Patent Application
20150140555
The present invention generally relates to methods for identifying and/or isolating at least one foetal primitive nucleated red blood cell. In particular, the invention relates to a method of identifying at least one foetal primitive nucleated red blood cell in a sample by detecting at least one membrane protein specific to the foetal primitive nucleated red blood cell.
Currently, prenatal diagnosis of chromosomal and single gene disorders rely on foetal cells obtained by invasive procedures such as amniocentesis, chorionic villous sampling (CVS) or foetal blood sampling (FBS) for cytogenetic and/or molecular analysis. These invasive tests carry a small but significant risk of foetal miscarriage. On the one hand this limits the uptake of the diagnostic test out of fear of foetal loss, and on the other hand causes the demise of an otherwise healthy foetus.
Non-invasive methods to diagnose the foetal genetic condition by enriching and analyzing foetal cells and foetal DNA that circulate in maternal blood have been studied.
Of the foetal cells that enter the first trimester maternal circulation, primitive foetal nucleated red blood cell (FPNRBC) is the preferred target cell. This is because of its short life-span and hence it is unlikely to persist from a previous pregnancy, unlike the situation with foetal lymphocyte where this phenomenon could be the basis for a misdiagnosis. First-trimester FPNRBC contain Epsilon-globin , an ideal foetal cell identifier which is highly specific as expression declines after the first trimester.
In humans, foetal primitive nucleated red blood cells (FPNRBCs, foetal primitive erythroblasts, first trimester foetal nucleated blood cells (FNRBCs)) generated in the yolk sac mesoderm remain the predominant blood cell type in the embryonic circulation until 10 weeks post-conception. Studies on this cell type in humans have been limited owing to limited access to pure populations of these cells for laboratory investigations; only recently has it been shown that these cells may enucleate within the first trimester human placenta, suggesting that may be terminally differentiated. Primitive erythroblasts differ from foetal definitive erythroblasts not only in their anatomical site of origin, but also in the types of haemoglobins contained within them.
Adult anucleate red blood cells (AARBCs, adult red blood cells (RBCs)) are smaller, discoid, readily deformable cells that are produced in the long bone marrow. Owing to their ready availability, these cells have been extensively studied in recent years. Using mass spectrometry, AARBC membrane and cytoplasmic proteins have been characterized, and differences demonstrated between mouse and human AARBCs.
Enrichment of first trimester FPNRBC from maternal blood for non-invasive prenatal diagnosis has been a difficult task due to the lack of unique antibodies against its surface proteins. While WBCs can be separated using anti-CD45 antibody from maternal blood samples, separation of FPNRBCs from overwhelming adult RBCs has been the challenge. The success of non-invasive prenatal diagnosis using first trimester FPNRBCs from maternal blood depends on the enrichment of these rare cells (one cell amongst a million nucleated maternal cells).
The goal of isolating and analyzing foetal DNA from as little as one FPNRBC recovered from amongst a million nucleated maternal cells is possible with the use of automated micromanipulation, laser capture microscopy systems and downstream analysis of foetal cell with single cell whole genomic amplification coupled with array CGH technologies. Therefore, it is not inconceivable that very small numbers of foetal cells (˜20 cells) enriched from maternal blood from an on-going euploid pregnancy may actually be sufficient for non-invasive prenatal diagnosis.
Accordingly, there is a need in the art for a method for detecting and/or isolating FNRBCs and provide methods as potential reliable approaches for future NIPD using FNRBCs present in maternal blood.
According to one aspect of the invention, there is provided a method for identifying and/or isolating at least one foetal erythroblast, the method comprising: detecting the expression of at least one foetal erythroblast specific marker selected from the group consisting of neutral amino acid transporter B (SLC1A5), solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 isoform A (SLC3A2), splice isoform A of chloride channel protein 6, transferrin receptor protein 1, splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, Splice Isoform 1 of Protein C9orf5, Cleft lip and palate transmembrane protein 1, BCG induced integral membrane protein BIGM103, antibacterial protein FALL-39 precursor, CAAX prenyl protease 1 homolog, splice isoform 2 of synaptophysin-like protein, vitamin K epoxide reductase complex subunit 1-like protein 1, splice isoform 1 of Protein C20orf22 (ABHD12), Hypothetical protein DKFZp564K247 (Hypoxia induced gene 1 protein) (IPI Accession No. IPI00295621), Hypothetical protein DKFZp586C1924 (IPI Accession No. IPI00031064), ALEX3 protein variant, hypothetical protein MGC14288 (IPI Accession No. IPI00176708), protein with IPI Accession No. IPI00639803, and protein with IPI Accession No. IPI00646289; wherein detection of the marker indicates the presence of the foetal erythroblast.
According to other aspects of the invention, there is also provided a marker or identifying foetal erythroblast selected from the foetal erythroblast specific marker according to any aspect of the present invention, a method of diagnosing at least one prenatal disorder in an individual using at least one foetal erythroblast specific marker, an antibody or antigen binding fragment thereof that is capable of binding to at least one foetal erythroblast specific marker, and a kit for identifying and/or isolating foetal erythroblast in a sample.
Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.
Reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” as used herein thus usually means “at least one”.
The term “comprising” is herein defined as “including principally, but not necessarily solely”. Furthermore, the term “comprising” will be automatically read by the person skilled in the art as including “consisting of”. The variations of the word “comprising”, such as “comprise” and “comprises”, have correspondingly varied meanings.
The term “fragment” is herein defined as an incomplete or isolated portion of the full sequence of a protein which comprises the active/binding site(s) that confers the sequence with the characteristics and function of the protein. In particular, it may be shorter by at least one amino acid. More in particular, the fragment comprises the binding site(s) that enable the protein to bind to at least one marker of the present invention.
The term “antigen binding fragment” is herein defined as an incomplete or isolated portion of the full sequence of an antibody which comprises the active/binding site(s) that confers the sequence with the characteristics and function of the antibody. In particular, it may be shorter by at least one amino acid. More in particular, the fragment comprises the binding site(s) that enable the antibody to bind to at least one marker of the present invention.
The term “erythroblast” as used herein refers to a red blood cell having a nucleus. In particular, an erythroblast refers to a nucleated precursor cell from which a reticulocyte develops into an erythrocyte. “Erythroblast” may be used interchangeably with a “Normoblast” and refers to a nucleated red blood cell, the immediate precursor of an erythrocyte. For example, the erythroblast may be of mammalian origin. In particular, the erythroblast may be a primitive or human foetal erythroblast. The term “foetal primitive nucleated red blood cell (FPNRBC)” is herein defined as cells generated in the yolk sac mesoderm that remain as the predominant blood cell type in the embryonic circulation until 10 weeks post-conception. The term “FPNRBC” may be used interchangeably with foetal primitive erythroblasts or first trimester foetal nucleated red blood cells (FNRBCs)).
The phrase “adult anucleate red blood cells (AARBCs, adult red blood cells (RBCs))” is herein defined as cells that are relatively smaller as compared to FPNRBC, discoid, readily deformable and produced in the long bone marrow. The term AARBCs may be used interchangeably with “adult red blood cells (RBCs)”.
The term “mammalian” is herein defined as a mammalian individual, in particular, a primate for example a human being. For purposes of research, the subject may be a non-human. For example the subject may be an animal suitable for use in an animal model, e.g., a pig, horse, mouse, rat, cow, dog, cat, cattle, non-human primate (e.g. chimpanzee) and the like.
