Centromeric Gene Research Articles

Multigenic phylogeny and analysis of tree incongruences in Triticeae (Poaceae)

BackgroundIntrogressive events (e.g., hybridization, gene flow, horizontal gene transfer) and incomplete lineage sorting of ancestral polymorphisms are a challenge for phylogenetic analyses since different genes may exhibit conflicting genealogical histories. Grasses of the Triticeae tribe provide a particularly striking example of incongruence among gene trees. Previous phylogenies, mostly inferred with one gene, are in conflict for several taxon positions. Therefore, obtaining a resolved picture of relationships among genera and species of this tribe has been a challenging task. Here, we obtain the most comprehensive molecular dataset to date in Triticeae, including one chloroplastic and 26 nuclear genes. We aim to test whether it is possible to infer phylogenetic relationships in the face of (potentially) large-scale introgressive events and/or incomplete lineage sorting; to identify parts of the evolutionary history that have not evolved in a tree-like manner; and to decipher the biological causes of gene-tree conflicts in this tribe.ResultsWe obtain resolved phylogenetic hypotheses using the supermatrix and Bayesian Concordance Factors (BCF) approaches despite numerous incongruences among gene trees. These phylogenies suggest the existence of 4-5 major clades within Triticeae, with Psathyrostachys and Hordeum being the deepest genera. In addition, we construct a multigenic network that highlights parts of the Triticeae history that have not evolved in a tree-like manner. Dasypyrum, Heteranthelium and genera of clade V, grouping Secale, Taeniatherum, Triticum and Aegilops, have evolved in a reticulated manner. Their relationships are thus better represented by the multigenic network than by the supermatrix or BCF trees. Noteworthy, we demonstrate that gene-tree incongruences increase with genetic distance and are greater in telomeric than centromeric genes. Together, our results suggest that recombination is the main factor decoupling gene trees from multigenic trees.ConclusionsOur study is the first to propose a comprehensive, multigenic phylogeny of Triticeae. It clarifies several aspects of the relationships among genera and species of this tribe, and pinpoints biological groups with likely reticulate evolution. Importantly, this study extends previous results obtained in Drosophila by demonstrating that recombination can exacerbate gene-tree conflicts in phylogenetic reconstructions.

LOHAS: loss‐of‐heterozygosity analysis suite

Detection of loss of heterozygosity (LOH) plays an important role in genetic, genomic and cancer research. We develop computational methods to estimate the proportion of homozygous SNP calls, identify samples with structural alterations and/or unusual genotypic patterns, cluster samples with close LOH structures and map the genomic segments bearing LOH by analyzing data of genome-wide SNP arrays or customized SNP arrays. In addition to cancer genetics/genomics, we also apply the methods to study long contiguous stretches of homozygosity (LCSH) in general populations. The LCSH analysis aids in the identification of samples with complex LCSH patterns indicative of nonrandom mating and/or meiotic recombination cold spots, separation of samples with different genetic backgrounds and sex, and mapping of regions of LCSH. Affymetrix Human Mapping 500K Set SNP data from an acute lymphoblastic leukemia study containing 304 cancer patients and 50 normal controls and from the HapMap Project containing 30 African trios, 30 Caucasian trios and 90 independent Asian samples were analyzed. We identified common gene regions of LOH, e.g., ETV6 and CDKN1B, and identified frequent regions of LCSH, e.g., the region that encompasses the centromeric gene desert region of chromosome 16. Unsupervised analysis separated cancer subtypes and ethnic subpopulations by patterns of LOH/LCSH. Simulation studies considering LOH width, effect size and heterozygous interference fraction were performed, and the results show that the proposed LOH association test has good test power and controls type 1 error well. The developed algorithms are packaged into LOHAS written in R and R GUI.

Donor KIR2DS1 and KIR3DS1 Are Associated With Improved Outcomes Following Unrelated Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia

Current concepts of NK alloreactivity are based on donor/recipient KIR ligand mismatching and on donor KIR genotype variants, while little is known about the role of specific activating KIR genes. Specifically, the importance of the telomeric KIR2DS1 and KIR3DS1genes, and the centromeric KIR2DS2 gene has not been studied in detail. We tested the hypothesis that individual donor activating KIR genes would influence the outcome of AML patients undergoing unrelated allogeneic HCT.

