A targeted association study in systemic lupus erythematosus identifies multiple susceptibility alleles.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Multiple genetic and environmental factors contribute to the pathogenesis of this disease. Recent genome-wide association studies have added substantially to the number of genes associated with SLE. To replicate some of these susceptibility loci, single-nucleotide polymorphisms reported to be associated to SLE were evaluated in a cohort of 245 well-phenotyped Canadian SLE trios. Our results replicate previously reported associations to alleles of interferon regulatory factor 5 (IRF5), major histocompatibility complex (MHC), tumor necrosis factor (ligand) superfamily member 4 (TNFSF4), Kell blood group complex subunit-related family member 6 (XKR6), B-cell scaffold protein with ankyrin repeats 1 (BANK1), protein tyrosine phosphatase non-receptor type 22 (PTPN22), ubiquitin-conjugating enzyme E2L 3 (UBE2L3) and islet cell autoantigen 1 (ICA1). We also identify putative associations to cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a gene associated with several autoimmune disorders, and ERBB3, a locus on 12q13 that was previously reported to be associated with type 1 diabetes. This study confirms the existence of multiple genetic risk factors for SLE, and supports the notion that some risk factors for SLE are shared with other inflammatory disorders.

Homogeneous assays for single-nucleotide polymorphism typing using AlphaScreen.

AlphaScreen technology allows the development of high-throughput homogeneous proximity assays. In these assays, signal is generated when 680 nm laser light irradiates a donor bead in close proximity to an acceptor bead. For the detection of nucleic acids, donor and acceptor beads are brought into proximity by two bridging probes that hybridize simultaneously to a common target and to the generic oligonucleotides attached covalently to the beads. This method allows the detection of as little as 10 amole of a single-stranded DNA target. The combination of AlphaScreen with allele-specific amplification (ASA) and allele-specific hybridization (ASH) has allowed the development of two homogenous single-nucleotide polymorphism (SNP) genotyping platforms. Both types of assay are very robust, routinely giving accurate genotyping results with < 2 ng of genomic DNA per genotype. An AlphaScreen validation study was performed for 12 SNPs by using ASA assays and seven SNPs by using ASH assays. More than 580 samples were genotyped with accuracy >99%. The two assays are remarkably simple, requiring no post-PCR manipulations. Genotyping has been performed successfully in 96- and 384-well formats with volumes as small as 2 microL, allowing a considerable reduction in the amount of reagents and genomic DNA necessary for genotyping. These results show that the AlphaScreen technology can be successfully adapted to high-throughput genotyping.

Structural analysis of the human pro-apoptotic gene Bik: chromosomal localization, genomic organization and localization of promoter sequences.

The human Bik gene codes for a strong pro-apoptotic protein BIK. We have used fluorescent in-situ hybridization to establish the chromosomal localization of the Bik gene to 22q13.3. Genomic clones of the Bik gene were identified from a cosmid library of chromosome 22. Detailed analysis of the Bik gene revealed that it spans a region of about 19kb and comprises of five exons. Sequence analysis indicated that the 5' flanking region of Bik lacks canonical TATA and CAAT boxes but directs transcriptional initiation from a single site. A 1.9kb region containing the promoter elements of the Bik gene was identified and was found to direct expression of the reporter cat gene in transient transfection studies. By mutational analysis, the minimal Bik promoter was localized to a region between -211 to +153. Northern blot analysis showed a ubiquitous expression profile of the Bik mRNA with elevated levels of expression in heart and skeletal muscle.

Tightly clustered 11q23 and 22q11 breakpoints permit PCR-based detection of the recurrent constitutional t(11;22).

