THE CAYAPA INDIANS OF ECUADOR: A GENETICALLY ISOLATED GROUP WITH UNEXPECTED COMPLEMENT C7 M/N ALLELE FREQUENCIES

The Cayapa Indians live in north-western Ecuador in close proximity to a Black population of African ancestry. C7 M/N allotyping has proved to be a good technique for plasma genetic analysis in several populations. Investigation of 124 Cayapa plasma samples revealed the highest allele frequency of C7*N observed in any population examined so far (0.36 versus 0.225 or lower). The marked difference in frequency compared with several Oriental populations, which are believed to have been derived from the same Asian population as native Amerindians, may reflect the effect of a small founder population followed by a high degree of genetic isolation. The allele frequency of 0.12 for C7*N determined for the neighbouring Black population supports the conclusion that there has been a lack of genetic admixture of Cayapas with other populations, confirming the results of ethnohistorical investigations and other protein polymorphism studies.     

C7 M/N protein polymorphism typing applied to inherited deficiencies of human complement proteins C6 and C7.

C7 M/N typing, the determination of the complement component C7 M/N phenotypes, was successfully used in family studies to trace haplotypes bearing C7 deficiency genes. Furthermore, it was shown to be preferable to C7 allotyping based on isoelectric focusing (IEF) since it distinguishes two common alleles (C7*M and C7*N), whereas one common C7 IEF allele (C7*1) predominates in most populations. It is also the more sensitive method, as it enabled detection of very low amounts of abnormal C7 molecules in the third generation of a combined subtotal C6/C7-deficient subject and thus confirmed that this partial deficiency gene is not silent in heterozygotes. In this respect C7 M/N typing is even more informative than DNA restriction fragment length polymorphism typing which will assess the presence but not necessarily the functional status of a gene. C6 and C7 genes are tightly linked and therefore C7 M/N typing was also applied to tracing C6 deficiency genes in families. C6/C7 haplotype analysis of South African C6-deficient (C6Q0) subjects revealed a strong allelic association of C6*Q0 and C7*M.     

Generation and characterisation of monoclonal antibodies specific to avian influenza H7N9 haemagglutinin protein

Introduction: Emerging virulent strains of influenza virus pose a serious public health threat with potential pandemic consequences. A novel avian influenza virus, H7N9, breached the species barrier from infected domestic poultry to humans in 2013 in China. Since then, it has caused numerous infections in humans with a close contact to poultry. Materials and Methods: In this study, we describe the preliminary characterisation of five murine monoclonal antibodies (MAbs) developed against recombinant haemagglutinin (rHA) protein of avian H7N9 A/Anhui/1/2013 virus by their Western blot and enzyme-linked immunosorbent assay (ELISA) reactivity and binding affinity. Results: Of the five MAbs, four were highly specific to H7N9 HA and did not show any cross-reactivity in ELISA with rHA protein from pandemic as well as seasonal H1N1, H2N2, H3N2, H5N1 and influenza virus B (B/Brisbane/60/2008). However, one of the MAbs, MA-24, in addition to HA protein of H7N9 also reacted strongly with HA protein of H3N2 and weakly with HA of pandemic and seasonal H1N1 and H2N2. All the five MAbs also reacted with H7N9 rHA in Western blot. The MAbs bound H7N9 rHA with an equilibrium dissociation constant (K_D) ranging between 0.14 and 25.20 nM, indicating their high affinity to HA. Conclusions: These antibodies may be useful in developing diagnostic tools for the detection of influenza H7N9 virus infections.     