The term “sample” as used herein refers to a subset of tissues, cells or component parts (for example fluids) that may include, but are not limited to, maternal tissue, maternal blood, cord blood, amniocenteses, chorionic villus sample, foetal blood, and/or foetal tissue/fluids. In particular, foetal tissue may be trophoblast tissue, placental tissue or a combination thereof. The sample as used in the present invention may have been previously subjected to a density gradient purification including, but not limited to, Ficoll gradient and Percoll gradient.
The term “CD45 negative” as used herein refers to any cell that expresses no signal or is negative for native, recombinant or synthetic forms of the CD45 molecule/marker. The presence of CD45 expression on a cell in a sample may be determined using any immunostaining method known in the art and using any anti-CD45 reagent. Any cells positively stained with anti-CD45 reagent may be excluded as these may include CD45 positive white blood cells (WBC).
The term “nucleated” as used herein refers to a cell that has a nucleus. Nucleated cells may be distinguished from red blood cells which are not nucleated based on any nuclear staining known in the art.
The term “prenatal disorder” as used herein refers to diseases or conditions in a foetus or embryo before it is born. The prenatal disorder may be selected from the group consisting of a chromosomal disorder, a genetic disorder, or a combination thereof. In particular, the prenatal disorder may be selected from the non-limiting group consisting of Down Syndrome, Edwards Syndrome, Patau Syndrome, a neural tube defect, spina bifida, cleft palate, Tay Sachs Disease, sickle-cell anemia, thalassemia, cystic fibrosis, fragile X syndrome, spinal muscular atrophy, myotonic dystrophy, Huntington's Disease, Charcot-Marie-Tooth disease, haemophilia, Duchenne muscular dystrophy, mitochondrial disorder, Hereditary multiple exostoses, osteogenesis imperfecta disorder, a combination thereof and the like.
At present, enrichment of FPNRBCs from maternal blood has been a challenge because of their rarity in maternal circulation and the lack of surface specific antigens for immunocell sorting of these cells. CD71 and GPA are commonly used to enrich these cells from maternal blood: as such use of CD71 may result in loss as this surface antigen is expressed only on ˜68% of FPNRBCs and GPA binds to both. AARBCs and FNRBCs making analyses of enriched sample difficult because of a very high background of AARBCs.
Cell surface membrane proteins have an integral role in maintaining health: when altered structurally or functionally, they are responsible for the more commonly known diseased states such as spherocytosis and sickle cell disease, and also the less commonly recognized conditions such as elliptocytosis, familial pseudohyperkalaemia, dehydrated hereditary stomatocytosis and membrane defects in β-thalassemia. Knowledge about cell membrane proteins and their functions in health and disease could lead to understanding mechanisms of disease processes such as the invasion of the malaria parasite into human erythrocytes and the possibility of developing therapeutic interventions.
In contrast to the large amount of information already available on the AARBC membrane proteome, no information is currently available on the proteome of human foetal primitive erythroblasts. Only very limited data on their cell surface antigens such as CD71 and Glycophorin A and some information on their cytoplasmic haemoglobin are known. The knowledge on the membrane proteome of the FPNRBC may be useful in two ways: to facilitate a deeper understanding of primitive erythropoiesis in humans, and to identify specific surface antigen(s) for the enrichment of ε-globin-positive foetal primitive erythroblasts from maternal blood for non-invasive prenatal diagnosis. It has been suggested that the ε-globin-positive foetal primitive erythroblast is the ideal foetal cell type for non-invasive prenatal diagnosis and identification of unique membrane proteins on either FPNRBC or AARBC may be exploited for non-invasive prenatal diagnosis in the future. Differences between human FPNRBCs and AARBCs are disclosed herein. Accordingly, there is a need in the art to provide markers that facilitate the identification and/or isolation of FPNRBCs.
The inventors of the present application made the first attempt to explore unique membrane proteins of FPNRBCs. They identified unique surface proteins with transmembrane domains that may be useful as markers for the separation of human FPNRBCs from adult RBCs by immuno-cell sorting protocols. Antibodies against these proteins may enable the immuno-cell sorting.
According to an aspect of the present invention, there is provided a method for identifying at least one foetal erythroblast the method comprising: detecting the expression of at least one foetal erythroblast specific marker selected from the group consisting of neutral amino acid transporter B (SLC1A5), solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 isoform A (SLC3A2), Splice Isoform A of Chloride channel protein 6, Transferrin receptor protein 1, Splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, Splice Isoform 1 of Protein C9orf5, Cleft lip and palate transmembrane protein 1, BCG induced integral membrane protein BIGM103, Antibacterial protein FALL-39 precursor, CAAX prenyl protease 1 homolog, Splice Isoform 2 of Synaptophysin-like protein, Vitamin K epoxide reductase complex subunit 1-like protein 1, Splice Isoform 1 of Protein C20orf22 (ABHD12), Hypothetical protein DKFZp564K247 (Hypoxia induced gene 1 protein) (IPI Accession No. IPI00295621), Hypothetical protein DKFZp586C1924 (IPI Accession No. IPI00031064), ALEX3 protein variant, Hypothetical protein MGC14288 (IPI Accession No. IPI00176708), protein with IPI Accession No. IPI00639803 and protein with IPI Accession No. IPI00646289; wherein detection of the marker indicates the presence of the foetal erythroblast. In particular, the foetal erythroblast is of mammalian origin. More in particular, the foetal erythroblast is of human origin. A brief description of individual foetal erythroblast specific markers, on their location, physiological roles (including those related to human foetal development), and diseases related to their mutations is provided below.
The identification of foetal erythroblast specific marker will facilitate the identification and isolation of FNRBCs from maternal blood, and thus provides for a reliable approach for future NIPD using FNRBCs present in maternal blood.
Brief Description of the Foetal Erythroblast Specific Markers
Amino Acid Transporters
Transporters of cells and organelles regulate the uptake and efflux of important compounds such as sugars, amino acids, nucleotides, ions and drugs. Solute carrier (SLC) series of transporters include genes encoding passive transporters, ion transporters and exchangers.
Two amino acid transporting SLC proteins (SLC1A5 and SLC3A2) were identified unique to plasma membrane of foetal primitive erythroblasts. SLC1A5, neutral amino acid transporter B0, is a Na+ dependent transporter of SLC1 family expressed in kidney and intestine. SLC1A5 amino acid transport protein identified in foetal erythroblasts belongs to ASCT2 system which can transport glutamine and asparagine with high affinity, and neutral amino acids methionine, leucine and glycine with low affinity.
SLC3A2 (CD98hc) is the heavy chain of the hetero-dimeric protein 4F2 (CD98). CD98 as a hetero-dimer, is involved in amino acid transportation where the substrate specificity varies with the nature of the light chain. Different domains of CD98hc are necessary for association with light chains. Studies on the amino acid transport in human placenta is correlated well with the expression of mRNAs of CD98hc, and a possible role for these proteins in materno-foetal transfer of amino acids and iodo-thyronines is also suggested. CD98hc is found to be co-localized with α4β3 integrin to promote adhesion and motility of extravillous trophoblasts suggesting the functional importance of CD98hc in human foetal development.
There is evidence for amino acid transport in matured human red blood cells too; It has been previously demonstrated that a Na+ dependent amino acid transport system, and recently, CD98hc associated with L-type amino acid transporter 1 (LAT1) or LAT2 light chain may be involved in the cellular uptake of S-nitroso-L-cysteine into human adult red blood cells. However, the absence of CD98hc in mass spectrometric studies of AARBCs may probably be due to smaller peptides generated during MS.
Anion Transporters
Chloride channel (Cc) genes (Clc1-10) are expressed in all phyla from bacteria to man. Clc mediated anion transport is considered to be the main function of most of the Clc proteins.