The optimization of aDNA extraction protocol and sex determination of Bronze Age individuals from Oylum Höyük (Kilis, Turkey)

Oylum Höyük falls within the borders of the province of Kilis, located in the southern part of Turkey. It is an important Bronze Age settlement dating back to 5500 BC. In this study, the sex of 36 individuals found in Oylum Höyük was examined using morphological and genetic methods. In the morphological method, sex was identified in 14 female and 13 male individuals. The remaining 8 individuals could not be identified due to the poor preservation of their skeletal remains. Genetic sex was determined with the amplification of sex-specific X, Y centromeric alphoid repeats and ZFX-ZYF gene regions of DNA, mostly extracted from teeth. Based on ZFX-ZYF gene regions, sex was successfully determined using the PCR-RFLP method. The amplification results of ZFX-ZYF gene regions were also tested by applying the PCR-RFLP method. The amplification of X, Y centromeric alphoid repeats was successful in 29 individuals, while sex determination based on ZFX-ZFY fragments was carried out in 22 individuals. The vast majority of the results determined by analysis of ancient DNA were consistent with the morphological method. However, these 2 methods produced slightly different results for 5 individuals. Finally, the results indicated that ancient DNA analysis is a reliable method for sex determination, especially in poorly preserved and fragmented skeletal remains.

Insights into genotype–phenotype correlations in spinal muscular atrophy: A retrospective study of 103 patients

Spinal muscular atrophy (SMA) is an autosomal recessive disorder associated with homozygous deletion of the survival motor neuron 1 gene (SMN1). Its centromeric copy gene, SMN2, is the major modifying factor. However, the genotype-phenotype correlation is incomplete and is therefore not useful in clinical practice. We studied a cohort of 103 patients in order to refine this correlation. In addition to standard disease severity data, we collected three additional criteria: age at death; brainstem involvement; and loss of ambulation. Gene dosage analysis was conducted by multiplex ligation-dependent probe amplification (MLPA). SMN2 copynumber was highly correlated with survival duration in SMA type I and ambulation conservation or loss in type III. Among SMA severity groups, it was not significantly different in cases with brainstem involvement. Although the SMN2 copynumber could provide prognostic indications, clinical discrepancies still exist among patients, suggesting the existence of unidentified modifying factors.

Gene expression following ionising radiation: Identification of biomarkers for dose estimation and prediction of individual response

Purpose: To establish a panel of highly radiation responsive genes suitable for biological dosimetry and to explore inter-individual variation in response to ionising radiation exposure.Materials and methods: Analysis of gene expression in response to radiation was carried out using three independent techniques (Microarray, Multiplex Quantitative Real-Time Polymerase Chain Reaction (MQRT- PCR) and nCounter® Analysis System) in human dividing lymphocytes in culture and peripheral blood leukocytes exposed ex vivo from the same donors.Results: Variations in transcriptional response to exposure to ionising radiation analysed by microarray allowed the identification of genes which can be measured accurately using MQRT PCR and another technique allowing direct count of mRNA copies. We have identified genes which are consistently up-regulated following exposure to 2 or 4 Gy of X-rays at different time points, for all individuals in blood and cultured lymphocytes. Down-regulated genes including cyclins, centromeric and mitotic checkpoint genes, particularly those associated with chromosome instability and cancer could be detected in dividing lymphocytes only.Conclusions: The data provide evidence that there are a number of genes which seem suitable for biological dosimetry using peripheral blood, including sestrin 1 (SESN1), growth arrest and DNA damage inducible 45 alpha (GADD45A), cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin G1 (CCNG1), ferredoxin reductase (FDXR), p53 up-regulated mediator of apoptosis (BBC3) and Mdm2 p53 binding protein homolog (MDM2). These biomarkers could potentially be used for triage after large-scale radiological incidents and for monitoring radiation exposure during radiotherapy.

Microsatellite-centromere mapping in walking catfish Clarias macrocephalus (Günther, 1864) using gynogenetic diploids

Thirty new microsatellite loci were isolated from a microsatellite-enriched genomic library of walking catfish Clarias macrocephalus. The CA motif was the most abundant, although several other motifs were also isolated. Two gynogenetic diploid families, each consisting of a female and 50 offspring, were genotyped at 56 microsatellite loci, including 30 loci developed in the present study and 26 loci reported in the previous study. Overall, 33 anonymous microsatellites derived from genomic library and 11 EST-linked microsatellites were mapped to their centromeres. High levels of chiasma interference were apparent in the walking catfish genome as indicated by an average frequency of second-division segregation (y) of 0.643 ± 0.248. Twenty-six loci (59%) showed a high microsatellite–centromere recombination, with a frequency >0.67, and three loci had recombination frequencies >0.9. This study demonstrated that gene–centromere mapping provided a rapid method for the expansion of the initial linkage map of walking catfish.