Elongation of the cytoplasmic domain,due to a point deletion at exon 7, results in an HLA-C null allele,Cw*0409 N

The development of molecular techniques for HLA typing has allowed the identification of genes previously assigned as serologic blank alleles. Lack or poor cell surface expression has been found for molecules coded by HLA-A, -B, -DRB4, -DRB5, and -DPB1 genes. In this report we describe the first HLA-C gene encoding for a null cell surface molecule. HLA-Cw*0409 N shows a point deletion at position 1095 within exon 7. This mutation provokes a codon reading shift, generating a new translation stop codon 97 bp downstream to that described in alleles normally expressed. This new stop codon location implies the presence of 32 extra amino acid residues in the cytoplasmic domain. Transfection experiments suggest that elongation of the cytoplasmic domain in Cw*0409 N would be the cause of cell surface expression failure, although Cw*0409 N heavy chain is able to create stable complexes with beta2-microglobulin. HLA-C fragment length analysis in a small selected group of samples with B44-Cblk haplotypic associations allowed us to identify two additional subjects showing both a serologic silent Cw*04 allele and a point base deletion at the 3' end of the HLA-C gene. This finding indicates that the allele frequency of Cw*0409 N within serologic C blank alleles would be appreciable, although basically restricted to the (A23)-Cw*0409 N-B*4403-DR7-DQ2 haplotype.     

Genetic detection of the silent allele (*Q0) in hereditary deficiencies of the human complement C6, C7, and C9 components

DNA polymorphisms (RFLPs) of the human complement component C6, C7, and C9 genes were studied in three C7-deficient (C7D) families, one C6-deficient (C6D) family, and one C9-deficient (C9D) family. The 3 loci are closely linked on human chromosome 5. The haplotypes carrying the “silent” allele (C7*Q0, C6*Q0, and C9*Q0) were defined in each family, allowing for the detection of carriers among asymptomatic relatives. This paper describes familial studies on a type of hereditary trait, characterized by recurrent Neisseria infections in individuals homozygous for “silent” alleles at the C6, C7, or C9 loci. © 1995 Wiley-Liss, Inc.     

HLA-G genotypes and pregnancy outcome in couples with unexplained recurrent miscarriage.

HLA-G is a non-classical human leukocyte antigen expressed primarily in fetal tissues at the maternal-fetal interface. This expression pattern is unique among HLA genes and suggests that HLA-G may be involved in interactions that are critical in establishing and/or maintaining pregnancy. To evaluate the role of polymorphisms at this locus in maternal-fetal interactions, 113 couples with unexplained recurrent miscarriage were genotyped for seven polymorphisms that define 12 HLA-G alleles. Logistic regression analysis was used to assess whether HLA-G genotypes were associated with an increased risk for a subsequent miscarriage. The presence of an HLA-G*0104 or HLA-G*0105N allele in either partner was significantly associated with an increased risk for miscarriage, after adjustment for maternal age, number of previous miscarriages, history of a previous liveborn, and treatment with paternal mononuclear cells. The *0104 and *0105N alleles are defined by polymorphisms in the alpha-2 domain and encode protein variants that are present only in the full-length HLA-G1 protein. The significant genotype-specific risk in this population suggests that allelic variation in the alpha-2 domain of the HLA-G1 isoforms contributes to recurrent miscarriage.     

Identification of a conserved epitope cluster in the N protein of porcine reproductive and respiratory syndrome virus

In this study, 4 overlapping fragments and 12 overlapping peptides of the nucleocapsid (N) protein from porcine reproductive and respiratory syndrome virus (PRRSV) were expressed as glutathione S-transferase (GST) fusion proteins and used to probe a panel of 16 anti-N protein monoclonal antibodies (mAbs) by ELISA. The minimal epitope sequence of the following seven mAbs was determined by sequential deletion of terminal amino acid residues from each peptide: N2H7 corresponded to H54FPLA58; N2F7 corresponded to K52PHFPLA58; and N1A2, N1E3, N1G4, and N2E5 were reactive against E51KPHFP56. Furthermore, a polypeptide containing this epitope cluster was recognized by PRRSV-immune pig serum by Western blot, suggesting that residues 51-58 represent an immunodominant region of the N protein. Sequence alignment revealed that these epitopes are well conserved among North American and European genotypes of PRRSV. These findings enhance our knowledge of the antigenic structure of N protein and may facilitate the development of better diagnostic methods for PRRSV.     

