Genome-wide studies of von Willebrand factor propeptide identify loci contributing to variation in propeptide levels and von Willebrand factor clearance.

UNLABELLED: Essentials Variants at ABO, von Willebrand Factor (VWF) and 2q12 contribute to the variation in plasma in VWF. We performed a genome-wide association study of plasma VWF propeptide in 3,238 individuals. ABO, VWF and 2q12 loci had weak or no association or linkage with plasma VWFpp levels. VWF associated variants at ABO, VWF and 2q12 loci primarily affect VWF clearance rates. SUMMARY: Background Previous studies identified common variants at the ABO and VWF loci and unknown variants in a chromosome 2q12 linkage interval that contributed to the variation in plasma von Willebrand factor (VWF) levels. Whereas the association with ABO haplotypes can be explained by differential VWF clearance, little is known about the mechanisms underlying the association with VWF single-nucleotide polymorphisms (SNPs) or with variants in the chromosome 2 linkage interval. VWF propeptide (VWFpp) and mature VWF are encoded by the VWF gene and secreted at the same rate, but have different plasma half-lives. Therefore, comparison of VWFpp and VWF association signals can be used to assess whether the variants are primarily affecting synthesis/secretion or clearance. Methods We measured plasma VWFpp levels and performed genome-wide linkage and association studies in 3238 young and healthy individuals for whom VWF levels had been analyzed previously. Results and conclusions Common variants in an intergenic region on chromosome 7q11 were associated with VWFpp levels. We found that ABO serotype-specific SNPs were associated with VWFpp levels in the same direction as for VWF, but with a much lower effect size. Neither the association at VWF nor the linkage on chromosome 2 previously reported for VWF was observed for VWFpp. Taken together, these results suggest that the major genetic factors affecting plasma VWF levels, i.e. variants at ABO, VWF and a locus on chromosome 2, operate primarily through their effects on VWF clearance.

Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening systemic illness of abrupt onset and unknown cause. Proteolysis of the blood-clotting protein von Willebrand factor (VWF) observed in normal plasma is decreased in TTP patients. However, the identity of the responsible protease and its role in the pathophysiology of TTP remain unknown. We performed genome-wide linkage analysis in four pedigrees of humans with congenital TTP and mapped the responsible genetic locus to chromosome 9q34. A predicted gene in the identified interval corresponds to a segment of a much larger transcript, identifying a new member of the ADAMTS family of zinc metalloproteinase genes (ADAMTS13). Analysis of patients' genomic DNA identified 12 mutations in the ADAMTS13 gene, accounting for 14 of the 15 disease alleles studied. We show that deficiency of ADAMTS13 is the molecular mechanism responsible for TTP, and suggest that physiologic proteolysis of VWF and/or other ADAMTS13 substrates is required for normal vascular homeostasis.

Genotyping of apolipoprotein E by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I (Clostridium formicoaceticum), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample and obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine analysis of apo E.

Localization of the gene for familial primary pulmonary hypertension to chromosome 2q31-32.

Primary pulmonary hypertension (PPH), an often fatal disease, is characterized by elevated pulmonary artery pressures in the absence of a secondary cause. Endovascular occlusion in the smallest pulmonary arteries occurs by proliferation of cells and matrix, with thrombus and vasospasm. Diagnosis is often delayed because the initial symptoms of fatigue and dyspnea on exertion are nonspecific and definitive diagnosis requires invasive procedures. The average life expectancy after diagnosis is two to three years with death usually due to progressive right heart failure. The aetiology of the disease is unknown. Although most cases appear to be sporadic, approximately 6% of cases recorded in the NIH Primary Pulmonary Hypertension Registry are inherited in an autosomal dominant manner with reduced penetrance. Following a genome-wide search using a set of highly polymorphic short tandem repeat (STR) markers and 19 affected individuals from six families, initial evidence for linkage was obtained with two chromosome 2q markers. We subsequently genotyped patients and all available family members for 19 additional markers spanning approximately 40 centiMorgans (cM) on the long arm of chromosome 2. We obtained a maximum two-point lod score of 6.97 at theta = 0 with the marker D2S389; multipoint linkage analysis yielded a maximum lod score of 7.86 with the marker D2S311. Haplotype analysis established a minimum candidate interval of approximately 25 cM.