Three isoforms of Clc6 are known. Mutations in Clc genes have been implicated in various human diseases such as myotonia, renal salt loss, deafness, urinary protein loss, kidney stones, osteoporosis, blindness, and lysosomal storage disease. Recent studies in animal models suggest that Ccl6 may predominantly reside intracellularly in endosomes. In AARBCs, in addition to a small chloride channel other inorganic ion transporters, such as urea transporter-B (SLC14A1) and bicarbonate/chloride exchanger (SLC4A1, Band 3), are known to be functional.
Binding Proteins
Membrane receptors which can bind hormones, growth factors and metabolites are important for cellular growth and function. Transferrin receptor protein 1, Splice isoform 2 of protein GPR107 precursor, and olfactory receptor 11H4 were identified as being unique to primitive foetal erythroblasts. Transferrin receptor was initially identified on maturing erythroid cells and placenta. Iron is an essential requirement for the synthesis of haemoglobin in all stages in erythroid cells to where iron is transported by the transferrin receptor which, however, is absent in AARBCs as it is lost from reticulocytes as they become mature.
Guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) have 7 transmembrane helices and are expressed on cell surface, and bind to almost all of the known neurotransmitters and hormones released synaptically or those that are secreted into the circulatory system controlling organ functions. G-proteins are predominant intracellular molecules that bind and link GPCRs to second messenger systems such as adenyl cyclase, phospholipases, and ionic conductance channels. GPCRs are targets for 40% of all approved drugs and are the main focus of intense pharmaceutical research due to their key roles in cell physiology and disease, and the presence of GPR107 in foetal erythroblasts does not exclude the possibility for potential research using this cell type for foetal therapy.
Olfactory receptor (OR) is the largest mammalian gene family that codes for odorant receptors. Identification of one of the ORs (OR family H subfamily 11) in primitive foetal erythroblasts supports the earlier reports of an OR in hematopoietic cells and tissues: low level expression of OR-mRNA in human erythroleukemia and myeloid cell lines, and in tissues containing cells of erythroid lineage, such as human bone marrow and foetal liver were reported by Feingold and his colleagues. There is evidence for the expression of OR in non-olfactory testicular tissue; in humans, expression of hOR 17-4 and its functional role in sperm chemotaxis is known. In addition, human prostate specific G-protein coupled receptor (PSGR) with properties characteristic of an olfactory receptor was also observed in olfactory zone and the medulla oblongata (human), liver (rat) and in brain and colon (mouse).
Catalytic
CAAX prenyl endopeptidase also known as FACE, farnesylated protein-converting enzyme, is important for prenylation of CAXX motif containing eukaryotic proteins for their function and membrane targeting. FACE-1 and FACE-2 are two human enzymes expressed in several tissues, for example, leukocytes, ovary, testis, kidney and placenta. Prelamin-A is the substrate for FACE-1 and mutations in prelamin A cleavage site or FACE-1 enzyme have been documented in genetic diseases such as Hutchinson-Gilford progeria and mandibuloacral dysplasia. The identification of CAAX prenyl protease 1 homologue, an integral membrane protein containing seven transmembrane domains in foetal erythroblasts, as in other human tissues, indicates a possible house-keeping role for this enzyme in the processing of prenylated proteins.
Vitamin K epoxide reductase complex subunit 1 like protein (VKORC1L1), identified in the present disclosure, is the first report in a human erythroid cell type membrane protein whose sub-cellular location is not yet defined. VKORC1 was reported to be warfarin-sensitive. Vitamin K-dependent clotting factor deficiency type 2 (VKCFD2) in humans showing warfarin resistance is the result of mutation in VKORC1. Foetal warfarin syndrome (warfarin embryopathy) due to warfarin exposure during pregnancy is well known. It has also been suggested that rare polymorphisms and interethnic differences in VKORC1 determines warfarin requirement.
Signaling Pathway
Splice isoform 1 of Protein C9ORF5 identified in primitive foetal erythroblasts is annotated to be involved in signalling pathways. A novel human transcript CG-2 (C9ORF5) was isolated from the familial dysautonomia candidate region on 9831 and its expression was seen in human adult and foetal tissues such as brain, lung, liver and kidney. C9ORF5 was also found to be upregulated in prostrate cancer where the role for this gene is unknown.
Vesicle Recycling
Synaptophysin-like protein, pantophysin, an isoform of synaptophysin identified in primitive erythroblasts was annotated to be located in plasma/vesicle membrane. It is highly conserved and considered as a novel pre-synaptic marker for neurons and neuroendocrine (NE) cells. Pantophysin is localized in cytoplasmic micro-vesicles of various secretory, shuttling, and endocytotic recycling pathways and are co-localized with synaptophysin in transfected non-neuroendocrine and neuroendocrine cells and in neuroendocrine tissues. Non-neuronal distribution of pantophysin in epithelial, muscle tissues and fibroblasts has already been documented.
Antimicrobial Proteins
Expression of BCG induced integral membrane protein BIGM 103 (BCG induced gene in monocyte, clone 103) in foetal erythroblasts is novel. This protein was first identified from cDNA library prepared from monocytes induced with BCG cell wall. BIGM103 has sequence similarity with Zip-like family of proteins and matched with hZIP2 and hZIP1 and is predicted to possess zinc transporter and metallo-protease activities. A possible role in phagocytosis-mediated elimination of microbial components in macrophages and dendritic cells has also been suggested. FALL39 identified in foetal erythroblasts is one of the antimicrobial peptides of neutrophil granules such as Azurocidin (CAP-37) and CAP-57. FALL39 was also identified from human bone marrow and testis. Contrary to the microbicidal function, a novel pro-tumorigenic role for mature FALL-39 (hCAP-18/LL-37) was also demonstrated in ovarian cancer, through activation of matrix metalloproteinases, and there is evidence for strong association between leukocyte infiltration and cancer progression.
Proteins with No Known Function but Candidates for Research Related to Foetal Development.
Cleft lip and palate transmembrane protein 1—To date, no functional role for CLPTM 1 is defined. CLPTM 1 is reported to be homologous with Cisplatin Resistance Related gene-9, and observed to be more expressed in clinical samples resistant to chemotherapy in breast cancer. Clinically, folate deficiency is known to be associated with cleft lip and/or palate and auto-antibodies against folate receptors are reported to be present in mothers of children with cleft lips. Folate is an important vitamin for several metabolic pathways including those leading to the synthesis of nucleic acids, and are considered vital during infancy and pregnancy. Functional role for CLPTM 1 in foetal erythroblast plasma membrane needs further investigation.
Hypoxia-inducible gene 1 protein, (HIG1 domain family member 1A, HIGD1A) is one of the genes expressed during hypoxia. HIGD1A gene expression was reported in human hematopoietic stem/progenitor cells and in human cervical cells cultured under hypoxic conditions. HIG1 expression in cytoplasmic vesicles and mitochondria appears to be induced by both hypoxia and tumour micro environmental stressor such as glucose deprivation. In humans, the normal foetal development depends on the availability of oxygen and nutrients to the foetus. Identification of HIGD1A protein expression in primitive foetal erythroblasts, but not in adult erythrocytes, correlates with the relatively hypoxic environment of the placenta as compared to that of adult blood circulation.
Others
Identification of ALEX3 protein variant in foetal erythroblasts is unique. The genes for ALEX1, ALEX2 and ALEX3 are localized in human X chromosome. Significantly reduced or loss of mRNA expression of ALEX1 and ALEX2 in epithelial carcinomas (human lung, prostate, colon, pancreas, and ovarian carcinomas) but not in cell lines from other types of tumours leads to a speculation that ALEX genes may play a role in suppression of tumours originating from epithelial tissue.
