The Mz variety of the St(a+) phenotype--a variant of glycophorin A exhibiting a deletion

The NeuNAc level of erythrocyte membranes from two related donors exhibiting the Mz variety of St(a+) phenotype within the MNSs blood group system was found to be decreased by about 16%. The quantity of glycophorin A was decreased by about 38%, whereas that of glycophorin B was not significantly different from normal. Mz erythrocyte membranes were also found to contain an abnormal component (molar ratio to glycophorin A about 0.89:1.0) with an apparent molecular mass of about 24,000 Da. Immunoblotting experiments and amino-acid sequence analysis revealed that the novel component (and glycophorin A in one of the donors) carries blood group M activity. Blood group N activity was demonstrable for glycophorin A and glycophorin B from both donors. Amino-acid sequence analysis of chymotryptic, tryptic and cyanogen bromide peptides demonstrated that the novel molecule exhibits the typical structure of a Sta-active molecule. However, since it exhibits blood group M activity, it appears to represent a variant of glycophorin A lacking the residues 27-58 (encoded by exon three of the glycophorin A gene) rather than a glycophorin B-glycophorin A-hybrid molecule of the anti-Lepore type. Since one of the Mz heterozygotes was found to exhibit both M- and N activity on glycophorin A, the Mz gene complex appears to encode a blood group N-active glycophorin A apart from the novel component and a blood group s-active glycophorin B, although the level of glycophorin A in the erythrocyte membranes is decreased by about half.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence diversification and exon inactivation in the glycophorin A gene family from chimpanzee to human

In humans, the allelic diversity of MNSs glycophorins (GP) occurs mainly through the recombinational modulation of silent exons (pseudoexons) in duplicated genes. To address the origin of such a mechanism, structures of GPA, GPB, and GPE were determined in chimpanzee, the only higher primate known to have achieved a three-gene framework as in humans. Pairwise comparison of the chimpanzee and human genes revealed a high degree of sequence identity and similar exon-intron organization. However, the chimpanzee GPA gene lacks a completely formed M- or N-defining sequence as well as a consensus sequence for the Asn-linked glycosylation. In the case of the GPB gene, exon III is expressed in the chimpanzee but silenced, as a pseudoexon, in the human. Therefore, the protein product in the chimpanzee bears a larger extracellular domain than in the human. For the GPE genes, exon III and exon IV have been inactivated by identical donor splice-site mutations in the two species. Nevertheless, the chimpanzee GPE-like mRNA appeared to be transcribed from a GPB/E composite gene containing no 24-bp insertion sequence in exon V for the transmembrane domain. These results suggest a divergent processing of exonic units from chimpanzee to human in which the inactivation of GPB exon III preserved a limited sequence repertoire for diversification of human glycophorins.Correspondence to: O.O. Blumenfeld     

A carbohydrate structural variant of MM glycoprotein (glycophorin A)