Linkage of combined factors V and VIII deficiency to chromosome 18q by homozygosity mapping.

Combined Factors V and VIII deficiency is an autosomal recessive bleeding disorder identified in at least 58 families comprising a number of different ethnic groups. Affected patients present with a moderate bleeding tendency and have Factor V and Factor VIII levels in the range of 5-30% of normal. The highest frequency of the mutant gene is found in Jews of Sephardic and Middle Eastern origin living in Israel with an estimated disease frequency of 1:100,000. We sought to identify the gene responsible for combined Factors V and VIII deficiency using a positional cloning approach. Of 14 affected individuals from 8 unrelated Jewish families, 12 were the offspring of first-cousin marriages. After a genome-wide search using 241 highly polymorphic short tandem repeat (STR) markers, 13 of the 14 affected patients were homozygous for two closely linked 18q markers. Patients and all available family members were genotyped for 11 additional STRs spanning approximately 11 cM on the long arm of chromosome 18. Multipoint linkage analysis yielded a maximal log of the odds (LOD) score of 13.22. Haplotype analysis identified a number of recombinant individuals and established a minimum candidate interval of 2.5 cM for the gene responsible for combined Factors V and VIII deficiency. The product of this locus is likely to operate at a common step in the biosynthetic pathway for these two functionally and structurally homologous coagulation proteins. Identification of this gene should provide new insight into the biology of Factor V and Factor VIII production.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a rapid screening method to detect mutations causing Tay-Sachs disease.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used as a rapid method for the detection of human genetic polymorphisms. In particular, the mutations in the human HEXA gene that cause the infantile Tay-Sachs disease have been studied using MALDI-MS to demonstrate the feasibility of this technique for use in clinical and diagnostic analysis. The protocols involved in this approach include, polymerase chain reaction for the amplification of the mutation site from buccal cell DNA, followed by restriction enzyme digestion of the amplified regions of the template cells. The products of amplification and digestion were studied using MALDI-MS. MALDI-MS experiments are shown to provide essentially the same information as obtained from gel electrophoresis but orders of magnitude faster.

Polymorphism of adhesion molecule CD31 is not a significant risk factor for graft-versus-host disease.

Mismatch between bone marrow transplant (BMT) patient and donor for an amino acid polymorphism within the adhesion molecule CD31 has recently been reported to increase risk for the development of graft-versus-host disease (GVHD). We further examined this association in a larger series of 301 BMT patients (227 with grade III/IV GVHD and 74 with grade 0 GVHD) and their HLA-identical sibling donors. CD31 genotypes were determined by polymerase chain reaction and restriction endonuclease digestion. The role of mismatch at the CD31 locus in the development of GVHD was assessed by analyzing the extent of CD31 identity and CD31 compatibility among the grade 0 GVHD and grade III/IV GVHD sibling pairs. No significant association between CD31 mismatch and the development of severe GVHD was detected in our overall patient population. Sixty-three percent of grade III/IV GVHD sibling pairs and 69% of grade 0 GVHD sibling pairs had CD31 genotypes that were identical (P = .36, odds ratio = 1.30). In addition, neither the grade 0 GVHD group (P = .10) nor the grade III/IV GVHD group (P = .27) differed significantly from the expected probability of identity between sibling pairs. Mismatch at the CD31 polymorphism between recipients and donors showed no consistent association with the development of GVHD. Current evidence does not support the value of CD31 mismatch in the selection of BMT donors.

Rapid screening of genetic polymorphisms using buccal cell DNA with detection by matrix-assisted laser desorption/ionization mass spectrometry.