Palindromic AT-rich repeats (PATRRs) on chromosomes 11q23 and 22q11 at the constitutional t(11;22) breakpoint are predicted to induce genomic instability, which mediates the translocation. A PCR-based translocation-detection system for the t(11;22) has been developed with PCR primers flanking the PATRRs of both chromosomes, to examine the involvement of the PATRRs in the recurrent rearrangement. Forty unrelated carriers of the t(11;22) balanced translocation, plus two additional, independent cases with the supernumerary-der(22) syndrome, were analyzed to compare their translocation breakpoints. Similar translocation-specific junction fragments were obtained from both derivative chromosomes in all 40 carriers of the t(11;22) balanced translocation and from the der(22) in both of the offspring with unbalanced supernumerary-der(22) syndrome, suggesting that the breakpoints in all cases localize within these PATRRs and that the translocation is generated by a similar mechanism. This PCR strategy provides a convenient technique for rapid diagnosis of the translocation, indicating its utility for prenatal and preimplantation diagnosis in families including carriers of the balanced translocation.

Regions of genomic instability on 22q11 and 11q23 as the etiology for the recurrent constitutional t(11;22).

The constitutional t(11;22)(q23;q11) is the only known recurrent, non-Robertsonian translocation. To analyze the genomic structure of the breakpoint, we have cloned the junction fragments from the der(11) and der(22) of a t(11;22) balanced carrier. On chromosome 11 the translocation occurs within a short, palindromic AT-rich region (ATRR). Likewise, the breakpoint on chromosome 22 has been localized within an ATRR that is part of a larger palindrome. Interestingly, the 22q11 breakpoint falls within one of the 'unclonable' gaps in the genomic sequence. Further, a sequenced chromosome 11 BAC clone, spanning the t(11;22) breakpoint in 11q23, is deleted within the palindromic ATRR, suggesting instability of this region in bacterial clones. Several unrelated t(11;22) families demonstrate similar breakpoints on both chromosomes, indicating that their translocations are within the same palindrome. It is likely that the palindromic ATRRs produce unstable DNA structures in 22q11 and 11q23 that are responsible for the recurrent t(11;22) translocation.

Chromosome 22-specific low copy repeats and the 22q11.2 deletion syndrome: genomic organization and deletion endpoint analysis.

The 22q11.2 deletion syndrome, which includes DiGeorge and velocardiofacial syndromes (DGS/VCFS), is the most common microdeletion syndrome. The majority of deleted patients share a common 3 Mb hemizygous deletion of 22q11.2. The remaining patients include those who have smaller deletions that are nested within the 3 Mb typically deleted region (TDR) and a few with rare deletions that have no overlap with the TDR. The identification of chromosome 22-specific duplicated sequences or low copy repeats (LCRs) near the end-points of the 3 Mb TDR has led to the hypothesis that they mediate deletions of 22q11.2. The entire 3 Mb TDR has been sequenced, permitting detailed investigation of the LCRs and their involvement in the 22q11.2 deletions. Sequence analysis has identified four LCRs within the 3 Mb TDR. Although the LCRs differ in content and organization of shared modules, those modules that are common between them share 97-98% sequence identity with one another. By fluorescence in situ hybridization (FISH) analysis, the end-points of four variant 22q11.2 deletions appear to localize to the LCRs. Pulsed-field gel electrophoresis and Southern hybridization have been used to identify rearranged junction fragments from three variant deletions. Analysis of junction fragments by PCR and sequencing of the PCR products implicate the LCRs directly in the formation of 22q11.2 deletions. The evolutionary origin of the duplications on chromosome 22 has been assessed by FISH analysis of non-human primates. Multiple signals in Old World monkeys suggest that the duplication events may have occurred at least 20-25 million years ago.

Comparative sequence analysis of 634 kb of the mouse chromosome 16 region of conserved synteny with the human velocardiofacial syndrome region on chromosome 22q11.2.