Reports on protein expression or functional identity of five of the identified proteins of foetal erythroblasts (with at least one transmembrane domain) are not available in any other cell/tissue; they are, Hypothetical protein DKFZp586C1924, 8 kDa protein, 25 Kda protein, Hypothetical protein MGC14288, and Splice Isoform 1 of Protein C20orf22 (ABHD12). Protein databases searches (UniProtKB/Swiss-Prot) did not reveal much information for these proteins. Recently, mRNA expression of Hypothetical protein DKFZp586C1924(TMEM 126A) in human foetal and adult tissues and immuno-localization in mouse mitochondria have been reported.
According to another aspect of the invention, there is provided a method for identifying at least one foetal erythroblast comprising detecting the expression of at least one foetal erythroblast specific marker selected from the group consisting of neutral amino acid transporter B (SLC1A5), solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 isoform A (SLC3A2), Splice Isoform A of Chloride channel protein 6, Transferrin receptor protein 1, Splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, Splice Isoform 1 of Protein C9orf5, Cleft lip and palate transmembrane protein 1, BCG induced integral membrane protein BIGM103, antibacterial protein FALL-39 precursor, CAAX prenyl protease 1 homolog, splice isoform 2 of synaptophysin-like protein, and splice isoform 1 of Protein C20orf22 (ABHD12), wherein detection of the marker indicates the presence of the foetal erythroblast. In particular, the detecting comprises detecting the expression of at least one foetal erythroblast specific marker selected from the group consisting of splice isoform 1 of Protein C20orf22 (ABHD12), Splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, and ALEX3 protein variant.
Alternatively, the foetal erythroblast specific marker may be detected by an antibody, antigen binding fragment thereof, or the like. In particular, the antibody may be polyclonal or monoclonal. A person skilled in the art would understand that any molecular or compound capable of recognizing and/or binding to the foetal erythroblast specific marker can be used to detect the foetal erythroblast specific marker.
According to another aspect of the invention, there is provided a method of isolating at least one foetal erythroblast from a sample, the method comprising: (a) contacting the sample with at least one antibody or antigen binding fragment thereof that is capable of binding to at least one marker selected from the group consisting of neutral amino acid transporter B (SLC1A5), solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 isoform A (SLC3A2), Splice Isoform A of Chloride channel protein 6, Transferrin receptor protein 1, Splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, Splice Isoform 1 of Protein C9orf5, Cleft lip and palate transmembrane protein 1, BCG induced integral membrane protein BIGM103, Antibacterial protein FALL-39 precursor, CAAX prenyl protease 1 homolog, Splice Isoform 2 of Synaptophysin-like protein, Vitamin K epoxide reductase complex subunit 1-like protein 1, Splice Isoform 1 of Protein C20orf22 (ABHD12), Hypothetical protein DKFZp564K247 (Hypoxia induced gene 1 protein) (IPI Accession No. IPI00295621), Hypothetical protein DKFZp586C1924 (IPI Accession No. IPI00031064), ALEX3 protein variant, Hypothetical protein MGC14288 (IPI Accession No. IPI00176708), protein with IPI Accession No. IPI00639803 and protein with IPI Accession No. IPI00646289; and (b) isolating the foetal erythroblast that binds to the antibody or antigen binding fragment thereof from the sample.
In particular, the antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody or a combination thereof. More in particular, the foetal erythroblast that binds to the antibody is isolated from the sample using immunomagnetic separation, flow cytometry or a combination thereof.
The isolation of the mammalian nucleated foetal cell from the sample may be performed using, but not limited to, a micromanipulator or any system that allows individual picking of a foetal cell. In particular, the foetal cell may be a mammalian foetal erythroblast. More in particular, the foetal cell may be a primitive or human foetal erythroblast.
Density gradients and flow sorting methods may be employed to enhance enrichment and purity of foetal erythroblasts from maternal blood.
According to yet another aspect of the invention, there is provided a method of diagnosing at least one prenatal disorder in an individual, the method comprising: a. identifying at least one foetal erythroblast in a sample of the individual according to the method described above; b. isolating the foetal erythroblast; and c. determining at least one genetic marker associated with the prenatal disorder in the foetal erythroblast. In particular, the prenatal disorder may be selected from the group consisting of Down Syndrome, Edwards Syndrome, Patau Syndrome, a neural tube defect, spina bifida, cleft palate, Tay Sachs disease, sickle-cell anemia, thalassemia, cystic fibrosis, fragile X syndrome, spinal muscular atrophy, myotonic dystrophy, Huntington's disease, Charcot-Marie-Tooth disease, haemophilia, Duchenne Muscular Dystrophy, mitochondrial disorder, hereditary multiple exostoses and osteogenesis imperfecta disorder. More in particular, the sample may be selected from the group consisting of maternal tissue, maternal blood, cord blood, amniocytes, chorionic villus sample, foetal blood, and foetal tissue. In particular, the method may be carried out in vitro.
According to an aspect of the invention, there is provided a marker for identifying foetal erythroblast selected from the group consisting of neutral amino acid transporter B (SLC1A5), solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 isoform A (SLC3A2), Splice Isoform A of Chloride channel protein 6, Transferrin receptor protein 1, Splice Isoform 3 of Protein GPR107 precursor, Olfactory receptor 11H4, Splice isoform 1 of Protein C9orf5, Cleft lip and palate transmembrane protein 1, BCG induced integral membrane protein BIGM103, Antibacterial protein FALL-39 precursor, CAAX prenyl protease 1 homolog, Splice Isoform 2 of Synaptophysin-like protein, Vitamin K epoxide reductase complex subunit 1-like protein 1, Splice Isoform 1 of Protein C20orf22 (ABHD12), Hypothetical protein DKFZp564K247 (Hypoxia induced gene 1 protein) (IPI Accession No. IPI00295621), Hypothetical protein DKFZp586C1924 (IPI Accession No. IPI00031064), ALEX3 protein variant, Hypothetical protein MGC14288 (IPI Accession No. IPI00176708), protein with IPI Accession No. IPI00639803 and protein with IPI Accession No. IPI00646289. There is further provided an antibody or antigen binding fragment thereof that is capable of binding at least one marker according to the present invention.
Also provided is a kit for use in a method of identifying and/or isolating foetal erythroblast according to any aspects of the present invention.
Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001).
The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.
An in-depth literature search was conducted on the presence and functional roles of unique plasma membrane proteins of FPNRBCs in various human tissues and cells, including that of foetus (trophoblasts/placenta). Short description of these proteins, on their location, physiological roles (including those related to human foetal development), and diseases related to their mutations have been provided above, together with available data on similar functions in AARBCs.
FPRNBCs can be separated from WBCs in maternal blood by negative depletion of CD45 positive cells, and if suitable surface antigen known on FPNRBCs available, these ideal cells for non-invasive prenatal diagnosis can be enriched from AARBCs. Membrane proteins of FPNRBCs were profiled by mass spectrometry, and compared this profile with that of the AARBC membrane proteome as known in the art to identify unique surface membrane proteins of FPNRBCs which are absent in AARBCs.
Membrane proteins of FPNRBCs profiled by mass spectrometry may be compared to known membrane proteome of AARBC. A shot-gun proteomics approach, two-dimensional liquid chromatography coupled with MALDI-TOF/TOF-MS (2D-LCMS/MS) was used to characterize the membrane proteome of foetal primitive erythroblasts. This is the first report on the membrane proteome of the foetal primitive erythroblasts. Details of all 273 proteins identified are provided including their annotated sub-cellular locations, molecular functions and number of transmembrane domains. 133 (48.7%) proteins were membrane proteins, of which 37 were plasma membrane proteins.