A new approach is developed for the rapid and cost-effective detection of human genetic polymorphisms based on matrix-assisted laser description/ionization mass spectrometric (MALDI MS) detection using a nitrocellulose film substrate. This method employs polymerase chain reaction (PCR) amplification using DNA extracted from buccal cells as templates, followed by direct digestion with restriction enzymes and subsequent analysis by MALDI MS. The extraction of DNA from buccal cells provides a rapid and convenient means for sampling PCR-based diagnostic analysis. The amount of DNA was sufficient as the template for both normal PCR amplifications, and amplifications involving the use of mismatched primers and multiple primers. The MALDI MS methodology has been successfully used for the analysis of such PCR products where restriction fragments generated directly in PCR reactions have been used for detection of carbonic anhydrase and cystic fibrosis transmembrane conductance regulator as model genes. The detection of genetic polymorphisms following routine biological and clinical procedures with the MALDI MS method is demonstrated. The results from MALDI MS analysis are shown to be comparable to those obtained from gel electrophoresis but the MALDI MS method is several orders of magnitude faster than gel electrophoretic techniques. The method described herein should also be readily extended to other areas involving DNA screening and testing.

The complete nucleotide sequence of cosmid vector pTL5: location and origin of its genetic components.

The complete nucleotide sequence (5793 bp) of the cosmid vector pTL5 and the origin of its genetic components has been determined. Cosmid pTL5, a derivative of cosmid vector pHC79, is composed of genetic components from pBR322, bacteriophage lambda and the hybrid lambdoid bacteriophage Charon (Ch) 4A cohesive ends (cos) region. The Ch4A cos region contains genetic components from two bacteriophages, the lambda cos-left arm and the phi 80 cos-right arm regions. The Ch4A cos region has been used in the construction of many other cosmid-type vectors, some of which have been sequenced and entered into the GenBank database.

Nucleotide sequencing double-stranded plasmids with primers selected from a nonamer library.

Nonamer primers, selected from a nonamer library, were tested by sequencing two plasmid subclones containing known insert sequences. These sequences were scanned (nonamer-mapped) against the 2391-member nonamer library to identify all members that share a 100% match at only one site. A total of 59 nonamers were tested using a slightly modified T7 polymerase sequencing procedure for double-stranded DNA. The success rate for nonamer primed reactions was about 60%, and single-stranded coverage was obtained for approximately 90% of each plasmid insert. The results presented demonstrate that a nonamer library, with as few as 2391 members, can greatly aid the completion of many sequencing projects by reducing the number of required custom primers. With the development of a technique for the rapid identification of all useful library primers for a particular sequencing project, one could envision a high-throughput shotgun-type sequencing procedure that would not require large numbers of subclones.

Matrix-assisted laser desorption/ionization mass spectrometry of restriction enzyme-digested plasmid DNA using an active Nafion substrate.

Matrix-assisted laser desorption/ionization using a 3-hydroxypicolinic acid matrix from an active Nafion substrate has been used for detection of restriction enzyme-digested double-stranded plasmid DNA using time-of-flight mass spectrometry. DNA strands of up to 267 base pairs were detected with minimal sample purification, although only as species corresponding to single-stranded DNA.

Nucleotide sequence analysis of 77.7 kb of the human V beta T-cell receptor gene locus: direct primer-walking using cosmid template DNAs.

The nucleotide sequence of 77.7 kb from the human T-cell receptor beta-chain locus was determined directly from three overlapping cosmid clones using the primer-walking approach. Computer-aided analyses of this sequence reveal the presence of at least 11 genic regions that are closely related to the human T-cell receptor beta variable region (TCRBV) gene family. These include five germline sequences that have previously been determined, V beta 21.2, V beta 8.1, V beta 8.2, V beta 8.3, and V beta 16, and four whose sequences have partially been determined at the mRNA level, V beta 6, V beta 23, V beta 12.2, V beta 24. The two remaining V beta Tcr-related sequences have eluded discovery by cDNA and RT-PCR cloning and genomic blot hybridization methods. These two V beta Tcr-related genes lack > 75% nucleotide sequence identity with any other V beta Tcr gene member and therefore, by convention, are referred to as new subfamily members V beta 25 and V beta 26. This lack of shared identity with other subfamily members explains why they were not detected by hybridization. The promoter regions of these V beta Tcr genes contain the conserved Tcr decamer element located between 80 and 110 bp 5' of the translation start site, generally near a putative TATAA promoter element. Our sequence analysis also reveals that a 3.3-kb duplication unit was involved in the recombination event that produced the closely related V beta 8.1 and 8.2 gene subfamily members. This sequenced region of the V beta locus contains an average number of repetitive DNA elements (21 Alu, three L1, three MER, and three retrovirus-related elements.

