Characterization of the leukemogenic potential of distal cytoplasmic CSF3R truncation and missense mutations.

An increasing number of variants of unknown significance are being identified in leukemia patients with the application of deep sequencing and these include CSF3R cytoplasmic mutations. Previous studies have demonstrated oncogenic potential of certain CSF3R truncation mutations prior to internalization motifs. However, the oncogenic potential of truncating the more distal region of CSF3R cytoplasmic domain as well as cytoplasmic missense mutations remains uncharacterized. Here we identified that CSF3R distal cytoplasmic truncation mutations (Q793-Q823) also harbored leukemogenic potential. Mechanistically, these distal cytoplasmic truncation mutations demonstrated markedly decreased receptor degradation, probably owing to loss of the de-phosphorylation domain (residues N818-F836). Furthermore, all truncations prior to Q823 demonstrated increased expression of the higher molecular weight CSF3R band, which is shown to be essential for the receptor surface expression and the oncogenic potential. We further demonstrated that sufficient STAT5 activation is essential for oncogenic potential. In addition, CSF3R K704A demonstrated transforming capacity due to interruption of receptor ubiquitination and degradation. In summary, we have expanded the region of the CSF3R cytoplasmic domain in which truncation or missense mutations exhibit leukemogenic capacity, which will be useful for evaluating the relevance of CSF3R mutations in patients and helpful in defining targeted therapy strategies.

Effects of selection for ethanol preference on gene expression in the nucleus accumbens of HS-CC mice.

Previous studies on changes in murine brain gene expression associated with the selection for ethanol preference have used F2 intercross or heterogeneous stock (HS) founders, derived from standard laboratory strains. However, these populations represent only a small proportion of the genetic variance available in Mus musculus. To investigate a wider range of genetic diversity, we selected mice for ethanol preference using an HS derived from the eight strains of the collaborative cross. These HS mice were selectively bred (four generations) for high and low ethanol preference. The nucleus accumbens shell of naive S4 mice was interrogated using RNA sequencing (RNA-Seq). Gene networks were constructed using the weighted gene coexpression network analysis assessing both coexpression and cosplicing. Selection targeted one of the network coexpression modules (greenyellow) that was significantly enriched in genes associated with receptor signaling activity including Chrna7, Grin2a, Htr2a and Oprd1. Connectivity in the module as measured by changes in the hub nodes was significantly reduced in the low preference line. Of particular interest was the observation that selection had marked effects on a large number of cell adhesion molecules, including cadherins and protocadherins. In addition, the coexpression data showed that selection had marked effects on long non-coding RNA hub nodes. Analysis of the cosplicing network data showed a significant effect of selection on a large cluster of Ras GTPase-binding genes including Cdkl5, Cyfip1, Ndrg1, Sod1 and Stxbp5. These data in part support the earlier observation that preference is linked to Ras/Mapk pathways.

Genes, behavior and next-generation RNA sequencing.

Advances in next-generation sequencing suggest that RNA-Seq is poised to supplant microarray-based approaches for transcriptome analysis. This article briefly reviews the use of microarrays in the brain-behavior context and then illustrates why RNA-Seq is a superior strategy. Compared with microarrays, RNA-Seq has a greater dynamic range, detects both coding and noncoding RNAs, is superior for gene network construction, detects alternative spliced transcripts, detects allele specific expression and can be used to extract genotype information, e.g. nonsynonymous coding single nucleotide polymorphisms. Examples of where RNA-Seq has been used to assess brain gene expression are provided. Despite the advantages of RNA-Seq, some disadvantages remain. These include the high cost of RNA-Seq and the computational complexities associated with data analysis. RNA-Seq embraces the complexity of the transcriptome and provides a mechanism to understand the underlying regulatory code; the potential to inform the brain-behavior relationship is substantial.

Immune response gene profiles in the term placenta depend upon maternal muscle mass.

