013-10-01 | Methods and tools for vel blood group typing |
ES13773677T ES2713162T3 (en) | 2012-10-01 | 2013-10-01 | Methods for blood group determination Vel |
US15/949,200 US10761098B2 (en) | 2012-10-01 | 2018-04-10 | Methods and tools for Vel blood group typing |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2018096043A1 (en) * | 2016-11-23 | 2018-05-31 | Blutspende Zürich | DETERMINATION OF THE GENOTYPE UNDERLYING THE S-s-U- PHENOTYPE OF THE MNSs BLOOD GROUP SYSTEM |
US11549137B2 (en) | 2016-11-23 | 2023-01-10 | Christoph Gassner | Determination of the genotype underlying the S-s-U-phenotype of the MNSs blood group system |
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