Unique, surface membrane proteins of FPRNBCs were identified by comparing the data of the present study with membrane proteins of AARBCs to identify common, and 12 plasma membrane proteins with transmembrane domains and 8 proteins with transmembrane domains but without known sub-cellular location were identified as unique-to-FPNRBCs. Except for the transferrin receptor, all other 19 unique-to-FPNRBC membrane proteins have never been described in red blood cells. Reverse-transcriptase PCR (RT-PCR) and immunocytochemistry validated the 2D-LCMS/MS data. The findings provide potential surface antigens for separation of FPNRBCs from maternal blood for non-invasive prenatal diagnosis, and help understand the biology these rare cells.
Proteomic analyses of FPNRBCs had not been attempted previously owing to the difficulty to obtain sufficient number of cells. Access to placental villi from patients undergoing termination of pregnancy enabled to pool cells for 2D-LCMS/MS analysis. In addition, the extraction of membrane proteins is yet another challenge in proteomics; recovery of more membrane proteins (48.7% of total) from a limited sample (5×107 cells) than those from AARBCs using similar protocol is encouraging, which also explains the structural complexity of these nucleated cells.
Sub-cellular localization and molecular functions annotated for most of the proteins of FPNRBCs are novel for this cell type. Identified FPNRBC membrane proteins show diverse physiological functions varying from transport, catalytic, binding to structural, while about 32% were transport and/or catalytic. Among the membrane proteins, most were identified from mitochondria (48 proteins) and plasma membrane (37 proteins).
Tissues
Placental tissue collection from women undergoing elective first trimester surgical termination of pregnancy was approved by the Institutional Review Board, and all patients gave written informed consent.
Extraction of FPNRBCs from Placental Villi
FPNRBCs were extracted from placental villi, and AARBCs were prepared from volunteer blood sample. Placental tissues were collected at the termination of pregnancy (7+0 to 9+3 weeks amenorrhoea). FPNRBCs were extracted from placental villi as per protocol known in the art. Placental villi were digested in trophoblast digestion buffer (146.3 ml HBSS containing 0.182 g trypsin and 3.75 ml 1M Hepes (Gibco®-Invitrogen-Life-Technologies, NY, USA) for 30 min at 37° C. in a shaking-water-bath, and digestion was stopped using foetal calf serum (Pierce, Ill., USA) (5 ml/45 ml digestion buffer). Single cell suspensions were centrifuged (3000 rpm, 20° C., 10 min). Red cell pellets containing FPNRBCs were suspended in PBS, and separated using Percoll 1083 (GE Healthcare, Uppsala, Sweden) (3000 rpm, 20° C., 20 min). FPNRBC purity was determined by basic staining of cytospun slides. Samples were stored for membrane preparation (if purity≧90% FPNRBCs) in HES buffer (20 mM HEPES, pH 7.4, 1 mM EDTA and 250 mM sucrose) with protease-inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) at −80° C. Morphologies of FPNRBCs and AARBCs are shown, in
Membrane Protein Preparation and Digestion
Membranes from pooled FPNRBCs (5×107 cells) were prepared as described in the art. Cells stored in HES buffer were lysed by thawing and sonication, and ultra-centrifuged at 100,000×g 4° C. (1 h) to obtain the membrane pellet which was then washed using high pH solution (0.1M Na2CO3, pH11), and twice with Milli-Q water. Proteins were extracted from FPNRBC membranes using methanol (MeOH)/50 mM NH4HCO3 (60:40, vol/vol), and protein reduction, alkylation and digestion were carried out as described by Blonder et al. Tryptic digestion was carried out using sequencing grade modified trypsin (Promega, Southampton, UK). Digested sample was centrifuged and the pellet washed in MeOH solution (60% MeOH in 50 mM NH4HCO3) twice. Supernatants were pooled (MeOH-derived digests), while the pellet was re-suspended in Trifluoroethanol (TFE)/50 mM NH4HCO3 (50:50 vol/vol) and the proteins extracted were then diluted 10 times with 50 mM NH4HCO3 for a second trypsin digestion to obtain supernatants (TFE-derived digests). Both digests were lyophilized and stored at −80° C.
Two-Dimensional Liquid Chromatography and Mass Spectrometry (2D-LCMS/MS)
2D-LCMS/MS was essentially the same described earlier by us (Zhang et al., 2007). Lyophilized digests were re-suspended in solvent [(98% H2O, 2% acetonitrile (CAN) and 0.05% trifluoroacetic acid (TFA)], and after centrifugation supernatants were separated using an Ultimate-Dual-HPLC system (Dionex, Sunnyvale, Calif., USA). All samples were first separated on a strong cation exchange (SCX) column (300 μm i.d., ×15 cm, packed with 10 μm POROS 10S) and eluted fractions were captured on the PepMap trap column (300 μm i.d., ×1 mm, packed with 5 μm C18 100 Å), and eluted by gradient elution to a reversed-phase column (Monolithic Capillary Column, 200 μm i.d., ×5 cm). LC fractions were mixed with matrix-assisted laser desorption/onization (MALDI) matrix (7 mg/ml α-cyano-4-hydroxycinnamic acid and 130 μg/ml ammonium citrate in 75% CAN) at a flow rate of 5.4 μl/min through a 25 nl mixing-tee (Upchurch Scientific, Oak Harbor, Wash., USA) before being spotted onto 192-well stainless steel MALDI target plates (AB SCIEX, Foster City, Calif., USA), at a rate of one well per 5 s, using a Probot Micro Fraction collector (Dionex).
Samples on the MALDI target plates were analyzed using an ABI 4700 Proteomics Analyzer (AB SCIEX) with a MALDI source and time of flight analyzer TOF/TOF™ optics. For MS analysis, typically 1000 shots were accumulated for each sample well. Tandem-MS_(MS/MS) analyses were performed using nitrogen, at collision energy of 1 kV and a collision gas pressure of ˜3.0×10−7 Torr. 3000 to 6000 shots were combined for each spectrum depending on the quality of the data.
Database Searching
MASCOT search engine (v2.0; Matrix Science) was used to search tandem mass spectra. GPS Explorer™ software (v3.6; AB SCIEX) was used to create and search files with the MASCOT search engine for peptide and protein identifications. The International Protein Index (IPI) human protein database (v3.10) was used for the search of tryptic peptides and 57478 entries were searched. All MS/MS spectra from the LC runs were combined for the search. Cysteine carbamidomethylation, N-terminal acetylation and pyroglutamination, and methionine oxidation were selected as variable modifications. Two missed cleavages were allowed. Precursor error tolerance was set to 200 ppm and MS/MS fragment error tolerance was 0.4 Da.
Estimation of False Positive Rate
The false positive rate was calculated by comparing the search results from a randomized database versus the actual database. The minimum ion score C.I. percent such that no more than 5% false discovery rate (FDR) was achieved and was used as the cut-off threshold at the peptide level. All the proteins identified from random database search were single peptide-matched. Proteins identified by this method from IPI human database were colour coded as red, green or black: those red coloured proteins are matched to at least two peptides and hence are statistically confident (FDR is zero); proteins that are green coloured are identified by single peptide where match scores are higher than the highest score in the decoy database and essentially the FDR is zero; black coloured proteins were identified based on single peptide match fall within the set threshold of 5% FDR. Top ranked peptides with Best Ion scores≧33 and 36 for TFE and MeOH extractions, respectively, were included for analysis as peptides counted for each protein. All the MS/MS spectra were further validated manually.
