Association between sarcoidosis and HLA polymorphisms in a Czech population from Central Europe: focus on a relationship with clinical outcome and treatment.

Background: Sarcoidosis is an immune-mediated systemic disease with unknown etiology affecting the lung predominantly. The clinical manifestation of sarcoidosis is rather diverse ranging from Löfgren's syndrome to fibrotic disease. Also, it differs among patients with distinct geographical and ethnic origins, consistent with environmental and genetic factors' role in its pathogenesis. Of those, the polymorphic genes of the HLA system have been previously implicated in sarcoidosis. Therefore, we have performed an association study in a well-defined cohort of Czech patients aiming to define how variation in HLA genes, may contribute to disease origin and development. Materials and methods: Total of the 301 Czech unrelated sarcoidosis patients were diagnosed according to international guidelines. In those, HLA typing was performed using next-generation sequencing. The allele frequencies at six HLA loci (HLA-A,-B,-C,-DRB1,-DQA1, and -DQB1) observed in the patients were compared with HLA allele distribution determined in 309 unrelated healthy Czech subjects; sub-analyses of relationships between HLA and distinct sarcoidosis clinical phenotypes were performed. Associations were assessed by two-tailed Fischer's exact test with correction for multiple comparisons. Results: We report two variants, HLA-DQB1*06:02, and HLA-DQB1*06:04, as risk factors for sarcoidosis, and three variants, HLA-DRB1*01:01, HLA-DQA1*03:01, and HLA-DQB1*03:02 as protective factors. HLA-B*08:01, HLA-C*07:01, HLA-DRB1*03:01, HLA-DQA1*05:01, and HLA-DQB1*02:01 variants associated with Löfgren's syndrome, a more benign phenotype. HLA- DRB1*03:01 and HLA-DQA1*05:01 alleles were connected with better prognosis-chest X-ray (CXR) stage 1, disease remission, and non-requirement of corticosteroid treatment. The alleles HLA-DRB1*11:01 and HLA-DQA1*05:05 are associated with more advanced disease represented by the CXR stages 2-4. HLA-DQB1*05:03 associated with sarcoidosis extrapulmonary manifestation. Conclusion: In our Czech cohort, we document some associations between sarcoidosis and HLA previously described in other populations. Further, we suggest novel susceptibility factors for sarcoidosis, such as HLA-DQB1*06:04, and characterize associations between HLA and sarcoidosis clinical phenotypes in Czech patients. Our study also extends the role of the 8.1 ancestral haplotype (HLA-A*01:01∼HLA-B*08:01∼HLA-C*07:01∼HLA-DRB1*03:01∼HLA-DQA1*05:01∼HLA-DQB1*02:01), already implicated in autoimmune diseases, as a possible predictor of better prognosis in sarcoidosis. The general translational application of our newly reported findings for personalized patient care should be validated by an independent study from another, international referral center.

A BAC-based physical map of the major autosomes of Drosophila melanogaster.

We constructed a bacterial artificial chromosome (BAC)-based physical map of chromosomes 2 and 3 of Drosophila melanogaster, which constitute 81% of the genome. Sequence tagged site (STS) content, restriction fingerprinting, and polytene chromosome in situ hybridization approaches were integrated to produce a map spanning the euchromatin. Three of five remaining gaps are in repeat-rich regions near the centromeres. A tiling path of clones spanning this map and STS maps of chromosomes X and 4 was sequenced to low coverage; the maps and tiling path sequence were used to support and verify the whole-genome sequence assembly, and tiling path BACs were used as templates in sequence finishing.

Identification of novel candidate genes associated with cleft lip and palate using array comparative genomic hybridisation.