A variant of the MM glycoprotein (glycophorin A) was isolated from erythrocyte membranes of two individual donors, a mother (L.G.) and daughter (V.W.). This glycoprotein was found to be a carbohydrate variant in which, for both donors, certain O-glycosidically linked saccharides retained the core structure consisting of NeuAc(alpha 2,3)Gal(beta 1,3)GalNAc that is common to all O-linked saccharides of the MN glycoproteins, and, in addition, contained substituents, of varying chain lengths, on the primary carbinol of GalNAc. These saccharides were released from the polypeptide by beta-elimination in the presence of sodium borohydride, and aspects of their structure were investigated by glycosidase digestion and periodate oxidation. Thus, the smallest variant structure was deduced to be NeuAc(alpha 2,3)Gal(beta 1,3)[GlcNAc(beta 1,6)]H2GalNAc. The 6-O-linked GlcNAc appears to serve as the focus of further chain elongation reactions, involving alternate additions of Gal and GlcNAc residues and leading to the formation of several homologous structures. Two such structures, NeuAc(alpha 2,3)Gal(beta 1,3)[GlcNAc(beta 1,?) Gal(beta 1,3/4)GlcNAc(beta 1,6)]H2GalNAc and NeuAc(alpha 2,3) Gal(beta 1,3)[Gal(beta 1,3/4)GlcNAc(beta 1,6)]H2GalNAc were the predominant species present. A larger saccharide was also isolated and its partial sequence was determined to be Gal(beta 1,3/4)GlcNAc(beta 1,?)[Gal(beta 1,3/4)Glc-NAc(beta 1,?)] Gal(beta 1,3/4)GlcNAc(beta 1,6)[NeuAc(alpha 2,3)Gal-(beta 1,3)]H2GalNAc. Because the peptide portion of these glycoproteins contains two methionine residues, it was possible to isolate two CNBr glycopeptides from separate regions of the molecule, and to assess the distribution of these variant structures in the polypeptide. The saccharides were linked to about 2-3 Ser and/or Thr residues in the donor LG glycoprotein and one of the attachment sites was located within the CNBr glycooctapeptide representing the NH2 terminus. Considerable heterogeneity in saccharide structure was documented for this site, and it is likely that such heterogeneity occurs also at other sites. The variant saccharides bear structural similarities to the core region of O-linked saccharides of certain blood group-active mucins and ovarian cyst secretions, and to the outer sequences of N-linked carbohydrate units (I-, i-active) of the major glycoprotein of human erythrocytes, band 3. The structures of the variant saccharides suggest that they may be potential precursors of H blood group-active carbohydrates, present in varying degrees of maturity, and attached to an integral protein of erythrocytes.     

Detection of internal exon deletion with exon Del

Background

Exome sequencing allows researchers to study the human genome in unprecedented detail. Among the many types of variants detectable through exome sequencing, one of the most over looked types of mutation is internal deletion of exons. Internal exon deletions are the absence of consecutive exons in a gene. Such deletions have potentially significant biological meaning, and they are often too short to be considered copy number variation. Therefore, to the need for efficient detection of such deletions using exome sequencing data exists.

Results

We present ExonDel, a tool specially designed to detect homozygous exon deletions efficiently. We tested ExonDel on exome sequencing data generated from 16 breast cancer cell lines and identified both novel and known IEDs. Subsequently, we verified our findings using RNAseq and PCR technologies. Further comparisons with multiple sequencing-based CNV tools showed that ExonDel is capable of detecting unique IEDs not found by other CNV tools.

Conclusions

ExonDel is an efficient way to screen for novel and known IEDs using exome sequencing data. ExonDel and its source code can be downloaded freely at https://github.com/slzhao/ExonDel.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-332) contains supplementary material, which is available to authorized users.     

Metachromatic leukodystrophy: a 12-bp deletion in exon 2 of the arylsulfatase A gene in a late infantile variant

Sequencing of the arylsulfatase A gene in a late infantile metachromatic leukodystrophy patient showed the presence of a 12-bp deletion in exon 2. This deletion was found in a compound heterozygous state with the previously described 287 CT transition.     

Gamma thalassemia resulting from the deletion of a gamma-globin gene.

The first example of a deletion of one of the two gamma globin genes has been characterized through an analysis of the DNA of the heterozygous parent of a homozygous newborn, using restriction endonuclease mapping techniques. A deletion of approximately 5 kb was observed which was probably caused by an unequal crossing-over between the -G gamma- and -A gamma- genes resulting in the formation of a -G gamma A gamma- hybrid gene. Data on proportions of G gamma and A gamma chains in newborn babies assumed to be heterozygous for the hybrid and normal genes suggest that this hybrid gene may be producing its A gamma chain at levels normally seen only for the G gamma chain.     

Miltenberger Class I and II erythrocytes carry a variant of glycophorin A.

The membranes from Miltenberger Class I (Mi I) and II (Mi II) erythrocytes, two rare variants at the blood group MNSs locus, exhibited an abnormal glycoprotein of 32 kDa apparent molecular mass sharply stained by the periodic acid/Schiff procedure and a decreased content of glycoprotein alpha (synonym glycophorin A, glycoprotein MN) as seen on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Purified 125I-labelled Vicia graminea lectin binds to the unusual 32 kDa glycoprotein separated from Mi I and Mi II erythrocyte membrane of blood group NN or MN, but no significant labelling of this band was observed with Mi samples typed MM. On the basis of such lectin-labelling experiments we have described two heterozygous MN, Mi I individuals that carry one copy of an M gene producing a normal alpha-glycoprotein with M-specificity and one copy of a MiI gene producing a 32 kDa glycoprotein with N-specificity. Further investigations have shown that the 32 kDa glycoprotein was immunoprecipitated by two mouse monoclonal antibodies (R18 and R10) reacting specifically with the external domain of glycoprotein alpha. These results demonstrate that Mi I and Mi II erythrocytes carry an unusual variant of glycoprotein alpha.     