Annotation
Sub cellular and functional categories of the identified proteins were obtained based on annotations of Gene Ontology using GoFig. (http://udgenome.ags.udel.edu/gofigure/index.html). Swiss-prot and TrEMBL data base were also used for functional annotation of unique proteins of FPNRBCs. The number of transmembrane domains (TMD) of the identified proteins was predicted using TMHMM Server (v2.0) (http://www.cbs.dtu.dk/services/TMHMM/).
Evaluation of the Identified Unique Proteins
a) Reverse Transcriptase PCR (RT-PCR) for mRNA Expression of Unique Proteins
RNA extraction—RNA from FPNRBCs was isolated using an RNeasy Mini Kit (Qiagen, Germany) according to manufacturer's instructions. Briefly, FPNRBCs (3×106 cells) were resuspended in 350 μl lysis buffer and passed through QIAshredder spin column. The lysate was mixed with 350 μl of 70% ethanol and pipetted onto an RNeasy mini column, and centrifuged at 15000×g for 15 sec. RNA trapped in the column was washed using 350 μl buffer RW1 and incubated with 10 μl of DNase in 70 μl RDD buffer at room temperature for 15 min. RNA was then washed twice with 350 μl of buffer RW1 and once with 500 μl buffer RPE and recovered by the addition of 50 μl RNase-free water onto the column and centrifugation at 15000×g for 1 min.
RT-PCR—cDNA template was synthesised using Sensiscript RT Kit (Qiagen, Germany). Briefly, 5 μl of RNA was mixed with oligo-dT, RNase inhibitor, dNTP mix and RNase-free water (as per manufacturer's instructions) and incubated at 70° C. for 5 min and chilled on ice. RT buffer and RT enzyme were added to the mixture and incubated at 25° C. (15 min), 42° C. (60 min) and 72° C. (15 min), and cooled on ice. PCR mixture contained 5 μl cDNA, 1×PCR buffer, 1 mM dNTP, 8 mM MgCl2, 2.5 U Taq polymerase and 0.6 μM primers. Denatured (94° C. 2 min) mixture was amplified by 45 cycles of 94° C. for 15 sec, ˜60° C. (depends on primer pairs) for 15 sec, 72° C. for 1 min. A final extension at 72° C. for 4 min was performed for each gene. RT control (no enzyme in RT step) and PCR control (Water-blanks) were also included. PCR products were separated by electrophoresis in a 2% agarose gel, stained with ethidium bromide (0.5 g/ml) and visualized under UV light. The images were captured using a digital imager (Alpha Innotech Corp., San Leandro, Calif.). Primer pairs (Sigma-Proligo) used for the amplification for individual gene are listed in Table 1.
b) Localisation of Unique Proteins on FPNRBCs by Alkaline Phosphatase Immunocytochemistry
8 commercially available antibodies against unique proteins of FPNRBCs annotated to be on plasma membrane, and also in other membranes or unique proteins with unknown sub-cellular location were used to localize their antigens in both FPNRBCs and AARBCs: Neutral amino acid transporter B (SLC1A5) (Chemicon-International, Temecula, Calif., USA), Solute carrier family 3, member 2, isoform A (SLC3A2), Olfactory receptor 11H4 (OR11H4) and Antibacterial protein FALL-39 precursor (Cathelicidin antimicrobial peptide, CAP-18) (all from Abcam, Cambridge, UK), Cleft lip and palate transmembrane protein1 (CLPTM1), Armadillo Repeat-Containing X-linked protein 3 (ARMCX3/ALEX3), and CAAX prenyl proteasel homolog (FACE1) (all from Novus-Biologicals, Littleton, Colo.), and Chloride channel protein 6 (CLCN6) (Santa-Cruz Biotechnology, Inc., CA, USA). Cells were fixed for 10 min either with 4% paraformaldehyde for SLC1A5, SLC3A2, OR11H4, CLCN6, CLPTM1, ARMCX3 or ice-cold methanol:acetone (1:1) for CAP-18 and FACE1; Following steps were common for all slides: Briefly, nonspecific binding was inhibited with diluted goat serum (Sigma-Diagnostics, MO, USA) (1:10 in PBS) for 120 min which was followed by incubation with respective primary-antibodies (1:100) for 60 min at room temperature or overnight at 4° C. Slides were then incubated with corresponding mouse or rabbit biotinylated secondary-antibody (1:100) for 60 min (Vector-Laboratories, CA, USA). This was followed by incubation with streptavidin conjugated alkaline phosphatase (Vector-Laboratories) (1:100). Immunoreaction was detected with freshly prepared Vector-Blue-substrate (Vector-Laboratories) for 10 min in dark. All incubations were performed in a humidifying chamber at room temperature and washes between incubations were in 1×PBST (5 min). Slides were rinsed in water and nuclei stained with nuclear-fast-stain (10 min), slides were rinsed in water and dehydrated with 100% ethanol (30 secs each). Air dried slides were mounted with Vectashield (Vector-Laboratories) and analysed by light microscopy. The staining intensity for each antibody tested was calculated as described by Lehr et al. Mean pixel intensities calculated from the luminosity histogram function on Adobe Photoshop CS4 software (Adobe Systems, Mountain View, Calif.) were compared for statistical significance.
Isolation of FPNRBCs in Spiked Blood Samples
Spiked model mixtures (1×105 FNRBCs in 2 ml peripheral blood) were sorted by CD45 depletion (Magnetic associated cell sorting) and NAT-B positive selection. The enriched mixture was tested for FPNRBCs recovery by haemocytometer, cytospun onto slides and identified by Wright staining.
Statistical Analysis
Mean staining intensities (Mean±SD) between FPNRBCs and AARBCs were compared using Mann-Whitney U test (GraphPad Prism software, GraphPad Prism Inc, CA). Differences were considered significant when P values were <0.05.
FPNRBC Membrane Proteins
Cell membrane protein extraction is challenging because many of these proteins have hydrophobic side chains. Furthermore, the significant quantity of protein needed for detailed proteomic analysis restricts studies on limited-access cells such as the human FPNRBCs. To overcome these difficulties, cell membrane protein material harvested from several trophoblastic villi were collected and pooled, and developed a protocol for maximal cell membrane protein recovery. Two organic solvents, MeOH and TFE, were used and recovered both hydrophilic and hydrophobic proteins using pooled samples of FPNRBCS. A total of 273 proteins were identified, with 144 recovered in MeOH and 199 proteins recovered in TFE digests respectively, while 70 proteins were common to both (Table 2;
As FPNRBCs are nucleated, and also contain other organelles, protein identification found not only plasma membrane proteins, but also membrane proteins from the nucleus, mitochondria, endoplasmic reticulum, Golgi, microsomes and peroxisomes.
Location Annotation of Identified Proteins
A total of 273 proteins were identified, and their locations within the cell annotated (Table 3): 133 were membrane proteins (Table 3) while 132 were non-membrane proteins including 16 that have been described as exclusively cytoplamic (Table 4). Locations of the remaining 8 are as yet unclassified (Table 5).