AIM AND METHOD: We analysed DNA samples isolated from individuals born with cleft lip and cleft palate to identify deletions and duplications of candidate gene loci using array comparative genomic hybridisation (array-CGH). RESULTS: Of 83 syndromic cases analysed we identified one subject with a previously unknown 2.7 Mb deletion at 22q11.21 coinciding with the DiGeorge syndrome region. Eighteen of the syndromic cases had clinical features of Van der Woude syndrome and deletions were identified in five of these, all of which encompassed the interferon regulatory factor 6 (IRF6) gene. In a series of 104 non-syndromic cases we found one subject with a 3.2 Mb deletion at chromosome 6q25.1-25.2 and another with a 2.2 Mb deletion at 10q26.11-26.13. Analyses of parental DNA demonstrated that the two deletion cases at 22q11.21 and 6q25.1-25.2 were de novo, while the deletion of 10q26.11-26.13 was inherited from the mother, who also has a cleft lip. These deletions appear likely to be causally associated with the phenotypes of the subjects. Estrogen receptor 1 (ESR1) and fibroblast growth factor receptor 2 (FGFR2) genes from the 6q25.1-25.2 and 10q26.11-26.13, respectively, were identified as likely causative genes using a gene prioritization software. CONCLUSION: We have shown that array-CGH analysis of DNA samples derived from cleft lip and palate subjects is an efficient and productive method for identifying candidate chromosomal loci and genes, complementing traditional genetic mapping strategies.

A 19-kb CpG island associated with single-minded gene 2 in Down syndrome chromosomal region.

To help in isolating the genes involved in Down syndrome, we sought CpG islands in 4 Mb cosmid/PAC contigs spanning most of the 21q.22.2 band using seven rare cutting enzymes. A striking feature was observed upstream of hSIM2 where at least 41 rare-cutting sites were clustered within a 20-kb region. To investigate the structure of the cluster, a cosmid containing hSIM2 was submitted to shotgun sequencing. Sequence analysis revealed that the cluster was a long CpG island extending 19, 128 nucleotides which includes in the first and second exons of hSIM2. Taken together with our observation in which the CpG islands were concentrated within 1.2 Mb around hSIM2, we propose that this region functions as an R-band, and the cluster provides a unique element for marking of DNA for the spatial and temporal expression of the hSIM2 locus.

A comparative map of bovine chromosome 19 based on a combination of mapping on a bacterial artificial chromosome scaffold map, a whole genome radiation hybrid panel and the human draft sequence.

We have constructed a medium density physical map of bovine chromosome 19 using a combination of mapping loci on both a bovine bacterial artificial chromosome (BAC) scaffold map and a whole genome radiation hybrid (WGRH) panel. The resulting map contains 70 loci spanning the length of bovine chromosome 19. Three contiguous groups of BACs were identified on the basis of multiple loci mapping to individual BAC clones. Bovine chromosome 19 was found in this study to be comprised almost entirely from regions of human chromosome 17, with a small region putatively assigned to human chromosome 10. Fourteen breakpoints between the bovine and human chromosomes were detected, with a possibility of five more based on ordering of the WGRH map.

Alphoid DNA from different chromosomes forms de novo minichromosomes with high efficiency.

Clones from one BAC and one PAC library carrying centromeric alphoid DNA were characterized and found to be stable but to differ according to the enzyme used to make the library. Five different clones with homogeneous alphoid DNA, derived from chromosomes 13/21, 14/22, 17 and 18, were all shown to form minichromosomes de novo after transfection into the human cell line HT1080 in greater than 29% of the cell lines analysed. Similarly sized alphoid arrays (110-160 kb) from chromosomes 17, 13/21 and 14/22 all formed minichromosomes in about 50% of the cell lines analysed while a smaller array (50 kb) of 14/22 alphoid was less efficient (29% of cell lines) and a larger array (200 kb) from chromosome 18 was more efficient (2/2 cell lines). Thus the larger arrays of alphoid DNA gave higher percentages of cell lines with minichromosomes. However, smaller arrays may be preferable for gene expression as there appeared to be more EGFP expression from these minichromosomes.

Streptomyces ATP nucleotide 3'-pyrophosphokinase and its gene.

Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular, ribosome-independent, and stringent factor-mimic ppGpp synthetase with an unusually broad acceptor spectrum. The gene-containing DNA fragments cloned from chromosomal DNA of a producer S. morookaensis into pIJ699 and pUC plasmids were found to express the active enzyme in the transformed S. lividans TK24 and enteric E. coli JM109 and nitrogen-fixing Klebsiella pneumoniae M5a1 and 5022, respectively. Base sequence of the structural gene and the deduced amino acid sequence exhibited little homology to those of E. coli stringent factor and related proteins. Growth retardation was seen in some transformants.

Construction of bacterial artificial chromosome (BAC/PAC) libraries.

This unit describes the construction of BAC and PAC libraries. Two vectors, pCYPAC2 and pPAC4 have been used for preparing PAC libraries, and a new BAC vector pBACe3.6 has been developed for construction of BAC libraries. A support protocol describes preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, simultaneous dephosphorylation with alkaline phosphatase, and subsequent purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, support protocols provide procedures for embedding total genomic DNA from lymphocytes or animal tissue cells, respectively, in InCert agarose. Another support protocol details the next steps for the genomic DNA: partial digestion with MboI or with a combination of EcoRI endonuclease and EcoRI methylase, and subsequent size fractionation by preparative PFGE. The final support protocol covers the isolation of BAC and PAC plasmid DNA for analyzing clones.

Construction of bacterial artificial chromosome (BAC/PAC) libraries.

Large-insert genomic libraries are necessary for physical mapping of large chromosomal regions, for isolation of complete genes, and for use as intermediates in DNA sequencing of entire genomes. Construction of BAC and PAC libraries is detailed in the unit, including preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, dephosphorylation with alkaline phosphatase, and purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, procedures for embedding total genomic DNA from lymphocytes or animal tissue cells are also provided. Other protocols detail partial digestion of genomic DNA with MboI or with a combination of EcoRI endonuclease and EcoRI methylase (including methods for optimizing the extent of digestion), and subsequent size fractionation by preparative PFGE. Finally, the isolation of BAC and PAC plasmid DNA for analyzing clones is also presented.

A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites.

To construct large-insert libraries for the sequencing, mapping, and functional studies of complex genomes, we have constructed a new modular bacterial artificial chromosome (BAC) vector, pBACe3.6 (GenBank Accession No. U80929). This vector contains multiple cloning sites located within the sacB gene, allowing positive selection for recombinant clones on sucrose-containing medium. A recognition site for the PI-SceI nuclease has also been included, which permits linearization of recombinant DNA irrespective of the characteristics of the insert sequences. An attTn7 sequence present in pBACe3.6 permits retrofitting of BAC clones by Tn7-mediated insertion of desirable sequence elements into the vector portion. The ability to retrofit BAC clones will be useful for functional analysis of genes carried on the cloned inserts. The pBACe3.6 vector has been used for the construction of many genomic libraries currently serving as resources for large-scale mapping and sequencing.

An improved approach for construction of bacterial artificial chromosome libraries.

Presented here are improved methodologies that enable the generation of highly redundant bacterial artificial chromosome/P1-derived artificial chromosome libraries, with larger and relatively uniform insert sizes. Improvements in vector preparation and enhanced ligation conditions reduce the number of background nonrecombinant clones. Preelectrophoresis of immobilized high-molecular-weight DNA removes inhibitors of the cloning process, while sizing DNA fragments twice within a single gel effectively eliminates small restriction fragments, thus increasing the average insert size of the clones. The size-fractionated DNA fragments are recovered by electroelution rather than the more common melting of gel slices with subsequent beta-agarase treatment. Concentration of the ligation products yields a 6- to 12-fold reduction in the number of electroporations required in preparing a library of desirable size. These improved methods have been applied to prepare PAC and BAC libraries from the human, murine, rat, canine, and baboon genomes with average insert sizes ranging between 160 and 235 kb.

A bacterial artificial chromosome library for sequencing the complete human genome.

