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.

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

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.

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 characterization of a serine/threonine kinase in the DiGeorge minimal critical region.

The majority of patients with DiGeorge, velocardiofacial or conotruncal anomaly facial syndromes share a common genetic etiology, deletion of chromosomal region 22q11.2. This report describes a computational approach toward the identification and molecular characterization of a newly identified serine/threonine kinase from the minimal critical deleted region (MDGCR). A cosmid contig of the minimal critical region has been assembled and sequenced in its entirety. Database searches and computer analysis of one cosmid (111f11) for coding sequences identified two regions with high similarity to the mouse serine/threonine kinase, Tsk1. Our investigations demonstrate that one of these regions contains a testis-specific gene that undergoes differential splicing, while the other region is most likely a pseudogene. Northern blot analysis and cDNA cloning demonstrate that there is alternate processing of the 3'UTR without altering the conserved kinase domains within the open reading frame. Serine/threonine kinases can play a regulatory role and have been found to be expressed during early embryogenesis. Based on its position in the MDGCR and possible function, the gene reported here is a candidate for the features seen in the 22q11 deletion syndrome.

Isolation and characterization of a gene from the DiGeorge chromosomal region homologous to the mouse Tbx1 gene.

DiGeorge syndrome, velocardiofacial syndrome, conotruncal anomaly face syndrome, and isolated and familial forms of conotruncal cardiac defects have been associated with deletions of chromosomal region 22q11.2. This report describes the identification, cloning, and characterization of the human TBX1 gene, which maps to the center of the DiGeorge chromosomal region. Further, we have extended the mouse cDNA sequence to permit comparisons between human and mouse Tbx1. TBX1 is a member of a phylogenetically conserved family of genes that share a common DNA-binding domain, the T-box. T-box genes are transcription factors involved in the regulation of developmental processes. There is 98% amino acid identity between human and mouse TBX1 proteins overall, and within the T-box domain, the proteins are identical except for two amino acids. Expression of human TBX1 in adult and fetal tissues, as determined by Northern blot analysis, is similar to that found in the mouse. Additionally, using 3 'RACE, we obtained a differentially spliced message in adult skeletal muscle. Mouse Tbx1 has been previously shown to be expressed during early embryogenesis in the pharyngeal arches, pouches, and otic vesicle. Later in development, expression is seen in the vertebral column and tooth bud. Thus, human TBX1 is a candidate for some of the features seen in the 22q11 deletion syndrome.

Interchromosomal duplications of the adrenoleukodystrophy locus: a phenomenon of pericentromeric plasticity.

A 9.7 kb segment encompassing exons 7-10 of the adrenoleukodystrophy (ALD) locus of the X chromosome has duplicated to specific locations near the pericentromeric regions of human chromosomes 2p11,10p11, 16p11 and 22q11. Comparative sequence analysis reveals 92-96% nucleotide identity, indicating that the autosomal ALD paralogs arose relatively recently during the course of higher primate evolution (5-10 million years ago). Analysis of sequences flanking the duplication region identifies the presence of an unusual GCTTTTTGC repeat which may be a sequence-specific integration site for the process of pericentromeric-directed transposition. The breakpoint sequence and phylogenetic analysis predict a two-step transposition model, in which a duplication from Xq28 to pericentromeric 2p11 occurred once, followed by a rapid distribution of a larger duplicon cassette among the pericentromeric regions. In addition to facilitating more effective mutation detection among ALD patients, these findings provide further insight into the molecular basis underlying a pericentromeric-directed mechanism for non-homologous interchromosomal exchange.

The DiGeorge syndrome minimal critical region contains a goosecoid-like (GSCL) homeobox gene that is expressed early in human development.

The majority of patients with DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) have deletions of chromosomal region 22q11.2. The abnormalities observed in these patients include conotruncal cardiac defects, thymic hypoplasia or aplasia, hypocalcemia, and characteristic facial features. To understand the genetic basis of these disorders, we have characterized genes within the region that is most consistently deleted in patients with DGS/VCFS, the minimal DiGeorge critical region (MDGCR). In this report, we present the identification and characterization of a novel gene, GSCL, in the MDGCR, with homology to the homeodomain family of transcription factors. Further, we provide evidence that this gene is expressed in a limited number of adult tissues as well as in early human development. The identification of GSCL required a genomic sequence-based approach because of its restricted expression and high GC content. The early expression, together with the known role of homeobox-containing proteins in development, make GSCL an outstanding candidate for some of the abnormalities seen in DGS/VCFS.

Disruption of the clathrin heavy chain-like gene (CLTCL) associated with features of DGS/VCFS: a balanced (21;22)(p12;q11) translocation.

The smallest region of deletion overlap in the patients we have studied defines a DIGeorge syndrome/velocardiofacial syndrome (DGS/VCFS) minimal critical region (MDGCR) of approximately 250 kb within 22q11. A de novo constitutional balanced translocation has been identified within the MDGCR. The patient has some features which have been reported in individuals with DGS/VCFS, including: facial dysmorphia, mental retardation, long slender digits and genital anomalies. We have cloned the breakpoint of his translocation and shown that it interrupts the clathrin heavy chain-like gene (CLTCL) within the MDGCR. The breakpoint of the translocation partner is in a repeated region telomeric to the rDNA cluster on chromosome 21p. Therefore, it is unlikely that the patient's findings are caused by interruption of sequences on 21p. The chromosome 22 breakpoint disrupts the 3' coding region of the CLTCL gene and leads to a truncated transcript, strongly suggesting a role for this gene in the features found in this patient. Further, the patient's partial DGS/VCFS phenotype suggests that additional features of DGS/VCFS may be attributed to other genes in the MDGCR. Thus, haploinsufficiency for more than one gene in the MDGCR may be etiologic for DGS/VCFS.

Structural and mutational analysis of a conserved gene (DGSI) from the minimal DiGeorge syndrome critical region.

The majority of patients with DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), conotruncal anomaly face syndrome (CTAFS) and some individuals with familial or sporadic conotruncal cardiac defects have hemizygous deletions of chromosome 22. Most patients with these disorders share a common large deletion, spanning > 1.5 Mb within 22q11.21-q11.23. Recently, the smallest region of deletion overlap has been narrowed to a 250 kb area, the minimal DGS critical region (MDGCR), which includes the locus D22S75 (N25). We have isolated and characterized a novel, highly conserved gene, DGSI, within the MDGCR. DGSI has 10 exons and nine introns encompassing 1702 bp of cDNA sequence and 11 kb of genomic DNA. The encoded protein has 476 amino acids with a predicted mol. wt of 52.6 kDa. The intron-exon boundaries have been analyzed and conform to the consensus GT/AG motif. The corresponding murine Dgsi has been isolated and localized to proximal mouse chromosome 16. The mouse gene contains the same number of exons and introns, and the predicted protein has 479 amino acids with 93.2% identity to that of the human DGSI gene. By database searching, both genes have significant homology to a Caenorhabditis elegans hypothetical protein, F42H10.7. Further, mutation analysis has been performed in 16 patients, who have no detectable 22q11.2 deletion and some of the characteristic clinical features of DGS/VCFS. We have detected eight sequence variants in DGSI. These occurred in the 5'-untranslated region, the coding region and the intronic regions adjacent to the intron-exon boundaries of the gene. Seven of the eight variants were also present in normal controls or unaffected family members, suggesting they may not be of etiologic significance.