The compact Brachypodium genome conserves centromeric regions of a common ancestor with wheat and rice

The evolution of five chromosomes of Brachypodium distachyon from a 12-chromosome ancestor of all grasses by dysploidy raises an interesting question about the fate of redundant centromeres. Three independent but complementary approaches were pursued to study centromeric region homologies among the chromosomes of Brachypodium, wheat, and rice. The genes present in pericentromeres of the basic set of seven chromosomes of wheat and the Triticeae, and the 80 rice centromeric genes spanning the CENH3 binding domain of centromeres 3, 4, 5, 7, and 8 were used as "anchor" markers to identify centromere locations in the B. distachyon chromosomes. A total of 53 B. distachyon bacterial artificial chromosome (BAC) clones anchored by wheat pericentromeric expressed sequence tags (ESTs) were used as probes for BAC-fluorescence in situ hybridization (FISH) analysis of B. distachyon mitotic chromosomes. Integrated sequence alignment and BAC-FISH data were used to determine the approximate positions of active and inactive centromeres in the five B. distachyon chromosomes. The following syntenic relationships of the centromeres for Brachypodium (Bd), rice (R), and wheat (W) were evident: Bd1-R6, Bd2-R5-W1, Bd3-R10, Bd4-R11-W4, and Bd5-R4. Six rice centromeres syntenic to five wheat centromeres were inactive in Brachypodium chromosomes. The conservation of centromere gene synteny among several sets of homologous centromeres of three species indicates that active genes can persist in ancient centromeres with more than 40 million years of shared evolutionary history. Annotation of a BAC contig spanning an inactive centromere in chromosome Bd3 which is syntenic to rice Cen8 and W7 pericentromeres, along with BAC FISH data from inactive centromeres revealed that the centromere inactivation was accompanied by the loss of centromeric retrotransposons and turnover of centromere-specific satellites during Bd chromosome evolution.

Reduced Survival of Motor Neuron (SMN) Protein in Motor Neuronal Progenitors Functions Cell Autonomously to Cause Spinal Muscular Atrophy in Model Mice Expressing the Human Centromeric (SMN2) Gene

Spinal muscular atrophy (SMA) is a common (approximately 1:6400) autosomal recessive neuromuscular disorder caused by a paucity of the survival of motor neuron (SMN) protein. Although widely recognized to cause selective spinal motor neuron loss when deficient, the precise cellular site of action of the SMN protein in SMA remains unclear. In this study we sought to determine the consequences of selectively depleting SMN in the motor neurons of model mice. Depleting but not abolishing the protein in motor neuronal progenitors causes an SMA-like phenotype. Neuromuscular weakness in the model mice is accompanied by peripheral as well as central synaptic defects, electrophysiological abnormalities of the neuromuscular junctions, muscle atrophy, and motor neuron degeneration. However, the disease phenotype is more modest than that observed in mice expressing ubiquitously low levels of the SMN protein, and both symptoms as well as early electrophysiological abnormalities that are readily apparent in neonates were attenuated in an age-dependent manner. We conclude that selective knock-down of SMN in motor neurons is sufficient but may not be necessary to cause a disease phenotype and that targeting these cells will be a requirement of any effective therapeutic strategy. This realization is tempered by the relatively mild SMA phenotype in our model mice, one explanation for which is the presence of normal SMN levels in non-neuronal tissue that serves to modulate disease severity.

Multi‐exon genotyping of SMN gene in spinal muscular atrophy by universal fluorescent PCR and capillary electrophoresis

In this study, we established the first method for simultaneous evaluation of nine exons in the survival motor neuron (SMN) genes for full-scale genotyping. This method was used not only to quantify the copy numbers of highly homogenous telomeric SMN (SMN1)/centromeric SMN genes in exons 7 and 8 but also to determine intragenic mutations in all nine exons for complete diagnosis of spinal muscular atrophy (SMA). Additionally, we utilized the "universal fluorescent PCR" for simultaneously fluorescent labeling of eleven gene fragments (nine exons in SMN and two internal standards). Such technique is very beneficial for multi-exon analysis due to only requirement of one universal fluorescent primer which could fluorescently amplify all gene fragments. Of all 262 detected individuals, three subjects possessing different ratios of SMN1/centromeric SMN in the two exons were determined as gene conversion, and we also detected three interesting intragenic mutations (c.1 -39A>G, c.22_23insA in exon 1, c.84C>T in exon 2a) which were associated with the SMA patients owning one copy of SMN1 including two mutations never reported previously. This high-resolved method provided better potential technique for genotyping and identifying SMA, carrier and normal controls in large population.