A 30-fold redundant human bacterial artificial chromosome (BAC) library with a large average insert size (178 kb) has been constructed to provide the intermediate substrate for the international genome sequencing effort. The DNA was obtained from a single anonymous volunteer, whose identity was protected through a double-blind donor selection protocol. DNA fragments were generated by partial digestion with EcoRI (library segments 1--4: 24-fold) and MboI (segment 5: sixfold) and cloned into the pBACe3.6 and pTARBAC1 vectors, respectively. The quality of the library was assessed by extensive analysis of 169 clones for rearrangements and artifacts. Eighteen BACs (11%) revealed minor insert rearrangements, and none was chimeric. This BAC library, designated as "RPCI-11," has been used widely as the central resource for insert-end sequencing, clone fingerprinting, high-throughput sequence analysis and as a source of mapped clones for diagnostic and functional studies.

Genomic sequence of a 320-kb segment of the Z chromosome of Bombyx mori containing a kettin ortholog.

The sex chromosome constitution of the silkworm, Bombyx mori, is ZW in the female and ZZ in the male. Very little molecular information is available about the Z chromosome in Lepidoptera, although the topic is interesting because of the absence of gene dosage compensation in this chromosome. We constructed a 320-kb BAC contig around the Bmkettin gene on the Z chromosome in Bombyx and determined its nucleotide sequence by the shotgun method. We found 13 novel protein-coding sequences in addition to Bmkettin. All the transposable elements detected in the region were truncated, and no LTR retrotransposons were found, in stark contrast to the situation on the W chromosome. In this 320-kb region, four genes for muscle proteins (Bmkettin, Bmtitin1, Bmtitin2, and Bmprojectin) are clustered, together with another gene (Bmmiple) on the Z chromosome in B. mori; their orthologs are also closely linked on chromosome 3 in Drosophila, suggesting a partial synteny. Real-time RT-PCR experiments demonstrated that transcripts of 13 genes of the 14 Z-linked genes found accumulated in larger amounts in males than in female moths, indicating the absence of gene dosage compensation. The implications of these findings for the evolution and function of the Z chromosome in Lepidoptera are discussed.

Source and component genes of a 6-200 Mb gene cluster in the house mouse.

We identified and analyzed the genes Sp100, Csprs, and Ifi75 in two members of the genus Mus, M. musculus and M. caroli. Sp100 is a nuclear dot gene; Csprs and Ifi75 are novel genes encoding a putative G-protein coupled receptor (GPCR) and a putative transcriptional coactivator, respectively. A fourth gene, Sp100-rs, occurs in M. musculus, but not in M. caroli. Sp100-rs is a chimeric gene which arose by fusion of Sp100 and Csprs copies. Sp100-rs and Ifi75 are components of a repeat cluster that extends over 6-200 Mb of the M. musculus genome. The Sp100-rs fusion gene arose only 1-2 million years ago and has become fixed and amplified in M. musculus. Although the gene is transcribed, it appears to have no function. The repeat cluster may have become fixed in the species as a 'hitchhiker' in a 'selective sweep'.

Streptomyces ATP nucleotide 3'-pyrophosphokinase-gene cloning and sequence analysis.

Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular enzyme that transfers 5'-beta, gamma-pyrophosphoryl groups of ATP to a variety of nucleotides at the 3'-OH site. The enzyme gene was cloned from partially Sau3AI-digested chromosomal DNA of S. morookaensis in S. lividans TK24/pIJ699 and then in E. coli JM83/pUC12. Some transformants produced the active enzyme. The gene was sequenced by the dideoxynucleotide termination procedure. Its GC content was 72%. Its putative promoter regions, showing little homology to that of the Streptomyces consensus type, were pointed out. No sequence homology was found between the pyrophosphokinase and any other known genes including those of the most mechanistically similar bacterial stringent factor and related proteins. Northern hybridization analysis showed that the gene is constitutionally polycistronic and expressed under transcriptional control. Nuclease S1 mapping indicated that the gene transcription starts from its translation initiation site.

Cosmid assembly and anchoring to human chromosome 21.