Maternal thinness leads to metabolic challenges in the offspring, but it is unclear whether reduced maternal fat mass or muscle mass drives these metabolic changes. Recently, it has been shown that low maternal muscle mass--as measured by arm muscle area (AMA)--is associated with depressed nutrient transport to the fetus. To determine the role of maternal muscle mass on placental function, we analyzed the gene expression profiles of 30 human placentas over the range of AMA (25.2-90.8 cm(2)) from uncomplicated term pregnancies from the Southampton Women's Survey cohort. Eighteen percent of the ∼60 genes that were highly expressed in less muscular women were related to immune system processes and the interferon-γ (IFNG) signaling pathway in particular. Those transcripts related to the IFNG pathway included IRF1, IFI27, IFI30, and GBP6. Placentas from women with low muscularity are, perhaps, more sensitive to the effects of inflammatory cytokines than those from more muscular women.

Gene networks and haloperidol-induced catalepsy.

The current study examined the changes in striatal gene network structure induced by short-term selective breeding from a heterogeneous stock for haloperidol response. Brain (striatum) gene expression data were obtained using the Illumina WG 8.2 array, and the datasets from responding and non-responding selected lines were independently interrogated using a weighted gene coexpression network analysis (WGCNA). We detected several gene modules (groups of coexpressed genes) in each dataset; the membership of the modules was found to be largely concordant, and a consensus network was constructed. Further validation of the network topology showed that using approximately 35 samples is sufficient to reliably infer the transcriptome network. An in-depth analysis showed significant changes in network structure and gene connectivity associated with the selected lines; these changes were validated using a bootstrapping procedure. The most dramatic changes were associated with a gene module richly annotated with neurobehavioral traits. The changes in network connectivity were concentrated in the links between this module and the rest of the network, in addition to changes within the module; this observation is consistent with recent results in protein and metabolic networks. These results suggest that a network-based strategy will help identify the genetic factors associated with haloperidol response.

Detection of reciprocal quantitative trait loci for acute ethanol withdrawal and ethanol consumption in heterogeneous stock mice.

RATIONALE: Previous studies have suggested that there is an inverse genetic relationship between ethanol consumption (two-bottle choice, continuous access) and ethanol withdrawal (e.g., Metten et al., Behav Brain Res 95:113-122, 1998a). OBJECTIVES: The current study used short-term selective breeding from heterogeneous stock (HS) animals to examine this relationship. The primary goal of the current study was to determine if reciprocal quantitative trait loci (QTLs) could be found in the selectively bred lines. The advantage of detecting QTLs in HS animals is that it is possible to extract a haplotype signature for the QTL, which in turn can be used to narrow the number of candidate genes generated from gene expression and sequence databases (see, e.g., Hitzemann et al., Mamm Genome 14:733-747, 2003). RESULTS: Seven reciprocal QTLs were detected on chromosomes (Chr) 1 (two), 3, 6, 11, 16, and 17 that exceeded the nominal LOD threshold of 10; genetic drift, which occurs during selection, dramatically increases the LOD threshold. The proximal Chr 1 QTL was examined in some detail. The haplotype structure of the QTL was such that the LP/J allele was associated with low withdrawal and high consumption. The QTL appears to be located in a gene-poor region between 170 and 173 Mbp. Based on available sequence data, two plausible candidate genes emerge-Nos1ap and Atf6alpha. CONCLUSIONS: The data presented here confirm some aspects of the negative genetic relationship between acute ethanol withdrawal and ethanol consumption. The QTL data point to the potential involvement of NO signaling and/or the unfolded protein response.

Characterization of the quantitative trait locus for haloperidol-induced catalepsy on distal mouse chromosome 1.

We report here the confirmation of the quantitative trait locus for haloperidol-induced catalepsy on distal chromosome (Chr) 1. We determined that this quantitative trait locus was captured in the B6.D2-Mtv7a/Ty congenic mouse strain, whose introgressed genomic interval extends from approximately 169.1 to 191.3 Mb. We then constructed a group of overlapping interval-specific congenic strains to further break up the interval and remapped the locus between 177.5 and 183.4 Mb. We next queried single nucleotide polymorphism (SNP) data sets and identified three genes with nonsynonymous coding SNPs in the quantitative trait locus. We also queried two brain gene expression data sets and found five known genes in this 5.9-Mb interval that are differentially expressed in both whole brain and striatum. Three of the candidate quantitative trait genes were differentially expressed using quantitative real-time polymerase chain reaction analyses. Overall, the current study illustrates how multiple approaches, including congenic fine mapping, SNP analysis and microarray gene expression screens, can be integrated both to reduce the quantitative trait locus interval significantly and to detect promising candidate quantitative trait genes.