복제수 증폭시스템과 염색체 분단기술을 이용한 Simultaneous YAC Manipulation-Amplification (SYMA) 시스템의 개발

복잡한 진핵생물에서의 물리적 지도 작성이나 기능해석에 효모인공염색체(YAC)를 이용하기 위해서는 원하는 target region의 인공염색체화 및 single-copy인 YAC의 복제수를 늘이는 것이 요구된다. 본 연구에서는 YAC manipulation system에 복제수 증폭시스템(copy number amplification system)을 도입한 Simultaneous YAC Manipulation-Amplification (SYMA) system을 구축하였다. 식물염색체를 가진 YAC clone의 splitting과 증폭을 위해 conditional centromere와 thymidine kinase (TK) 유전자를 가진 pBGTK plasmid를 구축하였고, splitting fragment의 PCR을 위한 주형으로 사용하였다. 590 kb의 YAC clone은 splitting과 동시에 copy number amplification element를 가진 100 kb YAC와 490 kb YAC로 분리되었고, 100 kb YAC는 유도기질로 3 mg/ml sulfanilamide와 <TEX>$50\;{\mu}g/ml$</TEX> methotrexate (S3/M50)의 첨가에 의해 14.4배로 그 복제수가 증가하였음을 확인할 수 있었다. Artificial chromosome manipulation and amplification of single-copy yeast artificial chromosome (YAC) are usually required in order to use YACs for applications such as physical mapping and functional analysis in eukaryotes. We designed and implemented a Simultaneous YAC Manipulation-Amplification (SYMA) system that combines the copy number amplification system of YAC with a convenient YAC manipulation system. To achieve the desired split and to amplify a YAC clone-harboring plant chromosome, a pBGTK plasmid containing a conditional centromere and thymidine kinase (TK) gene was constructed as a template to amplify the splitting fragment via PCR. By splitting, new 490-kb and 100-kb split YACs containing the elements for copy number amplification were simultaneously generated from a 590-kb YAC clone. The 100-kb split YAC was then successfully amplified 14.4-fold by adding 3 mg/ml sulfanilamide and <TEX>$50\;{\mu}g/ml$</TEX> methotrexate (S3/M50) as inducing substances.

RNAi and heterochromatin repress centromeric meiotic recombination

During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis.

Abstract 1600: Molecular Characterization of PAX5 alterations in Adult BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL)

Abstract Background: Recently, a genome-wide analysis using single nucleotide polymorphism (SNP) microarrays, identified genetic alterations targeting PAX5 in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, the occurrence of PAX5 alterations has not been investigated in adult BCR-ABL1-positive ALL. Aim: To characterize mutations and rearrangements on 9p involving PAX5 in adult BCR-ABL1-positive ALL. Patients and Methods: Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results: SNP array analysis identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was heterozygous. Four patterns of PAX5 deletion were observed: focal deletion involving only the PAX5 gene in one case (3%); deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in PAX5 haploinsufficiency or altered gene expression. In one patient the deletion of PAX5 involved only a subset of exons (2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used: genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Normal and deleted PAX5 subgroups were also investigated for point mutations, analyzing each exon by direct sequencing, but we did not found nucleotide substitutions. Conclusion: PAX5 deletions, but not point mutations, are frequent events in adult BCR-ABL1 positive ALL and are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1600.

