Convergent lines of evidence support CAMKK2 as a schizophrenia susceptibility gene.

Genes that are differentially expressed between schizophrenia patients and healthy controls may have key roles in the pathogenesis of schizophrenia. We analyzed two large-scale genome-wide expression studies, which examined changes in gene expression in schizophrenia patients and their matched controls. We found calcium/calmodulin (CAM)-dependent protein kinase kinase 2 (CAMKK2) is significantly downregulated in individuals with schizophrenia in both studies. To seek the potential genetic variants that may regulate the expression of CAMKK2, we investigated the association between single-nucleotide polymorphisms (SNPs) within CAMKK2 and the expression level of CAMKK2. We found one SNP, rs1063843, which is located in intron 17 of CAMKK2, is strongly associated with the expression level of CAMKK2 in human brains (P=1.1 × 10(-6)) and lymphoblastoid cell lines (the lowest P=8.4 × 10(-6)). We further investigated the association between rs1063843 and schizophrenia in multiple independent populations (a total of 130 623 subjects) and found rs1063843 is significantly associated with schizophrenia (P=5.17 × 10(-5)). Interestingly, we found the T allele of rs1063843, which is associated with lower expression level of CAMKK2, has a higher frequency in individuals with schizophrenia in all of the tested samples, suggesting rs1063843 may be a causal variant. We also found that rs1063843 is associated with cognitive function and personality in humans. In addition, protein-protein interaction (PPI) analysis revealed that CAMKK2 participates in a highly interconnected PPI network formed by top schizophrenia genes, which further supports the potential role of CAMKK2 in the pathogenesis of schizophrenia. Taken together, these converging lines of evidence strongly suggest that CAMKK2 may have pivotal roles in schizophrenia susceptibility.

Sequence variants of the DRD4 gene in autism: further evidence that rare DRD4 7R haplotypes are ADHD specific.

A high prevalence of rare dopamine receptor D4 (DRD4) alleles in children diagnosed with attention-deficit hyperactivity disorder (ADHD) has been reported [Grady et al., 2003]. In this prior study, extensive resequencing/haplotype data of the DRD4 locus was used to suggest that population stratification was not the explanation for the high prevalence of rare alleles. In the current study, DNA resequencing/haplotyping was conducted on 136 DRD4 alleles obtained from autism probands, collected from the same geographic population as the prior ADHD probands (Orange County, CA). A number of studies have suggested that the susceptibility genes underlying these two disorders might partially overlap. Rare DRD4 variants were not uncovered in this autism sample beyond that expected by chance. These results suggest strongly that the high prevalence of rare DRD4 alleles in ADHD probands is due to ascertainment of the sample by diagnosis of ADHD.

The genetic architecture of selection at the human dopamine receptor D4 (DRD4) gene locus.

Associations of the seven-repeat (7R) allele of the human dopamine receptor D4 (DRD4) gene with both the personality trait of novelty seeking and attention deficit/hyperactivity disorder have been reported. Recently, on the basis of the unusual DNA sequence organization of the DRD4 7R 48-bp tandem repeat (VNTR), we proposed that the 7R allele originated as a rare mutational event that increased to high frequency by positive selection. We now have resequenced the entire DRD4 locus from 103 individuals homozygous for 2R, 4R, or 7R variants of the VNTR, a method developed to directly estimate haplotype diversity. DNA from individuals of African, European, Asian, North and South American, and Pacific Island ancestry were used. 4R/4R homozygotes exhibit little linkage disequilibrium (LD) over the region examined, with more polymorphisms observed in DNA samples from African individuals. In contrast, the evidence for strong LD surrounding the 7R allele is dramatic, with all 7R/7R individuals (including those from Africa) exhibiting the same alleles at most polymorphic sites. By intra-allelic comparison at 18 high-heterozygosity sites spanning the locus, we estimate that the 7R allele arose prior to the upper Paleolithic era (approximately 40000-50000 years ago). Further, the pattern of recombination at these polymorphic sites is the pattern expected for selection acting at the 7R VNTR itself, rather than at an adjacent site. We propose a model for selection at the DRD4 locus consistent with these observed LD patterns and with the known biochemical and physiological differences between receptor variants.