Evidence for linkage and association to alcohol dependence on chromosome 19.

An association study on markers showing suggestive evidence for linkage to severe alcoholism was performed on the Collaborative Study on the Genetics of Alcoholism (COGA) data set. Our linkage study was restricted to the autosomal markers on chromosomes 2, 3, 4, 13, and 19, with low homozygosity (below 30%) and high identity-by-state sharing in affected sib pairs (ASPs). We used a strict phenotype definition, only individuals diagnosed as affected both on the ALDX1 (COGA criterion) and ALDX2 (ICD-10) scales were used in the analyses. Linkage was assessed by excess identity-by-descent allele sharing in ASPs. The strongest evidence of linkage was detected on chromosome 19, in particular at markers D19S49 (p < 0.0001) and D19S431 (p < 0.002). An association study of allele and haplotype data was then carried out on chromosome 19 markers using affected-family-based controls. A haplotype defined by alleles at markers D19S49, D19S43, and D19S200 in chromosome 19 shows significant association (p < 0.003, odds ratio 2.82). Further, significant differences were observed in the distribution of the harm avoidance subscale among genotypes defined by D19S49 (p < 0.0001). These results provide evidence for the existence of a locus in chromosome 19 potentially involved in alcohol dependence susceptibility.

Evolution of HLA class II molecules: Allelic and amino acid site variability across populations.

Analysis of the highly polymorphic beta1 domains of the HLA class II molecules encoded by the DRB1, DQB1, and DPB1 loci reveals contrasting levels of diversity at the allele and amino acid site levels. Statistics of allele frequency distributions, based on Watterson's homozygosity statistic F, reveal distinct evolutionary patterns for these loci in ethnically diverse samples (26 populations for DQB1 and DRB1 and 14 for DPB1). When examined over all populations, the DQB1 locus allelic variation exhibits striking balanced polymorphism (P < 10(-4)), DRB1 shows some evidence of balancing selection (P < 0.06), and while there is overall very little evidence for selection of DPB1 allele frequencies, there is a trend in the direction of balancing selection (P < 0.08). In contrast, at the amino acid level all three loci show strong evidence of balancing selection at some sites. Averaged over polymorphic amino acid sites, DQB1 and DPB1 show similar deviation from neutrality expectations, and both exhibit more balanced polymorphic amino acid sites than DRB1. Across ethnic groups, polymorphisms at many codons show evidence for balancing selection, yet data consistent with directional selection were observed at other codons. Both antigen-binding pocket- and non-pocket-forming amino acid sites show overall deviation from neutrality for all three loci. Only in the case of DRB1 was there a significant difference between pocket- and non-pocket-forming amino acid sites. Our findings indicate that balancing selection at the MHC occurs at the level of polymorphic amino acid residues, and that in many cases this selection is consistent across populations.

Codon usage bias and base composition in MHC genes in humans and common chimpanzees.

Codon bias and base composition in major histocompatibility complex (MHC) sequences have been studied for both class I and II loci in Homo sapiens and Pan troglodytes. There is low to moderate codon bias for the MHC of humans and chimpanzees. In the class I loci, the same level of moderate codon bias is seen for HLA-B, HLA-C, Patr-A, Patr-B, and Patr-C, while at HLA-A the level of codon bias is lower. There is a correlation between codon usage bias and G+C content in the A and B loci in humans and chimps, but not at the C locus. To examine the effect of diversifying selection on codon bias, we subdivided class I alleles into antigen recognition site (ARS) and non-ARS codons. ARS codons had lower bias than non-ARS codons. This may indicate that the constraint of codon bias on nucleotide substitution may be selected against in ARS codons. At the class II loci, there are distinct differences between alpha and beta chain genes with respect to codon usage, with the beta chain genes being much more biased. Species-specific differences in base composition were seen in exon 2 at the DRB1 locus, with lower GC content in chimpanzees. Considering the complex evolutionary history of MHC genes, the study of codon usage patterns provides us with a better understanding of both the evolutionary history of these genes and the evolution of synonymous codon usage in genes under natural selection.