Mouse genomic DNA sequence extending 634 kb on proximal mouse chromosome 16 was compared to the corresponding human sequence from chromosome 22q11.2. Haploinsufficiency for this region results in velocardiofacial syndrome (VCFS) in humans. The mouse region is rearranged into three conserved blocks relative to human, but gene content and position are highly conserved within these blocks. Examination of the boundaries of one of these blocks suggested that the evolutionary chromosomal rearrangement occurred in the mouse lineage, resulting in inactivation of the mouse orthologue of ZNF74. Sequence analysis identified 21 genes and 15 ESTs. These include 2 novel genes, Srec2 and Cals2, and previously undescribed splice variants of several other genes. Exon discovery was carried out using GRAIL2, MZEF, or comparative analysis across 491 kb of conserved mouse and human sequence. Sequence comparison was highly effective, identifying every gene and nearly every exon without the high frequency of false-positive predictions seen when algorithmic methods were used alone. In combination, these procedures identified every gene with no false-positive predictions. Comparative sequence analysis also revealed regions of extensive conservation among noncoding sequences, accounting for 6% of the sequence. A library of such sequences has been established to form a resource for generalized studies of regulatory and structural elements.

Clustered 11q23 and 22q11 breakpoints and 3:1 meiotic malsegregation in multiple unrelated t(11;22) families.

The t(11;22) is the only known recurrent, non-Robertsonian constitutional translocation. We have analyzed t(11;22) balanced-translocation carriers from multiple unrelated families by FISH, to localize the t(11;22) breakpoints on both chromosome 11 and chromosome 22. In 23 unrelated balanced-translocation carriers, the breakpoint was localized within a 400-kb interval between D22S788 (N41) and ZNF74, on 22q11. Also, 13 of these 23 carriers were tested with probes from chromosome 11, and, in each, the breakpoint was localized between D11S1340 and APOA1, on 11q23, to a region

Comparative mapping of the DiGeorge region in the dog and exclusion of linkage to inherited canine conotruncal heart defects.

Conotruncal defects (CTDs) of the heart are a frequent component of DiGeorge, velocardiofacial, or other syndromes caused by deletions of the human chromosome 22q11 region (HSA22q11). In addition, some human patients with isolated nonsyndromic CTDs have been reported to have deletions of this region. Taken together, these findings lead to the conclusion that deletions of an HSA22q11 locus or loci produce abnormalities in cardiac development leading to CTDs. A spontaneous model of isolated inherited conotruncal malformations occurs in the keeshond dog. We have previously shown in experimental matings that nonsyndromic CTDs in the keeshond are inherited in a manner consistent with a major underlying locus. In the studies described in this article we tested two hypotheses: (1) the region of HSA22q11 commonly deleted in DiGeorge and related syndromes is evolutionarily conserved in the dog, and (2) a locus in this region is linked to hereditary CTD in the keeshond. Two loci within the minimal DiGeorge critical region (MDGCR) and two loci that lie telomeric to the MDGCR, one of which is commonly deleted in DiGeorge patients, were mapped in the dog using a combination of linkage analysis and fluorescence in situ hybridization (FISH). The results confirm conserved synteny of the loci DGS-I, CTP, D22S788 (N41), and IGLC on the telomeric end of canine chromosome 26 (CFA26). The group of four syntenic gene loci, which spans a genetic distance of 2.5 cM is the first to be mapped to this small acrocentric canine chromosome and adds gene-associated polymorphic markers to the developing dog linkage map. Linkage of loci in this region to hereditary CTD in the keeshond was excluded.

Patient with a 22q11.2 deletion with no overlap of the minimal DiGeorge syndrome critical region (MDGCR).

The apparent lack of genotype/phenotype correlation in patients with the DiGeorge anomaly and velocardiofacial syndrome (DGA/VCFS; the "22q11 deletion syndrome") indicates a complex genetic condition. Most cases, whatever the phenotype, have a 1.5-3 Mb chromosomal deletion that includes the minimal DiGeorge critical region (MDGCR). Another potential critical region on 22q11 has been suggested based on two patients with distal deletions outside the MDGCR. We report on a patient with a VCFS phenotype who has a deletion, mapped by short tandem repeat polymorphic loci and fluorescence in situ hybridization analysis, distal to and not overlapping the MDGCR. This patient is deleted for several genes, including the T-box 1 gene (TBX1; a transcription regulator expressed early in embryogenesis) and catechol-O-methyltransferase (COMT; involved in neurotransmitter metabolism). We discuss the role these two genes may play in the clinical phenotype of the patient.

Sequence-ready physical map of the mouse chromosome 16 region with conserved synteny to the human velocardiofacial syndrome region on 22q11.2.