Sub-cellular localization and functional categories of the identified proteins were obtained based on the annotations of Gene Ontology using GoFig. (http://udgenome.ags.udel.edu/gofigure/index.html). Swiss-prot and TrEMBL data base were also used for the functional annotations of unique proteins of FPNRBCs. Sub-cellular localizations of the 133 membrane proteins were analyzed: of these proteins, 37 were noted to localize to the plasma membrane, 48 mitochondrial membranes, 10 endoplasmic reticular membranes, and the remaining 38 membrane proteins were annotated to be localized in more than one location of the cell (
Functional Annotation of Membrane Proteins
Molecular functions of the 133 membrane proteins identified are detailed in the
Proteins with Transmembrane Domains
Transmembrane domains (TMDs) of all the proteins are provided in the Table 2. The number of predicted transmembrane domains in the identified membrane proteins varied from 0 to 15: NADH dehydrogenase subunit 5 was found to possess the maximum number of TMD. Plasma membrane proteins of primitive FPNRBCs with at least one TMD (25 proteins) and the plasma membrane proteins known to be present on other membranes as well (14 proteins) are presented in Tables 6 and 7, respectively.
precursor
variant
, mitochondrial precursor
nuclear
carrier protein
bloob group glycoprotein
membrane
precursor
G precursor
catalytic activity
translocating
receptor 1
clone NT2RP3003258 highly
ortholog of mouse
1 isoform 1
3
protein CGI-51
protein
67 kDa subunit precursor
oxidase
isoform b
editing enzyme
antigens isoform 1
precursor
protein
synthase 1
1 isoform 5
in
dermatitis
precursor
membrane
-associated protein 29
precursor
transporting lycosomal V0 subunit a
like domain containing protein
milochondial
subunit isoform b
glutamate carrier protein
dehydrogenase (NADP) milochondial precursor
membrane protein 2
nucleotide binding protein alpha 13 subunit
domain adjacent to
patient comptate cds
associated actin dependent regulator of
subunit
cell-derived receptor-1 eta
indicates data missing or illegible when filed
carrier protein
protein, milochondial precursor
precursor
protein 12
domain containing protein 1 precursor
ortholog of mouse
1
-protein
67 kDa subunit precursor
protein
Rhesus blood group. CcEe antigens isoform 1
precursor
: Golgi membrane
precursor
):
-related protein 1
synthase 1
-like profellin
membrane
2
103
-associated protein delta subunit
membrane
and palate transmembrane protein 1
indicates data missing or illegible when filed
peroxicase precursor
precursor
indicates data missing or illegible when filed
Single Peptide Based Identification of Proteins
Colour coding of proteins based on the number of peptides for their identification shown in Table 2 indicated that only 23 of 273 total proteins were black coloured that were identified based on single peptide match which fall within the set threshold of 5% FDR, and the rest were red (≦2 peptides) or green coloured (by single peptide) where FDR was zero. Proteins identified based on single peptides from TFE and MeOH extractions, their peptide sequence and ion score are presented in
Comparison of Plasma Membrane Proteins of FPNRBCs and AARBCs to Identify Unique Membrane Proteins
Mass spectrometry-based identification of membrane proteins of AARBCs have so far been reported by only a few studies including ours. From the published literature, a comprehensive list of all AARBC membrane proteins identified by mass spectrometry to date was curated. In the final list, only those candidates annotated as membrane proteins by gene ontology using GoFig. were included. Redundant entries were removed by manually comparing the sequences of all membrane proteins. A total of 299 non-redundant AARBC membrane proteins were finally short-listed (data not shown); Out of this, 202 were short-listed to include only membrane proteins with known- and potential surface domains (e.g. membrane-associated extracellular proteins and integral membrane proteins) (Table 8). Membrane proteins of FPNRBCs were compared manually with this final list of AARBC membrane proteins to identify both common and unique membrane proteins.
Membrane Proteins Common to Both AARBCs and FPNRBCs
31 proteins were common to both cell types. These included: structural proteins such as the erythrocyte band 7 integral-membrane protein, ankyrin, spectrin, dematin, Protein 4.1; proteins with transport function such as band 3, aquaporin, calcium-transporting ATPase, sodium/potassium-transporting ATPase, solute carrier family 2, facilitated glucose transporter, member 1; and plasma membrane binding proteins like Kell blood group glycoprotein (CD238).
Plasma Membrane Proteins Unique to FPNRBCs
A comparison of membrane proteins with potential surface domains (as annotated) indicated that only 31 proteins were common membrane proteins to AARBCs and FPNRBCs. It was further revealed that 20 proteins were unique to FPNRBCs, and 171 unique to AARBCs, respectively (
Membrane proteins unique to FPNRBCs fall mainly under broad functional groups such as (a) transporter proteins: neutral amino acid transporter B, solute carrier family 3 (activators of dibasic and neutral amino acid transport), splice isoform A of chloride channel protein 6 (chloride ion transport); (b) binding proteins: transferrin receptor protein, splice isoform 3 of Protein GPR107 precursor, olfactory receptor 11H4; and (c) catalytic proteins: CAAX prenyl protease 1 homolog, Vitamin K epoxide reductase complex subunit 1-like protein 1 (VKORC1 L1), Splice Isoform 1 of Protein C20orf22 (ABHD12).
Reverse Transcriptase PCR (RT-PCR) to Confirm Expression of Unique Membrane Proteins within FPNRBCs
FPNRBCs from trophoblastic villi were obtained and all were used to perform the mass spectrometry experiments. To determine if the proteins identified as unique to FPNRBCs were indeed expressed within FPNRBCs, extracted total RNA from FPNRBCs was used to perform an RT-PCR.
mRNA expression of unique proteins of FPNRBCs using total RNA extracted from FPNRBCs and by RT-PCR using primers specific for genes tested (Table 1). The mRNA expression of 23 proteins including 13 proteins unique to FPNRBCs was evaluated (
mRNA expression of all the unique proteins on FPNRBCs tested, except olfactory receptor 11H4 (OR11H4), was detected. The absence of amplification of olfactory receptor could probably be due to the low levels of mRNA accumulated as suggested by Feingold and his colleagues.
Immunocytochemical Localization of Unique FPNRBC Proteins
In situ localization of the putative unique FPNRBC proteins was thought to be more informative than western blotting because the location of plasma membrane, cytoplasmic and nuclear proteins could be readily visualized. These were compared to AARBCs. Intensities of immunostaining for the five antibodies tested, FACE-1, SLC1A5, CAP-18, ARMCX3 and OR11H4 were significantly higher (≦0.001) on FPNRBCs than on AARBCs; in contrast, anti-CLCN6 antibody stained AARBCs much more intensely than FPNRBCs (<0.001). There was no significant difference in the staining between FPNRBCs and AARBCs for CLPTM1 and SLC3A2 (
Intensities of immunostaining of four out of eight antibodies tested were significantly higher (p<0.05;
In
FPNRBCs Recovery with Anti-NAT-B Antibody
To test the possibility of sorting FPNRBCs using any of the markers found in the present disclosure, adult blood sample was spiked with FPNRBCs. Spike recovery of FPNRBCs using NAT-B (SLC1A5) marker was about 62.5% (
Identification of 133 membrane proteins from various sub-cellular locations with different functions would help to explore the importance of FPNRBC in medicine. 132 non-membrane proteins including a few known cytoplasmic proteins (for example, haemoglobin chains ε,γ,δ) are also provided.
Proteomic analyses of FPNRBCs had not been attempted previously owing to the difficulty to obtain sufficient number of cells. Access to placental villi from patients undergoing termination of pregnancy enabled to pool cells for 2D-LCMS/MS analysis. In addition, the extraction of membrane proteins is yet another challenge in proteomics; recovery of more membrane proteins (48.7% of total) from a limited sample (5×107 cells) than those from AARBCs using similar protocol is encouraging, which also explains the structural complexity of these nucleated cells.
Sub-cellular localization and molecular functions annotated for most of the proteins of FPNRBCs are novel for this cell type, which may be useful for protein/developmental/structural biologists, pathologists, haematologists and others. Identified FPNRBC membrane proteins show diverse physiological functions varying from transport, catalytic, binding to structural, while about 32% were transport and/or catalytic. Among the membrane proteins, most were identified from mitochondria (48 proteins) and plasma membrane (37 proteins).