The coat protein genes of squash mosaic virus: cloning, sequence analysis, and expression in tobacco protoplasts.

Complementary DNA of the middle-component RNA of the melon strain of squash mosaic comovirus (SqMV) was cloned. Clones containing the coat protein genes were identified by hybridization with a degenerate oligonucleotide synthesized according to the amino acid sequence of a purified peptide fragment of the SqMV large coat protein. A clone containing of 2.5 kbp cDNA insert of SqMV M-RNA was sequenced. The total insert sequence of 2510 bp included a 2373 bp open reading frame (ORF) (encoding 791 amino acids), a 123 bp 3'-untranslated region, and a poly(A) region. This ORF is capable of encoding both the 42 and 22 k SqMV coat proteins. Direct N-terminal sequence analysis of the 22 k coat protein revealed its presence at the 3' end of this ORF and the position of the proteolytic cleavage site (Q/S) used to separate the large and small coat proteins from each other. A putative location of the N-terminal proteolytic cleavage site of the 42 k coat protein (Q/N) was predicted by comparisons with the corresponding coat proteins of cowpea mosaic virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little similarity, their encoded coat proteins shared about 47% identity. The identity of the encoded 42 k and 22 k peptides was confirmed by engineering the respective gene regions for expression followed by transfer into tobacco protoplasts using the polyethylene glycol method. Both SqMV coat proteins were expressed in vivo as determined by their reactivity to SqMV coat protein specific antibodies.

Isolation of a partial candidate gene for Menkes disease by positional cloning.

Menkes disease is an X-linked recessive disorder of copper metabolism resulting in death in early infancy. The gene has been mapped to band Xq13 based, in part, on a translocation breakpoint in a female with the disease, which was found to lie within 300 kilobases (kb) of the PGK-1 locus, allowing the isolation of a YAC clone spanning the breakpoint. Phage subclones from the breakpoint region were isolated and used to screen cDNA libraries. cDNA clones were found which detect an 8 kb transcript from normal individuals but show diminished or absent hybridization in Menkes disease patients. Partial sequence of the cDNA shows a unique open reading frame containing putative metal binding motifs which have been found in heavy metal resistance genes in bacteria. This gene is a strong candidate for the Menkes disease gene.

The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses.

The nucleotide sequence of the beta globin gene cluster of the prosimian Galago crassicaudatus has been determined. A total sequence spanning 41,101 bp contains and links together previously published sequences of the five galago beta-like globin genes (5'-epsilon-gamma-psi eta-delta-beta-3'). A computer-aided search for middle interspersed repetitive sequences identified 10 LINE (L1) elements, including a 5' truncated repeat that is orthologous to the full-length L1 element found in the human epsilon-gamma intergenic region. SINE elements that were identified included one Alu type I repeat, four Alu type II repeats, and two methionine tRNA-derived Monomer (type III) elements. Alu type II and Monomer sequences are unique to the galago genome. Structural analyses of the cluster sequence reveals that it is relatively A+T rich (about 62%) and regions with high G+C content are associated primarily with globin coding regions. Comparative analyses with the beta globin cluster sequences of human, rabbit, and mouse reveal extensive sequence homologies in their genic regions, but only human, galago, and rabbit sequences share extensive intergenic sequence homologies. Divergence analyses of aligned intergenic and flanking sequences from orthologous human, galago, and rabbit sequences show a gradation in the rate of nucleotide sequence evolution along the cluster where sequences 5' of the epsilon globin gene region show the least sequence divergence and sequences just 5' of the beta globin gene region show the greatest sequence divergence.

Sequence analysis of linked maize 22 kDa alpha-zein genes.