High prevalence of rare dopamine receptor D4 alleles in children diagnosed with attention-deficit hyperactivity disorder.

Associations have been reported of the 7-repeat (7R) allele of the human dopamine receptor D4 (DRD4) gene with both the personality trait of novelty seeking and attention-deficit/hyperactivity disorder (ADHD). The increased prevalence of the 7R allele in ADHD probands is consistent with the common variant-common disorder hypothesis, which proposes that the high frequency of many complex genetic disorders is related to common DNA variants. Recently, based on the unusual DNA sequence organization and strong linkage disequilibrium surrounding the DRD4 7R allele, we proposed that this allele originated as a rare mutational event, which nevertheless increased to high prevalence in human populations by positive selection. We have now determined, by DNA resequencing of 250 DRD4 alleles obtained from 132 ADHD probands, that most ADHD 7R alleles are of the conserved haplotype found in our previous 600 allele worldwide DNA sample. Interestingly, however, half of the 24 haplotypes uncovered in ADHD probands were novel (not one of the 56 haplotypes found in our prior population studies). Over 10 percent of the ADHD probands had these novel haplotypes, most of which were 7R allele derived. The probability that this high incidence of novel alleles occurred by chance in our ADHD sample is much less than 0.0001. These results suggest that allelic heterogeneity at the DRD4 locus may also contribute to the observed association with ADHD.

A sequence-ready map of the human chromosome 1q telomere.

A 260-kb half-YAC clone derived from human chromosome 1q was mapped at high resolution using cosmid subclone fingerprint analysis and was integrated with overlapping clones from the telomeric end of a separately derived 1q44 BAC contig to create a sequence-ready map extending to the molecular telomere of 1q. Analysis of 100 kb of sample sequences from across the 260-kb region encompassed by the half-YAC revealed the presence of EST sequence matches corresponding to 12 separate Unigene clusters and to 12 separate unclustered EST sequences. Low-copy subtelomeric repeats typical of many human telomere regions are present within the distal-most 30 kb of 1q. The previously isolated and radiation hybrid-mapped markers Bda84F03, 1QTEL019, and WI11861 localized at distances approximately 32, 88, and 99 kb, respectively, from the 1q terminus. This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 1q and will enable telomeric closure of the human chromosome 1q DNA reference sequence by connecting the molecular 1q telomere to an internal BAC contig.

Integration of telomere sequences with the draft human genome sequence.

Telomeres are the ends of linear eukaryotic chromosomes. To ensure that no large stretches of uncharacterized DNA remain between the ends of the human working draft sequence and the ends of each chromosome, we would need to connect the sequences of the telomeres to the working draft sequence. But telomeres have an unusual DNA sequence composition and organization that makes them particularly difficult to isolate and analyse. Here we use specialized linear yeast artificial chromosome clones, each carrying a large telomere-terminal fragment of human DNA, to integrate most human telomeres with the working draft sequence. Subtelomeric sequence structure appears to vary widely, mainly as a result of large differences in subtelomeric repeat sequence abundance and organization at individual telomeres. Many subtelomeric regions appear to be gene-rich, matching both known and unknown expressed genes. This indicates that human subtelomeric regions are not simply buffers of nonfunctional 'junk DNA' next to the molecular telomere, but are instead functional parts of the expressed genome.

Population structure and history in East Asia.

Archaeological, anatomical, linguistic, and genetic data have suggested that there is an old and significant boundary between the populations of north and south China. We use three human genetic marker systems and one human-carried virus to examine the north/south distinction. We find no support for a major north/south division in these markers; rather, the marker patterns suggest simple isolation by distance.

Attention deficit/hyperactivity disorder children with a 7-repeat allele of the dopamine receptor D4 gene have extreme behavior but normal performance on critical neuropsychological tests of attention.