Unique membrane proteins of FPNRBCs were identified to be potential candidates as surface antigens for future separation of this cell type by antibody based techniques. A list of human AARBC membrane proteins prepared based on publications was used for comparison of membrane proteins of FPNRBCs with that of AARBCs: 12 membrane proteins annotated to be in plasma membranes and eight without known sub-cellular locations were found to be unique to FPNRBCs. Proteins with transmembrane domains without known sub-cellular location and molecular function may contain novel antigens of biological significance. This comparison also revealed that 171 proteins are unique to AARBCs which are not found in the data set of FPNRBCs.
A few proteins were found to be common in both the cell types, which included major structural and transport proteins of plasma membrane such as band 3, erythrocyte band 7, facilitated glucose transporter (SLCA2A1), Kell blood group glycoprotein (CD238), aquaporin, ATP-binding cassette half-transporter 1 and glycophorin C, suggesting similar functions for these proteins in FPNRBCs as of their adult counterpart.
In the present disclosure, plasma membrane proteins which are developmental-stage specific to immature red cells but not to AARBCs, such as transferrin receptor and ferritin heavy chain were identified unique to FPNRBCs; similarly, absence of leukocyte specific antigen in the data set also confirms the purity of the samples used.
Indirect validation of unique proteins of FPNRBCs by mRNA expression analysis using RT-PCR revealed the presence of all candidates tested except the olfactory receptor (OR11H4); and the reason for the failure of this protein may probably be due to the low level of the template present in the sample. RT-PCR results for unique proteins confirm their identifications by mass spectrometry. Such validation is not possible for AARBCs as they are mature cells without nuclei or RNA.
Proteomic identification followed by confirmation of their expression in tissues and cells by immunological techniques has been an useful tool in areas such as biomarker discovery, drug discovery and disease biology for example, tumour heterogeneity studies in bladder cancer. Stronger expression levels of unique proteins of FPNRBCs as identified by immunostaining for four of eight antibodies (FACE-1, SLC1A5, CAP-18 and OR11H4) on these cells compared to AARBCs, do support their mass spectrometric identifications. However, expression of chloride channel protein (CLCN6) was found to be opposite (stronger in AARBCs) and two other proteins (SLC3A2 and CLPTM1) did not reveal any difference in their immunostaining in the present study, and such observations may probably be due to the specificity and reactivity of the antibodies used or due to the expression levels and the isoforms of proteins identified. As mentioned earlier, FACE-1 and CAP-18 are also annotated to be present in other locations in addition to their presence in the plasma membrane.
Potential surface antigens for separation of FPNRBCs from maternal blood for non-invasive prenatal diagnosis were identified: these cells in maternal blood, can be separated easily from WBCs using leukocyte specific anti-CD 45 antibody, whereas, it is still challenging to select FPNRBCs from overwhelming AARBCs due to the absence of specific surface antigen present only in any one of these cell types. Identification of unique membrane proteins with transmembrane domains such as FACE-1, SLC1A5, CAP-18 and OR11H4 by mass spectrometry and their intense expressions in FNRBCs, as shown by immunocytochemistry have been done. These potential candidates may be used for separation of this cell type from AARBCs by positive selection by means of immuno-cell sorting techniques such as magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS). Similarly, the absence of immunoreaction of the chloride channel protein in FPNRBCs may also be useful for depletion from AARBCs by such strategies.
Biological Significance of the Unique Plasma Membrane Proteins of FPNRBCs
Briefly, 20 unique membrane proteins could be categorized under seven functional sub-groups: Transportes/Channel molecules: two amino acid transporting Solute Carrier (SLC) proteins, neutral amino acid transporter B0 (NAT-B; SLC1A5, ATB (0), ASCT2), SLC3A2; and an anion transporter, splice isoform A of chloride channel protein 6. Binding proteins: Transferrin receptor protein 1, Splice isoform 3 of protein GPR107 precursor and olfactory receptor 11H4. Catalytic: CAAX prenyl endopeptidase also known as farnesylated protein-converting enzyme (FACE), Vitamin K epoxide reductase complex subunit 1 like protein (VKORC1L1), Splice isoform 1 of protein C20orf22 (ABHD12); Signaling pathway: Splice isoform 1 of Protein C9ORF5; vesicle recycling: Pantopysin; Anti-microbial proteins: BCG induced integral membrane protein BIGM 103 (BCG induced gene in monocyte, clone 103), FALL39; Proteins with no known function: Cleft lip and palate transmembrane protein 1.
Proteins of unknown location and function—reports on protein expression or functional identity of five of the identified proteins of FPNRBCs (with at least one transmembrane domain) are not available in any other cell/tissue; they are, Hypothetical protein DKFZp586C1924, Splice isoform 1 of protein C20orf22 (ABHD12), Hypothetical protein MGC14288, 8 KDa protein and 25 KDa protein. Protein databases searches (UniProtKB/Swiss-Prot) did not reveal much information for these proteins.
These studies on human foetal primitive erythroblasts enables the understanding of the biology of these cells, including haemoglobin switching and regulation of their expression, and, to some extent, on the enrichment of these ideal cells from maternal blood for non-invasive prenatal diagnosis. The proteomic information on the membrane proteins of these cells would help to understand the biology and develop technology for enrichment of these cells from maternal blood for non-invasive prenatal diagnosis.
10 mls of post-TOP maternal blood was collected from two patients. Blood samples were processed using three-step enrichment protocol of our laboratory. Briefly, diluted blood sample was layered over Percoll 1118 density medium and centrifuged. The interface was collected and white blood cells were depleted by magnetic activated cell sorting (MACS) using anti-CD45 magnetic beads. Cells from negative fraction were incubated with anti-ASCT2 antibody for 30 minutes and washed and again incubated with anti-rabbit IgG-magnetic beads for indirect MACS (positive) selection of FPNRBCs. 20 FPNRBCs could be recovered from each sample (Table 11).
Recovery of Fetal Nucleated Erythroblasts from Model Mixture Experiments Using Antibodies Against ABHD12, GPR107, ORH114 and ALEX3
Fetal nucleated red blood cells were extracted from placental villi and stored in IMDM medium overnight. FPNRBCs and AARBCs in the sample were counted using haemocytometer. Fresh AARBCs were obtained by Ficoll-Plague centrifugation of diluted whole blood at 3,000 rpm for 20 minutes. The pelleted RBCs were collected and washed with 1×PBS and also stored in IMDM medium. AARBCs were spiked into the FPNRBCs-containing tubes such that the concentration of FPNRBCs was maintained at 1-9%. Either 0.5×105 or 1×105 FPNRBCs (depending on the availability of FPNRBCs extracted) were used in the mixtures. Each experiment was carried out in duplicates or triplicates depending on the availability of FPNRBCs extracted.
The cell mixture was pelleted by centrifuging at 2,200 rpm for 5 minutes. Supernatant was removed and appropriate volume of MACS buffer added. The concentration of antibodies for incubation with cell mixture was 1:50 for GPR107; OR11H4 and ABHD12, and 1:100 for ALEX3. After incubation at 4° C. for 30 minutes, cells were washed once at 2,200 rpm for 5 minutes and the buffer supernatant was discarded. 60 μl of MACS buffer and 40 μl of anti-rabbit IgG or anti-mouse IgG beads (Miltenyi) as appropriate were added and incubated at 4° C. for 30 minutes. After washing, the cells were separated using Miltenyi MS columns. The recovery of FPNRBCs from model mixture using anto-GPR107 appeared to be higher (29.4%) than that of OR11H4 and ABHD12, or ALEX3.
5. Choolani M et al. Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization. Blood 2001, 98:554-557.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201203910-3 | May 2012 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2013/000212 | 5/23/2013 | WO | 00 |