Hardy-Weinberg testing for HLA class II (DRB1, DQA1, DQB1, and DPB1) loci in 26 human ethnic groups.

Testing the fit of population data to Hardy-Weinberg proportions is crucial in the validation of many current approaches in population genetic studies. In this paper, we tested fit to Hardy-Weinberg proportions using exact approaches for both the overall and individual heterozygote genotype data of four HLA Class II loci: DRB1, DQA1, DQB1, and DPB1, from 26 human populations. Eighty of 99 overall tests fit the Hardy-Weinberg expectation (73% for DRB1, 89% for DQA1, 81% for DQB1 and 81% for DPB1). Deviations from Hardy-Weinberg proportions were both locus and group specific. Although we could not rule out other mechanisms at work, the individual test results indicated that the departure was possibly partly due to recent admixture. Evidence for selection and other sources of deviation are also discussed.

Locus and population specific evolution in HLA class II genes.

The population genetics of the HLA class II loci was studied with reference to variation in the frequency of (a) alleles at a locus and (b) amino acids at specific sites. Variation was surveyed at 4 loci (DRB1, DQA1, DQB1, and DPB1) in 22 populations from the Twelfth International Histocompatibility Workshop (Saint-Malo, 1996). Allele and amino acid variation was measured by computing heterozygosity and the effective number of alleles. Substantial variations in polymorphism were observed among the various populations and loci studied. In the majority of the populations, DRB1 has the highest heterozygosity and effective number of alleles. As previously shown, the Amerindian populations have lower levels of allelic diversity when compared to other populations. At the amino acid level, DRB1 antigen recognition sites (ARS) have the highest heterozygosities and effective number of alleles. For the other loci (DPB1, DQA1, and DQB1) for which there is no crystal structure and for which ARS sites were inferred from DRB1, non-ARS sites were often among the sites with highest levels of variation. It is possible that these putative non-ARS sites do play a role in antigen presentation. The homozygosity test for neutrality was applied to allele and amino acid data. Of the four HLA class II loci studied, only DPB1 failed to show evidence of balancing selection. DQB1 and DQA1 depart significantly from neutrality in the largest number of populations. Genetic distances between populations were computed based on frequency of alleles and amino acids at ARS sites.

HLA class II DR-DQ amino acids and insulin-dependent diabetes mellitus: application of the haplotype method.

Insulin-dependent diabetes mellitus (IDDM) HLA class II DRB1-DQA1-DQB1 data from four populations (Norwegian, Sardinian, Mexican American, and Taiwanese) have been analyzed to detect the amino acids involved in the disease process. The combination of sites DRB1#67 and 86; DQA1#47; and DQB1#9, 26, 57, and 70 predicts the IDDM component in these four populations, when the results and criteria of the haplotype method for amino acids, developed in the companion paper in this issue of the Journal, are used. The following sites, either individually, or in various combinations, previously have been suggested as IDDM components: DRB1#57, 70, 71, and 86; DQA1#52; and DQB1#13, 45, and 57 (DQB1#13 and 45 correlates 100% with DQB1#9 and 26). We propose that DQA1#47 is a better predictor of IDDM than is the previously suggested DQA1#52, and we add DRB1#67 and DQB1#70 to the HLA DR-DQ IDDM amino acids. We do not claim to have identified all HLA DR-DQ amino acids-or highly correlated sites-involved in IDDM. The frequencies and predisposing/protective effects of the haplotypes defined by these seven sites have been compared, and the effects on IDDM are consistent across the populations. The strongest susceptible effects came from haplotypes DRB1 *0301/DQA1 *0501/ DQB1*0201 and DRB1*0401-5-7-8/DQA1*0301/ DQB1*0302. The number of strong protective haplotypes observed was larger than the number of susceptible ones; some of the predisposing haplotypes were present in only one or two populations. Although the sites under consideration do not necessarily have a functional involvement in IDDM, they should be highly associated with such sites and should prove to be useful in risk assessment.