An association of the dopamine receptor D4 (DRD4) gene located on chromosome 11p15.5 and attention deficit/hyperactivity disorder (ADHD) has been demonstrated and replicated by multiple investigators. A specific allele [the 7-repeat of a 48-bp variable number of tandem repeats (VNTR) in exon 3] has been proposed as an etiological factor in attentional deficits manifested in some children diagnosed with this disorder. In the current study, we evaluated ADHD subgroups defined by the presence or absence of the 7-repeat allele of the DRD4 gene, using neuropsychological tests with reaction time measures designed to probe attentional networks with neuroanatomical foci in D4-rich brain regions. Despite the same severity of symptoms on parent and teacher ratings for the ADHD subgroups, the average reaction times of the 7-present subgroup showed normal speed and variability of response whereas the average reaction times of the 7-absent subgroup showed the expected abnormalities (slow and variable responses). This was opposite the primary prediction of the study. The 7-present subgroup seemed to be free of some of the neuropsychological abnormalities thought to characterize ADHD.

Dopamine genes and ADHD.

Family, twin, and adoption studies have documented a strong genetic basis for ADHD/HKD, but these studies do not identify specific genes linked to the disorder. Molecular genetic studies can identify allelic variations of specific genes that are functionally associated with ADHD/HKD, and dopamine genes have been the initial candidates based on the site of action of the stimulants drugs, which for a half century have provided the primary pharmacological treatment for ADHD/HKD. Two candidate dopamine genes have been investigated and reported to be associated with ADHD/HKD: the dopamine transporter (DAT1) gene [Cook et al., American Journal of Human Genetics 1995;56:993-998, Gill et al., Molecular Psychiatry 1997;2:311-313] and the dopamine receptor D4 (DRD4) gene [LaHoste et al., Molecular Psychiatry 1996;1:121-124: Smalley et al., 1998;3:427-430; Swanson et al., Molecular Psychiatry 1998;3:38-41]. Speculative hypotheses [Swanson and Castellanos, NIH Consensus Development Conference: Diagnosis and Treatment of Attention Deficit Hyperactivity Disorder, November 1998. p. 37-42] have suggested that specific alleles of these dopamine genes may alter dopamine transmission in the neural networks implicated in ADHD/HKD (e.g. that the 10-repeat allele of the DAT1 gene may be associated with hyperactive re-uptake of dopamine or that the 7-repeat allele of the DRD4 gene may be associated with a subsensitive postsynaptic receptor). These and other variants of the dopamine hypothesis of ADHD will be discussed.

An integrated physical map for the short arm of human chromosome 5.

The short arm of human chromosome 5 contains approximately 48 Mb of DNA and comprises 1.5% of the genome. We have constructed a mega-YAC/ STS map of this region that includes 436 YACs anchored by 216 STSs. By combining and integrating our map with the 5p maps of other groups using the same recombinant DNA library, a comprehensive map was constructed that includes 552 YACs and 504 markers. The YAC map covers >94% of 5p in four YAC contigs, bridges the centromere, and includes an additional 5 Mb of 5q DNA. The average marker density is 95 kb. This integrated 5p map will serve as a resource for the continuing localization of genes on the short arm of human chromosome 5 and as a framework for both generating and aligning the DNA sequence of this region.

Association between outcome and telomere DNA content in prostate cancer.

PURPOSE: To perform an initial retrospective investigation of the relationship between outcome in patients with organ confined prostate adenocarcinoma and the tumor cells' content of telomere DNA. MATERIALS AND METHODS: The case-controlled study group was composed of eighteen men diagnosed with prostatic adenocarcinoma prior to 1993. The group was selected so that approximately one half died within ten years of diagnosis and one half survived ten years or longer. Archival, paraffin-embedded tumor tissue was recovered for each patient. DNA was extracted from newly cut sections, fixed to nylon membranes and hybridized with P32-labeled centromere- and telomere-specific probes. Telomere DNA contents were quantitated from the hybridized radioactivities. The relationships between telomere DNA content and survival, and telomere DNA content and disease recurrence in men receiving prostatectomies were determined. RESULTS: Death and disease recurrence were associated with reduced telomere DNA content (p <0.0001, p <0.0001, respectively). CONCLUSIONS: Telomere DNA content may differentiate high-risk patients with metastatic prostate cancer from men with indolent disease who can be spared the unnecessary side effects and expense of treatment by management with "watchful waiting."

Reduced telomere DNA content is correlated with genomic instability and metastasis in invasive human breast carcinoma.