Complement component C6 and C7 haplotypes associated with deficiencies of C6

Both complete C6-deficiency (C6*Q0) and subtotal C6-deficiency (C6*SD) have been described as simple recessive traits and C6*SD has been described in combination with subtotal deficiency of the C7 coded at an adjacent locus. The trace of C6 protein found in both C6*SD traits is phenotypically indistinguishable, being smaller than normal C6 and having different isoelectric properties. A defect has been found in the C6 gene which plausibly explains the C6*SD phenotype, and this defect is also common to both C6*SD traits. We present data from seven DNA markers of the C6 and C7 genes which show that although at least four haplotypes are associated with C6*Q0, most South African C6*Q0 patients carry a common defective haplotype. The most common haplotype associated with C6*Q0 has been observed only once among unaffected haplotypes of relatives. In one family, the cases of C6*SD share a complete haplotype with both cases of combined deficiency and are probably heterozygous for this condition and complete deficiency of C6. In another family, the C6*SD is on a slightly different haplotype and C7 is normally expressed. Thus, the C6 defect is not sufficient on its own to explain the C7 deficiency in the combined deficient haplotype. The haplotype associated with the combined deficiency is found not only in normal control subjects, but also in one case of complete C6 deficiency. In this case the molecular defect seen in combined or C6*SD cases is absent.     

C4 polymorphism and extended HLA haplotypes in Namibian San and Khoi and in South African Xhosa

We studied C4A and C4B polymorphisms and HLA-B and -DR associations in the San, Khoi and Xhosa. C4A and C4B alleles were determined using conventional protein allotyping methods. The C4A*3, C4B*1 haplotype had a high frequency (30–55%) in all populations. The frequency of C4A*3, C4B*Q0 was 7–19%. The C4A*Q0, C4B*1 haplotype was frequent (15%) in the Khoi but very rare in the San (P0.001). C4A*12 A*91, C4B*Q0 was frequent in the Xhosa (15%) but rare in the San and Khoi (P0.001). Alleles C4A*5 and C4A*6, and the C4B*2 B*92 duplication were only found in the Xhosa. C4A alleles A*4, A*45, A*58, A*12, A*14, A*19 and the C4A*3 A*91 duplication were only found in the San/Khoi population group. In the San, fourteen extended haplotypes were found in a relatively high frequency (2–7%). In the Xhosa, one extended haplotype (B42, C4A*12 A*91, C4B*Q0, DR18) was found in a very high frequency (13%) and was characteristic for this group; five other extended haplotypes were found with a low frequency (3%).     

SLC7A7 genomic structure and novel variants in three Japanese lysinuric protein intolerance families

Lysinuric protein intolerance (LPI) is a rare inherited disease caused by defective transport of the dibasic amino acids at the basolateral membranes of epithelial cells in the renal tubules and small intestine. The metabolic defect leads to brain dysfunction caused by hyperammonemia with a functional impairment of the urea cycle. Recently, mutations in the human SLC7A7 cDNA coding for y(+)LAT-1, which express dibasic amino acid transport activity, were reported to be responsible for LPI. In the present study, we examined the genomic structure of SLC7A7 by DNA sequencing of PCR products, and determined that the gene had 11 exons and 10 introns spanning about 18 kb of genomic DNA. We also identified an alternative RNA splicing at the 5' untranslated region of the SLC7A7 mRNA in human peripheral blood leukocytes, cultured lymphoblasts, and fibroblasts. As a result of mutational analysis of SLC7A7 in three Japanese LPI families, we found a nonsense mutation (R410X), a splicing mutation(911+1G>A) in intron 4, and four silent polymorphisms (201C/T, 445A/G, 784C/T, 946T/C). Identification of the genomic structure of SLC7A7 may provide a molecular basis for a genetic survey for LPI.     