A Molecular-Cytogenetic Method for Locating Genes to Pericentromeric Regions Facilitates a Genomewide Comparison of Synteny Between the Centromeric Regions of Wheat and Rice

Centromeres, because of their repeat structure and lack of sequence conservation, are difficult to assemble and compare across organisms. It was recently discovered that rice centromeres often contain genes. This suggested a method for studying centromere homologies between wheat and rice chromosomes by mapping rice centromeric genes onto wheat aneuploid stocks. Three of the seven cDNA clones of centromeric genes from rice centromere 8 (Cen8), 6729.t09, 6729.t10, and 6730.t11 which lie in the Cen8 kinetochore region, and three wheat ESTs, BJ301191, BJ305475, and BJ280500, with similarity to sequences of rice centromeric genes, were mapped to the centromeric regions of the wheat group-7 (W7) chromosomes. A possible pericentric inversion in chromosome 7D was detected. Genomewide comparison of wheat ESTs that mapped to centromeric regions against rice genome sequences revealed high conservation and a one-to-one correspondence of centromeric regions between wheat and rice chromosome pairs W1-R5, W2-R7, W3-R1, W5-R12, W6-R2, and W7-R8. The W4 centromere may share homology with R3 only or with R3 + R11. Wheat ESTs that mapped to the pericentromeric region of the group-5 long arm anchored to the rice BACs located in the recently duplicated region at the distal ends of the short arms of rice chromosomes 11 and 12. A pericentric inversion specific to the rice lineage was detected. The depicted framework provides a working model for further studies on the structure and evolution of cereal chromosome centromeres.

PAX5 Wild-Type without IKZF1 (Ikaros) Deletion Is Associated with Prolonged Disease-Free Survival and Low Rate of Cumulative Incidence of Relapse in Adult BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL): On Behalf of GIMEMA AL Working Party.

Abstract 12 Background:Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism (SNP) microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, so far, the occurrence of PAX5 alterations and their clinical correlation has not been investigated in adults with BCR-ABL1-positive ALL. Aim:To characterize the rearrangements on 9p involving PAX5 and their clinical significance in adult BCR-ABL1-positive ALL. Patients and Methods:Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results:By SNP array analysis we identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was hemizygous. Four patterns of PAX5 deletion were observed: 1) focal deletion involving only the PAX5 gene in one case (3%); 2) deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); 3) broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); 4) deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in either PAX5 haploinsufficiency or expression of PAX5 alleles with impaired DNA-binding or transcriptional activation activity. In one patient the deletion of PAX5 involved only a subset of exons (exons 2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used, demonstrating that genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Furthermore, both normal and deleted PAX5 subgroups were investigated for point mutations by sequencing analysis but we did not found nucleotide substitutions, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. However, when we investigated the association of PAX5 deletions with clinical outcome, no association with PAX5 status was detected (overall survival: p=0.3294; disease free-survival,DFS: p=0.9249 and cumulative incidence of relapse, CIR: p=0.945), suggesting that PAX5 deletions are not associated with outcome. Twenty-three patients (26%) had deletion of both PAX5 and IKZF1, encoding for the transcription factor Ikaros. We assessed the contribution of both PAX5 and IKZF1 alterations on clinical outcome finding out that the occurrence of both normal PAX5 and IKZF1 loss is associated with a shorter DFS (p=0.04) and higher CIR cumulative incidence of relapse in comparison to patients with both normal IKZF1 and PAX5 (p = 0.009). These results were confirmed by multivariate analysis. Conclusion:PAX5 deletions are frequent events in adult BCR-ABL1 positive ALL and are often associated with IKZF1 loss. The genomic status of both PAX5 and IKZF1 can be useful to identify at diagnosis groups of patients with worse prognosis.Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Disclosures:No relevant conflicts of interest to declare.

Selection of Donors with Favorable KIR B Genotypes for Unrelated Hematopoietic Cell Transplantation Results in Superior Relapse Protection and Better Relapse-Free Survival for Patients with AML.