Telomere shortening leads to genomic instability and has been correlated with poor outcome in several types of cancer. A recently described, robust titration assay was used to quantify telomere DNA content in frozen and paraffin-embedded specimens of 49 invasive human breast carcinomas, including tumors with normal or abnormal contents of genomic DNA, which produced regional, distant, or local disease. Telomere DNA contents ranged from 53% to 370% of the content in a reference DNA purified from normal placenta. Tumors were divided into three groups of approximately equal size based on increasing telomere DNA content. All of 16 tumors in the group with the least telomere DNA (Group I), were aneuploid compared to 9/17 tumors in the group with the most telomere DNA (Group III). The Chi-square test for trend indicated that tumors with the least telomere DNA were significantly more likely to be aneuploid than tumors with the most telomere DNA (p < 0.002). Twelve of 14 tumors in Group I also produced metastatic disease compared to 8/15 tumors in Group III. The Fischer Exact Test indicated that tumors with the least telomere DNA were significantly more likely to be metastatic than tumors with the most telomere DNA (p < 0.05). There was no association between telomere DNA content and patients' age, tumors' size, grade, stage, or fraction of cells in S-phase. The correlation of reduced telomere DNA content with aneuploidy and metastasis, both of which are associated with poor outcome in invasive breast carcinoma, implies that telomere DNA content also could have prognostic value.

Inhibition of human telomerase by a retrovirus expressing telomeric antisense RNA.

Human telomerase, the RNA-dependent DNA polymerase that adds TTAGGG repeats to chromosome ends, is selectively expressed in immortalised cells and most tumours, suggesting a potential role for telomerase inhibitors in cancer therapy. Replication-deficient retroviruses were used to determine whether mRNA containing UUAGGG, the complementary sequence to the template region of the hTR telomerase RNA, is sufficient to inhibit telomerase activity. Telomerase activities measured by the telomeric repeat amplification protocol (TRAP) assay in extracts prepared from immortalised mouse fibroblasts, human HeLa cells and human kidney carcinoma cells were inhibited by 75% or greater in 26 of 56 cell clones expressing UUAGGG. Telomerase activity was not inhibited by expression of mRNA containing a transposed sequence, GGGAUU. Telomerase activities in vivo were inferred from changes in cellular morphology, proliferation capacity, growth rate and measurement of the content of telomere DNA. Giant senescent-like cells emerged shortly after cloning mouse PA317 and human HeLa cells expressing UUAGGG. The fraction of giant cells varied from 100% at the fifth population doubling (PD) in one culture to 2-6% at 50 PD in several other cultures. Giant cells were absent in all parental cells and clones expressing GGGAUU. The average cellular content of telomere DNA was independent of telomerase activity over 50 PD. The results indicate that expression of RNA complementary to the template region of hTR is sufficient to inhibit telomerase in vitro and in vivo, but that the effect of inhibition on individual cells is highly variable.

Construction of an approximately 700-kb transcript map around the familial Mediterranean fever locus on human chromosome 16p13.3.

We used a combination of cDNA selection, exon amplification, and computational prediction from genomic sequence to isolate transcribed sequences from genomic DNA surrounding the familial Mediterranean fever (FMF) locus. Eighty-seven kb of genomic DNA around D16S3370, a marker showing a high degree of linkage disequilibrium with FMF, was sequenced to completion, and the sequence annotated. A transcript map reflecting the minimal number of genes encoded within the approximately 700 kb of genomic DNA surrounding the FMF locus was assembled. This map consists of 27 genes with discreet messages detectable on Northerns, in addition to three olfactory-receptor genes, a cluster of 18 tRNA genes, and two putative transcriptional units that have typical intron-exon splice junctions yet do not detect messages on Northerns. Four of the transcripts are identical to genes described previously, seven have been independently identified by the French FMF Consortium, and the others are novel. Six related zinc-finger genes, a cluster of tRNAs, and three olfactory receptors account for the majority of transcribed sequences isolated from a 315-kb FMF central region (between D16S468/D16S3070 and cosmid 377A12). Interspersed among them are several genes that may be important in inflammation. This transcript map not only has permitted the identification of the FMF gene (MEFV), but also has provided us an opportunity to probe the structural and functional features of this region of chromosome 16.