The effect of chemotherapy on the in vivo frequency of glycophorin A 'null' variant erythrocytes

A human in vivo somatic cell assay based on the enumeration of variant erythrocytes lacking expression of an allelic form of the cell-surface sialoglycoprotein, glycophorin A, was applied to the study of blood samples from patients obtained prior to, during, and following chemotherapy for malignant disease in order to determine the effect of mutagenic chemical agents on the frequency of variant cells. In 22 patients assayed prior to therapy, the mean variant cell frequency was 11.9 per million, which was not significantly different from that observed in healthy controls. In an initial cross-sectional survey, blood samples were obtained at various times during and after therapy from 30 patients diagnosed with a variety of malignancies who were treated with one or more known mutagenic agents including adriamycin, bleomycin, cis-platinum, cyclophosphamide, dacarbazine, etoposide, lomustine, mechlorethamine, melphalan, mitomycin C, and procarbazine. Significant elevations in the mean frequency of variant cells over pre-therapy and normal levels were observed in samples obtained during and after therapy. In a time-series study, 14 breast cancer patients treated with CAF (cyclophosphamide, adriamycin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-fluorouracil), or VMF (vinblastine, methotrexate, 5-fluorouracil) adjuvant chemotherapy were sampled repeatedly during and after therapy. For the CAF and CMF patients an increase in the frequency of variant cells was observed with a lag in the appearance of induced variants after initiation of therapy; variant frequencies gradually increased during therapy reaching a maximum at or shortly after the end of therapy, then declined to near pre-therapy levels within 6 months. The maximum level of induced variants ranged from 2- to 7-fold over pre-therapy or normal levels depending on the combination of agents used. The breast cancer patients treated with both adriamycin and cyclophosphamide showed consistent elevations in the frequency of variant cells; patients treated only with cyclophosphamide showed lower and more variable elevations. The data demonstrate that mutagenic chemotherapy agents induce elevated levels of glycophorin A variant erythrocytes consistent with the hypothesis that variant cells result from somatic mutation. The elevations in variant cells were transient, suggesting that these agents primarily affect the rapidly cycling committed erythroid cell population.     

Moderately severe hemophilia A resulting from Glu----Gly substitution in exon 7 of the factor VIII gene.

To define the molecular basis of a TaqI site alteration in the factor VIII gene of a patient with moderately severe hemophilia A, we used a combination of genomic amplification followed by direct sequencing and oligonucleotide hybridization, to demonstrate an A-to-G substitution in exon 7 (codon 291) of this gene. This mutation generates a Gly in place of Glu at amino acid 272 of the mature factor VIII protein. The mutation arose de novo in a germ cell of the patient's mother.     

Different familial adenomatous polyposis phenotypes resulting from deletions of the entire APC exon 15

Germline mutations of the adenomatous polyposis coli ( APC) gene cause familial adenomatous polyposis (FAP), an autosomal, dominantly inherited disease that predisposes patients to colorectal cancer. The APC gene is composed of 15 coding exons and encodes an open reading frame of 8.5 kb. The 3' 6.5 kb of the APCopen reading frame is encoded by a single exon, exon 15. Most identified APC mutations are at the 5' half of the APC open reading frame and are nucleotide substitutions and small deletions or insertions that result in truncation of the APC protein. Very few well-characterized gross alterations of APC have been reported. Patients with FAP typically develop hundreds to thousands of colorectal tumors beginning in their adolescence. A subgroup of patients with FAP who develop fewer tumors at an older age have what is called attenuated FAP (AFAP). Accumulating evidence indicates that patients carrying germline APC mutations in the first four coding exons, in the alternatively spliced region of exon 9, or in the 3' half of the coding region usually develop AFAP. We characterized two germline APC alterations that deleted the entire APC exon 15 as the result of 56-kb and 73-kb deletions at the APC locus. A surprising finding was that one proband had the typical FAP phenotype, whereas the other had a phenotype consistent with that of AFAP.     