We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa alpha-zein genes. The 5' gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5' and 3' flanking regions that are typical of zein genes. In contrast, the 3' gene (psi gZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5' and 3' flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.

Further characterization of the 5'-flanking DNA of the gene encoding human plasminogen activator inhibitor-1.

Previous nucleotide (nt) sequence analysis of the 5'-flanking DNA of the gene (PAI-1) encoding plasminogen activator inhibitor-1 revealed an extensive region of shared nt sequence identity with the 5'-flanking region of the gene (t-PA) encoding tissue-type plasminogen activator [Bosma et al., J. Biol. Chem. 263 (1988) 9129-9141]. Additional sequence (1642 bp) from the PAI-1 gene 5'-flanking region reveals that these 'PAI-1/t-PA' sequence elements share an alignment that contains a total of 575 positions. This additional PAI-1 5'-flanking sequence also contains two Alu elements that form inverted repeats. Southern-blot analysis using the PAI-1/t-PA element as a probe indicates that this element is repeated in the human genome. which supports the classification of this element as a medium reiteration frequency sequence [Jurka, Nucleic Acids Res. 18 (1990) 137-141].

Strategy and methods for directly sequencing cosmid clones.

The primer-directed enzymatic sequencing method for sequencing double-stranded DNA templates has made possible the development of new strategies for directly sequencing large DNA molecules. Toward this goal, we have developed a strategy and the necessary techniques to obtain the complete sequence of cosmid clones (double-stranded DNA molecules in the size range of 50 kb). Our present strategy uses the chemical sequencing method to obtain sequence initiation points internal to a cosmid insert and the primer-directed enzymatic DNA sequencing method to extend these sequence contigs. As part of this development we added a nucleotide "chase" solution to the standard T7 sequencing protocol and included the use of both [alpha-32P]-dATP and -dCTP for labeling. With these modifications our double-stranded cosmid DNA sequencing reactions routinely extend well beyond 1000 bp, and film exposure times are kept to a minimum (24 to 48 h). We can routinely separate sequenced DNA fragments, using a 1-m gel system, which can be accurately read (with less than 0.5% error) to distances of 800 bp or more, from the oligomer primer. The strategy and procedures presented here allow the complete sequence of a cosmid clone to be obtained without subcloning.

A library of 3342 useful nonamer primers for genome sequencing.

Primer-directed sequencing, of double-stranded large recombinant DNA molecules, has not been accepted because of the delay and expenses involved in the synthesis of oligodeoxyribonucleotide primers. A potential solution to this problem was proposed by Studier [Proc. Natl. Acad. Sci. USA 86 (1989) 6917-6921] for using a library of short oligomer primers. Szybalski [Gene 90 (1990) 177-178] has made a complementary proposal using ligated hexamers to reduce the number of oligomers needed. We have used a set of rules for a computer-aided selection of a library consisting of 3342 specific nonamers. The effectiveness of this library of nonamers to sequence specific genes was studied using human sequences available in GenBank.

Watermelon mosaic virus II and zucchini yellow mosaic virus: cloning of 3'-terminal regions, nucleotide sequences, and phylogenetic comparisons.

The 3'-terminal genomic regions of an isolate of watermelon mosaic virus II (WMVII) and a Florida isolate of zucchini yellow mosaic virus (ZYMV-F) have been cloned. The nucleotide sequence of the WMVII cDNA clone shows the presence of the large nuclear inclusion protein gene, the coat protein gene and 3' untranslated region. The nucleotide sequence of a ZYMV-F cDNA clone shows the presence of the coat protein gene and 3' untranslated region. Comparisons of the nucleotide and deduced amino acid sequences of these clones with those from other potyviruses show that WMVII and the soybean mosaic virus N strain are closely related, thus supporting their classification as different strains of the same virus. Our comparisons also indicate that ZYMV-F is a distinct potyvirus type and that its closest relative is WMVII. Phylogenetic analysis using the most-parsimonious branching arrangement derived from the alignment of coat protein gene sequences suggests the existence of two major potyvirus groupings.