Integrated physical and transcript map of 5q31.3-qter.

We have constructed a physical and transcript map of 5q31.3-qter. The contig comprises 173 yeast artificial chromosomes (YACs) to which 159 sequence tagged sites (STSs), 47 expressed sequence tags (ESTs), and 32 genes were assigned. Previously published partial YAC contigs of the region have been refined and integrated. Given that the region contains 25 Mbp of DNA the average spacing of markers is approximately 100 kb.

Construction of human chromosome 16- and 5-specific circular YAC/BAC libraries by in vivo recombination in yeast (TAR cloning).

Transformation-associated recombination (TAR) in yeast was exploited for the selective isolation of human DNAs as large circular yeast artificial chromosomes (YACs) from two rodent/human hybrid cell lines containing human chromosomes 5 and 16. TAR cloning vectors containing the F-factor origin of replication were constructed for use in these experiments. Presence of the F-factor origin in TAR vectors provides the capability of transferring the YACs generated by in vivo recombination in yeast into Escherichia coli cells and propagating them as bacterial artificial chromosomes (BACs). A high enrichment of human versus rodent YACs was observed during isolation of human DNA from the rodent/human hybrid cell lines. Although <3% of the DNA content in the hybrid cells was human, as many as 75% of the transformants contained human YACs. In contrast to the standard YAC cloning method based on in vitro ligation, no human/mouse chimeras were observed during TAR cloning. The constructed human chromosome 16 YAC library had approximately 2.6x coverage, represented by 4320 YAC clones with an average insert size of 80 kb. YAC clones generated from chromosome 16 were successfully converted into BACs by electroporation of DNA isolated from yeast transformants into E. coli. The BAC clones represent approximately 0.6x chromosomal coverage. Pilot YAC and BAC libraries of chromosome 5 have been also constructed. The chromosomal distribution of YAC/BACs from chromosome 5 and chromosome 16 was evaluated by fluorescence in situ hybridization (FISH). The distribution of FISH signals appeared random along the length of each chromosome. We conclude that TAR cloning provides an efficient means for generating representative chromosome-specific YAC/BAC libraries.

Intercalated cytosine motif and novel adenine clusters in the crystal structure of the Tetrahymena telomere.

The cytosine-rich strand of the Tetrahymena telomere consists of multiple repeats of sequence d(AACCCC). We have solved the crystal structure of the crystalline repeat sequence at 2.5 A resolution. The adenines form two different and previously unknown clusters (A clusters) in orthogonal directions with their counterparts from other strands, each containing a total of eight adenines. The clusters appear to be stable aggregates held together by base stacking and three different base-pairing modes. Two different types of cytosine tetraplexes are found in the crystal. Each four-stranded complex is composed of two intercalated parallel-stranded duplexes pointing in opposite directions, with hemiprotonated cytosine-cytosine (C.C+) base pairs. The outermost C.C+base pairs are from the 5'-end of each strand in one cytosine tetraplex and from the 3'-end of each strand in the other. The A clusters and the cytosine tetraplexes form two alternating stacking patterns, creating continuous base stacking in two perpendicular directions along the x - and z -axes. The adenine clusters could be organizational motifs for macromolecular RNA.

Centromeric protein B null mice are viable with no apparent abnormalities.

The centromere protein B (CENP-B) is a centromeric DNA/binding protein. It recognizes a 17-bp sequence motif called the CENP-B box, which is found in the centromeric region of most chromosomes. It binds DNA through its amino terminus and dimerizes through its carboxy terminus. CENP-B protein has been proposed to perform a vital role in organizing chromatin structures at centromeres. However, other evidence does not agree with this view. For example, CENP-B is found at inactive centromeres on stable dicentric chromosomes, and also mitotically stable chromosomes lacking alpha-satellite DNA have been reported. To address the biological function of CENP-B, we generated mouse null mutants of CENP-B by homologous recombination. Mice lacking CENP-B were viable and fertile, indicating that mice without CENP-B undergo normal somatic and germline development. Thus, both mitosis and meiosis are able to proceed normally in the absence of CENP-B.

Solution structures of the Huntington's disease DNA triplets, (CAG)n.