Proximal mouse Chromosome (Chr) 16 shows conserved synteny with human Chrs 16, 8, 22, and 3. The mouse Chr 16/human Chr 22 conserved synteny region includes the DiGeorge/Velocardiofacial syndrome region of human Chr 22q11.2. A physical map of the entire mouse Chr 16/human Chr 22 region of conserved synteny has been constructed to provide a substrate for gene discovery, genomic sequencing, and animal model development. A YAC contig was constructed that extends ca. 5.4 Mb from a region of conserved synteny with human Chr 8 at Prkdc through the region conserved with human Chr 3 at DVL3. Sixty-one markers including 37 genes are mapped with average marker spacing of 90 kb. Physical distance was determined across the 2.6-Mb region from D16Mit74 to Hira with YAC fragmentation. The central region from D16Jhu28 to Igl-C1 was converted into BAC and PAC clones, further refining the physical map and providing sequence-ready template. The gene content and borders of three blocks of conserved linkage between human Chr 22q11.2 mouse Chr 16 are refined.

Cat eye syndrome chromosome breakpoint clustering: identification of two intervals also associated with 22q11 deletion syndrome breakpoints.

The supernumerary cat eye syndrome (CES) chromosome is dicentric, containing two copies of 22pter-->q11.2. We have found that the duplication breakpoints are clustered in two intervals. The more proximal, most common interval is the 450-650 kb region between D22S427 and D22S36, which corresponds to the proximal deletion breakpoint interval found in the 22q11 deletion syndrome (DiGeorge/velocardiofacial syndrome). The more distal duplication breakpoint interval falls between CRKL and D22S112, which overlaps with the common distal deletion interval of the 22q11 deletion syndrome. We have therefore classified CES chromosomes into two types based on the location of the two breakpoints required to generate them. The smaller type I CES chromosomes are symmetrical, with both breakpoints located within the proximal interval. The larger type II CES chromosomes are either asymmetrical, with one breakpoint located in each of the two intervals, or symmetrical, with both breakpoints located in the distal interval. The co-localization of the breakpoints of these different syndromes, plus the presence of low-copy repeats adjacent to each interval, suggests the existence of several specific regions of chromosomal instability in 22q11.2 which are involved in the production of both deletions and duplications. Since the phenotype associated with the larger duplication does not appear to be more severe than that of the smaller duplication, determination of the type of CES chromosome does not currently have prognostic value.

Goosecoid-like, a gene deleted in DiGeorge and velocardiofacial syndromes, recognizes DNA with a bicoid-like specificity and is expressed in the developing mouse brain.

The vast majority of patients with DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) have deletions of chromosomal region 22q11.2. These patients exhibit broad and variable phenotypes that include conotruncal cardiac defects, hypocalcemia, palatal and facial anomalies and developmental delay. Most of these abnormalities are thought to be due to defects in neural crest cell migration or differentiation. We have identified a homeobox-containing gene, Goosecoid-like (GSCL), that is in the region within 22q11 that is deleted most consistently in patients with DGS/VCFS. The GSCL gene is expressed in a limited number of adult tissues as well as in early human development, and is a member of a family of homeobox genes in vertebrates that includes Goosecoid and GSX. In this report, we present functional studies of the GSCL protein and determine the expression pattern of the GSCL gene in mouse embryos. We demonstrate that GSCL exhibits DNA sequence-specific recognition of sites bound by the Drosophila anterior morphogen, Bicoid. Several of these sites (TAATCCC) were found in the 5' upstream region of the GSCL gene itself, and we present evidence suggesting that GSCL might regulate its own transcription. In situ hybridization revealed that the mouse ortholog of GSCL, Gscl, is expressed in the brain starting as early as embryonic day 9.5, and expression continues in adults. This expression pattern is consistent with GSCL having either an indirect role in the development of neural crest-derived structures or a direct role in a subset of the phenotype observed in DGS/VCFS, such as learning disorders or psychiatric disease.

Conserved gene structure and genomic linkage for D-dopachrome tautomerase (DDT) and MIF.