A single mutation in the glycophorin A binding site of hepatitis A virus enhances virus clearance from the blood and results in a lower fitness variant

Hepatitis A virus (HAV) has previously been reported to bind to human red blood cells through interaction with glycophorin A. Residue K221 of VP1 and the surrounding VP3 residues are involved in such an interaction. This capsid region is specifically recognized by the monoclonal antibody H7C27. A monoclonal antibody-resistant mutant with the mutation G1217D has been isolated. In the present study, the G1217D mutant was characterized physically and biologically in comparison with the parental HM175 43c strain. The G1217D mutant is more sensitive to acid pH and binds more efficiently to human and rat erythrocytes than the parental 43c strain. In a rat model, it is eliminated from serum more rapidly and consequently reaches the liver with a certain delay compared to the parental 43c strain. In competition experiments performed in vivo in the rat model, the G1217D mutant was efficiently outcompeted by the parental 43c strain. Only in the presence of antibodies reacting specifically with the parental 43c strain could the G1217D mutant outcompete the parental 43c strain in serum, although the latter still showed a remarkable ability to reach the liver. Altogether, these results indicate that the G1217D mutation induces a low fitness phenotype which could explain the lack of natural antigenic variants of the glycophorin A binding site.     

A very small frame-shifting deletion within exon 19 of the Duchenne muscular dystrophy gene

We report the molecular characterization of a Japanese Duchenne muscular dystrophy (DMD) patient. The analysis of genomic gene by polymerase chain reaction indicates that the individuals have a limited deletion within an amplified region, which encompasses exon 19 of DMD gene. The amplified region was sequenced. Comparison of the deletion joint sequence with the normal amplified region sequence indicates that both 5' and 3' deletion end points are present within exon 19 and the deletion removes total 52 bp out of 88 bp of exon 19. Both his mother and sister are carriers of the deletion-containing allele. The mutation introduces a termination codon at residue 791 in exon 20, and is predicted to result in the production of a severely truncated protein. This sort of deletion (designated as DMD-Kobe) might be classified as a new type of DMD gene abnormality.     

A normal male with an inherited deletion of one exon within the DMD gene

Summary We describe two brothers with identical inherited deletions of one single exon within the middle of the DMD gene; one brother has Becker muscular dystrophy diagnosed at 11 years of age, whereas the older brother is normal at 18. These results have implications for genetic counselling and prenatal diagnosis in families with Becker muscular dystrophy.     

Allele-specific deletion in exon I of the HRAS1 gene

We have detected a 6-bp deletion in the untranslated first exon of a unique HRAS1 gene cloned from lymphocyte DNA of a familial melanoma patient. The deletion is without apparent functional consequence. Using an RNase protection assay, we have demonstrated the deletion in leukocyte DNAs of individuals unrelated to the patient. In these cases, the deletion marker is specifically associated with one class of common HRAS1 allele, thereby establishing the origin of the unique allele. We discuss the means by which DNA sequence heterogeneity at other loci may be rapidly analyzed.     

Flow cytometric characterization of normal and variant cells with monoclonal antibodies specific for glycophorin A