Highly polymorphic DNA triplet repeats, (CAG)n, are located inside the first exon of the Huntington's disease gene. Inordinate expansion of this repeat is correlated with the onset and progression of the disease. NMR spectroscopy, gel electrophoresis, digestion by single-strand specific P1 enzyme, and in vitro replication assay have been used to investigate the structural basis of (CAG)n expansion. Nondenaturing gel electrophoresis and 1D 1H NMR studies of (CAG)5 and (CAG)6 reveal the presence of hairpins and mismatched duplexes as the major and minor populations respectively. However, at high DNA concentrations (i.e., 1.0-2.0 mM that is typically required for 2D NMR experiments) both (CAG)5 and (CAG)6 exist predominantly in mismatched duplex forms. Mismatched duplex structures of (CAG)5 and (CAG)6 are useful, because they adequately model the stem of the biologically relevant hairpins formed by (CAG)n. We, therefore, performed detailed NMR spectroscopic studies on the duplexes of (CAG)5 and (CAG)6. We also studied a model duplex, (CGCAGCG)2 that contains the underlined building block of the duplex. This duplex shows the following structural characteristics: (i) all the nucleotides are in (C2'-endo, anti) conformations, (ii) mismatched A x A base pairs are flanked by two Watson-Crick G x C base pairs and (iii) A x A base pairs are stably stacked (and intra-helical) and are formed by a single N6-H--N1 hydrogen bond. The nature of A x A pairing is confirmed by temperature-dependent HMQC and HMQC-NOESY experiments on the [(CA*G)5]2 duplex where the adenines are 15N-labeled at N6. Temperature- and pH-dependent imino proton spectra, nondenaturing electrophoresis, and P1 digestion data demonstrate that under a wide range of solution conditions longer (CAG)n repeats (n> or =10) exist exclusively in hairpin conformation with two single-stranded loops. Finally, an in vitro replication assay with (CAG)8,21 inserts in the M13 single-stranded DNA templates shows a replication bypass for the (CAG)21 insert but not for the (CAG)8 insert in the template. This demonstrates that for a sufficiently long insert (n=21 in this case), a hairpin is formed by the (CAG)n even in presence of its complementary strand. This observation implies that the formation of hairpin by the (CAG)n may cause slippage during replication and thus may explain the observed length polymorphism.

Hairpin induced slippage and hyper-methylation of the fragile X DNA triplets.

The fragile X triplet repeats, (GCC)n x (GGC)n are located at the 5' untranslated region of the FMR-1 gene. Inordinate repeat expansion and hyper-methylation of the CpG islands inside the repeat lead to the suppression of the FMR-1 gene and the subsequent onset and progression of the disease. Previously, we have shown that the (GCC)n strand of the fragile X repeat readily forms hairpin structures under physiological conditions (Chen et al., Proc. Natl. Acad. Sci. USA, 92:5199-5203, 1995: Mariappan et al., Nucl. Acid Res. 24:784-792, 1996). Here, we show by an in vitro assay that formation of the (GCC)n hairpins leads to slippage during replication. The slippage structure is a three-way junction with two Watson-Crick, (GCC)n x (GGC)n, arms and a third (GCC)n hairpin arm. Formation of such slippage structures during replication may explain the observed length polymorphism of the fragile X repeat. We have also studied the substrate efficiency of these three-way junctions toward the human methyltransferase. the enzyme that methylates the CpG sites in DNA. These methylation studies show that the slippage structures induced by the (GCC)n hairpins are 10-15 times more efficient substrates than either the corresponding Watson-Crick duplexes or the (GCC)n hairpins. We demonstrate by appropriate designs that the exceptional substrate efficiency of the three-way junction slippage structures is due to two factors: (i) the presence of the (GCC)n hairpin in which CpG sites are more accessible for methylation than the CpG sites in the Watson-Crick duplex and (ii) the ability of the (GCC)n hairpin in these three-way junctions to move along the Watson-Crick arms that facilitates conversion of low-affinity Watson-Crick CpG sites into high-affinity hairpin CpG sites. Therefore, we suggest that the formation of the (GCC)n hairpins during replication can explain both length polymorphism and hyper-methylation of the fragile X repeats.