An HLB-B null allele (B*0808N) caused by a nucleotide deletion in exon 3, found in the family of a bone marrow transplant recipient

We have identified a variant HLA-B allele, B*0808N, segregating through two generations of healthy individuals, whilst HLA typing the family of a bone marrow patient. Serological typing identified a disparity between the father (A1, A3 B7 DR7) and the brother (A1, A2 B56 DR1, DR7) of the patient. Low/medium resolution polymerase chain reaction using sequence-specific primers (PCR-SSP) revealed a B*08 allele undetectable by serological methods. High resolution DNA typing by polymerase chain reaction-sequencing based typing (PCR-SBT), revealed a nucleotide deletion at position 131 (C) in exon 3, the only difference between the new allele and B*0801. The deletion results in a frame shift in the protein coding sequence, introducing a premature termination codon (TGA) in exon 4. Although a B*08 allele is present in these individuals, the deletion prevents correct expression of the antigen on the cell surface.     

抗SARS病毒N蛋白单克隆抗体的制备和初步应用

目的 制备抗SARS病毒N蛋白的单克隆抗体并研究其初步应用。方法 用基因重组N蛋白免疫小鼠，取免疫后的鼠脾细胞与骨髓瘤细胞融合，筛选分泌抗SAPS病毒N蛋白单克隆抗体细胞株。将阳性细胞株接种小鼠腹腔制备单克隆抗体腹水并对抗体进行纯化，分析纯化抗体的相对亲和力。选择亲和力较高的抗体制备检测SARS病毒抗原的酶联免疫诊断试剂，并对其敏感性和特异性进行分析。结果 共获得11株单克隆抗体细胞株，其中3株单抗与N蛋白具有较高的亲和力，4株纯化单抗与N蛋白反应很弱，其余4株单抗介于两者之间。用亲和力较高的单抗制备检测SARS病毒抗原的诊断试剂，其敏感性可达31PFU／ml，而且与其他呼吸道病毒无交叉反应。结论 该试剂特异性较好，可用于SARS病毒抗原的检测，其敏感性仍需用临床急性期样品进行评价。     

Identification of the null HLA-A2 allele, A*0232N

We have identified a null HLA-A*02 allele, HLA-A*0232N, by using a combination of serology, flow cytometry, polymerase chain reaction using sequence-specific primers (PCR-SSP) and full-length sequencing. The null HLA-A2 allele was identified in an Asian individual originally typed by serology as an apparently homozygous HLA-A3, B51. Subsequent genotyping by PCR-SSP identified the genotype as HLA-A*0201, *0301, B*51, Cw*1402. The serological type and lack of detectable HLA-A2 was confirmed using monoclonal antibody typing reagents. Flow cytometry studies failed to identify any cell surface HLA-A2 expression on the patient's peripheral blood lymphocytes. Genotyping using a PCR-SSP set designed to detect null alleles revealed the mutation had not been previously described. Full-length sequencing of the allele identified an allele which was subsequently named HLA-A*0232N. This allele is identical to HLA-A*0201 except for a novel point mutation (T for C) at position 493 which creates a premature stop codon. The sequencing enabled the development of a monospecific A*0232N PCR-SSP reaction which was used to screen 973 DNA samples: no further examples of A*0232N were identified.     

A new intronic polymorphism in the C7 gene 36 bp from the common expressed C7 M/N polymorphism

We report a new polymorphism in the complement C7 gene that results from an A-C transversion in intron 12, 27 bp upstream of exon 13 (C712.-27) and 36 bp upstream of the point mutation that underlies the C7 M/N antigenic polymorphism. The C7 12.-27 polymorphism subdivides C7 M haplotypes, but not C7 N. It also sheds light on the evolution of the various types of deficiency genes at the adjacent C6 locus.     