A human chromosome 21-specific cosmid library from the Lawrence Livermore National Laboratory has been analyzed by two complementary methods, fingerprinting and hybridization; 40% coverage of the entire chromosome 21 has been achieved. To prepare a contig pool, approximately 9300 cosmid clones randomly selected from the library were fingerprinted and automatically assembled into 467 overlapping sets by the fluorescence-tagged restriction fragment method. The average size of the overlapping sets was 9.5 cosmids with minimal tiling paths consisting of 5.4 cosmids with a 10-kb extension each. However, as many as 10% of overlaps within members were estimated to be false. For regional localization, we hybridized gridded arrays of cosmids with inter-Alu-PCR probes obtained from YAC clones and somatic cell hybrids and assigned 592 cosmids to 26 subregions of 21q. Of these, 371 clones were incorporated into 139 contigs, anchoring the total 1864 cosmids to the subregion. The remaining 221 clones were mapped as orphans. To correlate the cytogenetic, YAC, and cosmid maps on 21q, the translocation breakpoints of the chromosomes contained in the somatic cell hybrids were mapped with respect to the STS content of the YACs. From the gene cluster regions, 176 ribosomal and 25 alphoid clones were isolated by hybridization. Together, these sets of anchored contigs and cosmids will provide a valuable resource for construction of a high-resolution map and for isolation of genes of interest from chromosome 21.

Bacterial artificial chromosome libraries for mouse sequencing and functional analysis.

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9-14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, approximately 1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research.

Modular bacterial artificial chromosome vectors for transfer of large inserts into mammalian cells.

To facilitate the use of large-insert bacterial clones for functional analysis, we have constructed new bacterial artificial chromosome vectors, pPAC4 and pBACe4. These vectors contain two genetic elements that enable stable maintenance of the clones in mammalian cells: (1) The Epstein-Barr virus replicon, oriP, is included to ensure stable episomal propagation of the large insert clones upon transfection into mammalian cells. (2) The blasticidin deaminase gene is placed in a eukaryotic expression cassette to enable selection for the desired mammalian clones by using the nucleoside antibiotic blasticidin. Sequences important to select for loxP-specific genome targeting in mammalian chromosomes are also present. In addition, we demonstrate that the attTn7 sequence present on the vectors permits specific addition of selected features to the library clones. Unique sites have also been included in the vector to enable linearization of the large-insert clones, e. g., for optical mapping studies. The pPAC4 vector has been used to generate libraries from the human, mouse, and rat genomes. We believe that clones from these libraries would serve as an important reagent in functional experiments, including the identification or validation of candidate disease genes, by transferring a particular clone containing the relevant wildtype gene into mutant cells or transgenic or knock-out animals.

Isolation of a cosmid clone corresponding to an inv(21) breakpoint of a patient with transient abnormal myelopoiesis.

Transient abnormal myelopoiesis (TAM) is a leukemoid reaction occurring occasionally on Down syndrome (DS) newborn infants. It has been hypothesized that "disomic homozygosity" in 21-trisomic cells plays an important role in the genesis of TAM, and the putative TAM gene was suggested to be mapped at a 21q11 region. We encountered a DS-associated TAM infant with a 47,XY,inv(21)(q11.1q22.13),+inv(21)(q11.1q22.13) karyotype. On the basis of another presumption that in this patient the putative TAM gene is disrupted by the break, we tried to isolate a breakpoint DNA. FISH analysis with cosmid clones corresponding to various sequence-tagged-site (STS) markers mapped at around 21q11.1-q11.2, we confirmed that the proximal breakpoint of the inv(21) was located between two STSs, G51E07 and D21S215, the latter locus being consistent with the previous tentative mapping. After construction of a cosmid contig encompassing between the two markers, we have isolated a cosmid clone corresponding to the proximal breakpoint of the inversion. This breakpoint was located near a previously identified duplicated region that is homologous to the sequence at 21q22.1. The isolated cosmid clone is useful for analysis of other TAM patients and for a search for a transcript at or flanking the breakpoint.