Quantitative immunofluorescence measurements were performed on erythrocytes labeled with monoclonal antibodies to glycophorin A (GPA) to assess the level of binding of these antibodies to normal and variant cell types. The seven antibodies used in this study include two that bind preferentially to the M form of GPA, three that bind preferentially to the N form, and two that bind equally well to both. Flow cytometric analysis of mixtures of cells differing in M,N type showed binding specificities of greater than 100-fold for most of the antibodies, and showed that three antibodies bind cell-bound GPA with an affinity of approximately 10(9) M(-1). These data also showed that the level of expression of GPA varies by less than 10% from cell to cell and from individual to individual. Flow measurements were also done on human erythrocytes with the following variant forms of glycophorin: Mc, Mg, Mk, En(F), En(UK), Mi-I, Mi-II, Mi-III, S-s-U-, Tn+, and St(a+). Other cell types analyzed included erythrocytes from chimpanzee, rhesus, African green, and capuchin monkeys, and cells from the human erythroleukemia cell line, K562. Flow analysis with our seven antibodies showed these cell types have distinctive labeling patterns consistent with the known or inferred altered glycophorins presented on these cells. In most cases, variant alleles were expressed at normal levels. Our results support other observations that the variants En(UK) and St(a+) contain hybrid GPA-GPB proteins, and suggest that their level of expression is largely determined by the 3' end of the hybrid genes.     

Identification and characterization of a novel human APH-1b splice variant lacking exon 4

APH-1 is one of the four essential components of the presenilin-gamma-secretase complex and has two human homologs, APH-1a, and APH-1b, both of which are seven-pass membrane proteins. Here, we identified a novel splice variant of human APH-1b. This variant lacks exon 4, which encodes the entire fourth transmembrane domain. The mRNA expression of this variant was detected in most tissues at low levels. In transiently transfected cells, protein expression of the APH-1b variant was much lower than that of the wild-type. Furthermore, exogenous expression of the APH-1-interacting protein, nicastrin, significantly increased the variant protein levels. These data suggest that the APH-1b variant protein is destabilized, and implies that the fourth transmembrane domain plays an important role in the protein stability and function of APH-1.     

Ornithine transcarbamylase deficiency resulting from a C-to-T substitution in exon 5 of the ornithine transcarbamylase gene.

To define the molecular basis for the TaqI site alteration in the ornithine transcarbamylase (OTC) (E.C.2.1.3.3) gene of a female patient with mild OTC deficiency, we used a combination of genomic amplification followed by direct sequencing and oligodeoxyribonucleotide hybridization. We obtained evidence for a C-to-T substitution in exon 5 (codon 141) of this gene. This mutation generates a stop codon, in place of Arg, at amino acid 109 of the mature OTC protein. The mutation arose, de novo, in a germ cell of one of the parents.     

Lipoprotein lipase deficiency resulting from a nonsense mutation in exon 3 of the lipoprotein lipase gene.

The gene for erythroid 5-aminolevulinate synthase has been mapped to Xpter-Xq26 by Southern blot hybridization analysis of a mouse/human hybrid cell panel. In situ hybridization maps the gene to Xp21-Xq21, with the most likely location being on band Xp11.2. The mapping of the erythroid 5-amino-levulinate synthase gene to the X chromosome suggests that a defect in this gene may be the primary cause of X-linked sideroblastic anemia.     

Molecular analysis of a hybrid gene encoding human glycophorin variant Miltenberger V-like molecule

The genomic structure of a human glycophorin variant, Miltenberger class V-like molecule (MiV*), was examined. Southern blot analysis of total genomic DNA revealed that the 5' half of the MiV* gene derived from glycophorin A (GPA) gene whereas the 3' half derived from glycophorin B (GPB) gene. This structure is reciprocal to another glycophorin variant, Sta, which has a GPB-GPA hybrid structure. The genomic sequences around the crossing-over point were amplified by polymerase chain reaction, and the sequences were determined. Comparison of the nucleotide sequences of the GPA, GPB, and MiV* genes indicates that the crossing-over point is located in the region around the 3' end of intron 3 of the GPA gene. This place is different from the crossing-over point for Sta, which was found to be highly homologous to that for haptoglobin-related genes. However, the nucleotide sequences within the presumptive crossing-over point for the MiV* gene were found to be homologous in a reverse orientation to the crossing-over point proposed for haptoglobin-related genes. These results suggest strongly that homologous recombination through unequal crossing over can be facilitated by specific genomic elements such as those in common for formation of MiV*, Sta, and haptoglobin-related genes. The present study also localized the gene of the third glycophorin, GPE, at chromosome 4, q31.1 band, the same locus as for the GPA and GPB genes. The results indicate that GPE was not involved in generating MiV* or Sta hybrid gene despite the fact that it is localized adjacent to the GPA and GPB genes.