MUC7 Haplotype Analysis: Results from a Longitudinal Birth Cohort Support Protective Effect of the MUC7*5 Allele on Respiratory Function

The mucin MUC7 is a glycoprotein that plays a role in bacterial clearance and has candidacidal activity. There are two common allelic forms with 5 or 6 tandem repeats (TR) of a 23 amino acid motif within the highly glycosylated (mucin) domain. The MUC7 *5 allele has previously been shown to be less prevalent in patients with asthma, suggesting a protective role in respiratory function. Here we report the characterisation of other frequent genetic variation within and in the vicinity of the gene MUC7 . A total of 26 polymorphisms were identified of which 5 are located in transcribed regions. A subset of 8 polymorphisms was selected to represent the major haplotypes, and allelic association was studied in individuals of Northern European ancestry, including known asthmatics. There was low haplotype diversity and strong association between each of the loci, and the MUC7 * 5 allele-carrying haplotype remained the one most strongly associated with asthma. Five of these polymorphisms have also been tested in the 1946 longitudinal birth cohort, for whom developmental, environmental and respiratory health data are available. We show that the haplotype carrying MUC7 *5 is associated with higher FEV₁ at 53 years, reduced age-related decline of FEV₁, and also reduced incidence of wheeze.     

Genetic interactions between CDC7 and CDC28: growth inhibition of cdc28-1N by Cdc7 point mutants

Background: Cdc7 kinase of Saccharomyces cerevisiae , a nuclear phosphoprotein, regulates initiation of chromosomal DNA replication. Overexpression of kinase-negative Cdc7 point mutants (T281E, D182N and D163N) arrests the cell cycle of the wild-type Saccharomyces cerevisiae cells at the G1/S boundary. This is caused by titration of a regulatory protein, Dbf4, from the wild-type Cdc7, which leads to inactivation of its kinase activity.
Results: We report here that kinase-negative Cdc7 mutants, when overexpressed in cdc28-1N (ts) at a permissive temperature, not only inhibit DNA replication by inactivating the wild-type Cdc7 but may also disturb coordination between DNA replication and cell division. Suppression of growth inhibition under this condition requires co-expression of both Dbf4 and Cdc28, whereas Dbf4 alone can counteract the growth inhibition in the wild-type cells. In cdc28-1N (ts), co-expression of the wild-type Dbf4 rescues only the G1/S defect and results in accumulation of those cells with less than 1C DNA as well as 2C DNA. On the other hand, co-expression of Cdc28 alone leads to increase of those cells arrested at the G1/S boundary, as found typically in the wild-type. We also report that overexpression of T281A, a 'weak' allele of Cdc7, causes growth arrest in cdc28-1N (ts) cells, but not in the CDC28 wild-type cells. This suggests that T281A is inactive in cdc28-1N (ts) and is consistent with the idea that Cdc28 activates Cdc7 by phosphorylation.
Conclusion: We conclude that two essential serine-threonine kinases, Cdc28 and Cdc7, genetically inter-act for initiation of the S phase and possibly for G2/M progression and/or S phase checkpoint control.     

从噬菌体随机环七肽库筛选可模拟C1qR的C1q结合肽

为获得能结合C1q并模拟C1q受体 (C1qR )配体结合位点的短肽 ,以C1q为钓饵蛋白筛选噬菌体环七肽库 ,采用C1q结合ELISA、U937细胞配体结合抑制试验、多聚IgG (AIgG )竞争抑制试验鉴定阳性克隆 ,再进行单链DNA测序和分析。结果经 3轮筛选后 ,随机挑选 2 3个噬菌体克隆进行ELISA鉴定 ,10个克隆与C1q有较强的结合 ;利用U937细胞配体结合抑制试验 ,得到了 7个阳性克隆 ;从其展示肽DNA测序结果推导氨基酸序列 ,获得 7个短肽序列 :QTPFQLW、NPFNWTS、SPFXLTS、FLTWLDP、FSTFLYP、GPMWWSY和NPFXLIL。     