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (DDT) are small proteins, which are related both by sequence and by in vitro enzyme activity. Here we show that the gene for DDT in human and mouse is identical in exon structure to MIF. Both genes have two introns that are located at equivalent positions, relative to a twofold repeat in protein structure. Although in similar positions, the introns are in different phases relative to the open reading frame. Other members of this superfamily exist in nematodes and a plant, and a related gene in C. elegans shares an intron position with MIF and DDT. In addition to similarities in structure, the genes for DDT and MIF are closely linked on human Chromosome (Chr) 22 and mouse Chr 10.

t(11;22)(q23;q11.2) In acute myeloid leukemia of infant twins fuses MLL with hCDCrel, a cell division cycle gene in the genomic region of deletion in DiGeorge and velocardiofacial syndromes.

We examined the MLL genomic translocation breakpoint in acute myeloid leukemia of infant twins. Southern blot analysis in both cases showed two identical MLL gene rearrangements indicating chromosomal translocation. The rearrangements were detectable in the second twin before signs of clinical disease and the intensity relative to the normal fragment indicated that the translocation was not constitutional. Fluorescence in situ hybridization with an MLL-specific probe and karyotype analyses suggested t(11;22)(q23;q11. 2) disrupting MLL. Known 5' sequence from MLL but unknown 3' sequence from chromosome band 22q11.2 formed the breakpoint junction on the der(11) chromosome. We used panhandle variant PCR to clone the translocation breakpoint. By ligating a single-stranded oligonucleotide that was homologous to known 5' MLL genomic sequence to the 5' ends of BamHI-digested DNA through a bridging oligonucleotide, we formed the stem-loop template for panhandle variant PCR which yielded products of 3.9 kb. The MLL genomic breakpoint was in intron 7. The sequence of the partner DNA from band 22q11.2 was identical to the hCDCrel (human cell division cycle related) gene that maps to the region commonly deleted in DiGeorge and velocardiofacial syndromes. Both MLL and hCDCrel contained homologous CT, TTTGTG, and GAA sequences within a few base pairs of their respective breakpoints, which may have been important in uniting these two genes by translocation. Reverse transcriptase-PCR amplified an in-frame fusion of MLL exon 7 to hCDCrel exon 3, indicating that an MLL-hCDCrel chimeric mRNA had been transcribed. Panhandle variant PCR is a powerful strategy for cloning translocation breakpoints where the partner gene is undetermined. This application of the method identified a region of chromosome band 22q11.2 involved in both leukemia and a constitutional disorder.

Molecular cloning and characterization of the bovine and human tuftelin genes.

The bovine tuftelin gene was cloned and its structure determined by DNA sequence analysis and comparison to bovine tuftelin cDNA. The analyses demonstrated that the cDNA contains a 1014 bp open reading frame encoding a protein of 338 residues with a calculated molecular weight of 38,630 kDa and an isoelectric point of 5.85. Although similar, these results differ from those previously published [Deutsch et al. (1991) J. Biol. Chem. 266, 16021-16028] which contained a different conceptual amino acid sequence for the carboxy terminal region and identification of a different termination codon because of the absence of a single guanine residue in the published sequence. The protein does not appear to share homology or domain motifs with any other known protein. The bovine gene consists of 13 exons ranging in size from 66 to 1531 bp, the latter containing the encoded carboxy terminal and 3' untranslated regions. These exons are embedded in greater than 28 kbp of genomic DNA and codons are generally not divided at exon/intron borders. Sequence analysis of the cDNA and products produced by reverse transcriptase/polymerase chain reaction demonstrated that exons 2, 5 and 6 are alternatively spliced. The 3' portion of the human gene was also isolated and characterized by DNA sequencing, which demonstrated agreement between the bovine and human sequences in the segment in question. The difference between the presently reported sequence and that of the previously published one suggests the possibility of an unusual type of polymorphism which would result in markedly different amino acid sequences at the carboxy terminal region of the protein. The human tuftelin gene was localized to chromosome 1q21 by in situ hybridization.