人乳头瘤病毒16型L1/E7融合蛋白在原核细胞中的表达及免疫效果观察

目的构建能表达L1E7融合蛋白的原核表达菌株,纯化蛋白,并观察其免疫效果。方法用PCR方法分别扩增出C末端部分缺失的HPV16L1基因和HPV16E7编码基因N端部分序列。将上述基因连接,构建融合基因L1ΔCE7N并将其插到原核表达载体pGEX-2T中进行融合蛋白表达纯化,然后观察其免疫效果。结果L1ΔCE7N融合基因测序结果表明,序列与设计相符,读码框架正确。将其插入原核表达质粒在大肠埃希菌中获得高效表达;经Wester-Blot鉴定在相对分子质量约85×103处有特异性表达带,与预期相符。用亲和层析和分子筛可纯化L1ΔCE7N融合蛋白,将其免疫C57BL/6小鼠,结果表明融合蛋白能诱发高滴度L1、E7抗体,并能保护小鼠免受TC-1肿瘤细胞的攻击。结论本实验在原核系统中高效表达并纯化了L1ΔCE7N融合蛋白,该蛋白可作为预防和治疗HPV16感染以及相关肿瘤的候选疫苗株。为研制HPV16预防治疗性疫苗探索一条经济、易普及的途径。     

Novel reassortant H10N7 avian influenza viruses isolated from chickens in Eastern China

BackgroundSince 2004, the H10N7 subtype avian influenza virus (AIV) has caused sporadic human infections with variable clinical symptoms world-wide. However, there is limited information pertaining to the molecular characteristics of H10N7 AIVs in China.ObjectiveTo more fully characterize the genetic relationships between three novel H10N7 strains isolated from chickens in Eastern China and the strains isolated from birds throughout Asia, and to determine the pathogenicity of the H10N7 isolates in vivo.Study designAll eight gene segments from the Chinese H10N7 strains were sequenced and compared with AIV strains available in GenBank. The virulence of the three isolates was determined in chickens and mice.ResultsThree H10N7 subtype avian influenza viruses were isolated from chickens in live poultry markets in Eastern China in 2014: (1) A/chicken/Zhejiang/2C66/2014(H10N7) (ZJ-2C66), (2) A/chicken/Zhejiang/2CP2/2014(H10N7) (ZJ-2CP2), and (3) A/chicken/Zhejiang/2CP8/2014(H10N7) (ZJ-2CP8). Phylogenetic analysis indicated that the viruses contained genetic material from H10, H2, H7, and H3 AIV strains that were circulating at the same time. The reassortant H10N7 viruses were found to be minimally pathogenic in chickens and moderately pathogenic in mice. The viruses were able to replicate in mice without prior adaptation.ConclusionThese results suggest that H10N7 surveillance in poultry should be used as an early warning system for avian influenza outbreaks. The novel strains identified here may post a threat to human health in the future if they continue to circulate.     

中国蒙古族群体和北方汉族群体C1R遗传多态性研究

为了解中国人群Ｃ１Ｒ的遗传多态性，并评估和展望其在法医学和群体遗传学中的应用价值，运用作者建立的测定Ｃ１Ｒ表型的等电聚焦免疫印迹技术，调查了中国蒙古族群体和北方汉族群体补体Ｃ１ｒ亚单位（Ｃ１Ｒ）的遗传多态性，这两个群体中Ｃ１Ｒ等位基因的频率分布为：蒙古族：Ｃ１Ｒ＊１＝０．５３１７、Ｃ１Ｒ＊２＝０．２８１７、Ｃ１Ｒ＊５＝０．１７２５、Ｃ１Ｒ＊Ｖ＝０．０１４１；北方汉族：Ｃ１Ｒ＊１＝０．５３８１、Ｃ１Ｒ＊２＝０．２６１９、Ｃ１Ｒ＊５＝０．１７１４、Ｃ１Ｒ＊６＝０．００４８、Ｃ１Ｒ＊７＝０．００４８、Ｃ１Ｒ＊Ｖ＝０．０１９０。与其他群体Ｃ１Ｒ的资料相比，中国这两个群体Ｃ１Ｒ的基因频率分布与白种人、黑人有很大差异，提示Ｃ１Ｒ是群体遗传学研究较理想的遗传标记之一。