Abstract 665Unrelated donor (URD) transplants from donors with KIR group B/x genotypes (vs. A/A) confer a significant relapse-free survival (RFS) benefit for patients with acute myeloid leukemia (AML) (RR: 0.70 [95% CI: 0.55-0.88]; P = .002; Blood 2009; 113[3]). This new analysis was designed to investigate the beneficial effect of KIR B donors and to develop a donor selection strategy to improve clinical outcomes after hematopoietic cell transplantation (HCT) for leukemia. Based on an analysis of 27 unique KIR haplotype sequences we identified common centromeric and telomeric gene content motifs. KIR A haplotypes contain a Cen-A motif (defined by the presence of the inhibitory KIR gene 2DL3) and a Tel-A motif (defined by the presence of the activating gene 2DS4). The B haplotypes were defined as containing Cen-B (presence of 2DS2 and/or 2DL2) and/or Tel-B (presence of 2DS1), with further subdivisions possible at the allelic level. Thus, based on gene content alone, donor KIR genotypes can be classified as homogyzous A/A or defined by the type (Cen-B or Tel-B) or number (0, 1, 2 or ≥3; B domain content score) of B domains. Multivariate models were used to evaluate the effect of donor KIR genotypes on clinical outcomes after URD transplants facilitated by the National Marrow Donor Program for AML (n=1086) and acute lymphoblastic leukemia (ALL: n=334) between 1988 and 2006. The improved RFS associated with donor KIR B genotypes (Cen-A/B, Cen-B/B, Tel-A/B or Tel-B/B) vs. KIR A genotypes (Cen-A/A or Tel-A/A) in AML was most evident in the 115 donors (10.6%) who were homogyzous for the Cen-B motif (RR 0.72 [95% CI 0.55-0.94]; p=0.014). Likewise, Cen-B/B donors conferred significant protection against relapse (RR 0.34 [95% CI 0.2-0.57]; p < 0.0001); with absolute relapse rates of only 10% (Cen-B/B) vs. ∼31% (A/A). Similarly, multivariate models demonstrated that compared to KIR A/A donors, donors with higher KIR B domain content scores resulted in improved RFS (2B motifs: (RR 0.78 [95% CI 0.63-0.95]; p=0.013; n=244) or ≥3 B motifs: (RR 0.76 [95% CI 0.57-1.02]; p=0.07; n=84) and less relapse (2B motifs: (RR 0.54 [95% CI 0.39-0.74]; p=0.0001) or ≥3 B motifs: (RR 0.45 [95% CI 0.27-0.74]; p=0.0017). Donor KIR genotype had no effect on rates of graft vs. host disease or treatment related mortality. Importantly, the use of KIR B donors of any type was not associated with any improvement in clinical outcomes for patients with ALL. These data suggest that AML blasts may be particularly sensitive to killing by NK cells and raises the possibility that activating genes present in the donor KIR B haplotypes may uniquely recognize ligands on AML blasts. The KIR B genotype effects were not affected by the degree of HLA matching. Therefore, these data support the consideration of KIR genotyping with HLA typing into the unrelated donor search criteria for patients with AML. To capture the benefit of Cen-B and/or higher B domain content scores we propose that the ∼30% of donors who have ≥2 B domains (includes all Cen-B/B donors) be given preference over donors with 0 or 1 KIR B domains. In this large retrospective dataset, assignment by that rule resulted in significant improvements in RFS (RR 0.80 [95% CI 0.68-0.94]; p=0.0063) and relapse (RR 0.53 [95% CI 0.41-0.69]; p<0.0001). A prospective trial to test this strategy is planned. Disclosures:No relevant conflicts of interest to declare.

The NRG1 gene is frequently silenced by methylation in breast cancers and is a strong candidate for the 8p tumour suppressor gene

Neuregulin-1 (NRG1) is both a candidate oncogene and candidate tumour suppressor gene. It encodes the heregulins and other mitogenic ligands for the ERBB family, but it also causes apoptosis in NRG1-expressing cells. We found that most breast cancer cell lines had reduced or undetectable expression of NRG1. This included cell lines that had translocation breaks in the gene. Similarly, expression in cancers was generally comparable to or less than various normal breast samples. Many non-expressing cell lines had extensive methylation of the CpG island at the principal transcription start site at exon 2 of NRG1. Expression was reactivated by demethylation. Many tumours also showed methylation, while normal mammary epithelial fragments had none. Lower NRG1 expression correlated with higher methylation. siRNA-mediated depletion of NRG1 increased net proliferation, in a normal breast cell line and a breast cancer cell line that expressed NRG1. The short arm of chromosome 8 is frequently lost in epithelial cancers, and NRG1 is the most centromeric gene that is always affected. NRG1 may therefore be the major tumour suppressor gene postulated to be on 8p: it is in the correct location, is anti-proliferative, and is silenced in many breast cancers.

Fazioskapulohumerale Muskeldystrophie

Zusammenfassung Die fazioskapulohumerale Muskeldystrophie (FSHD, MIM 158900) zählt zu den häufigsten Muskeldystrophien mit einer Prävalenz von mindestens 1:20.000. Bei den meisten Patienten manifestiert sich die langsam progrediente Myopathie in der 2. Lebensdekade mit einer Schwäche der Gesichts- und Schultermuskulatur sowie der Oberarme. Typischerweise sind die unteren Extremitäten erst im weiteren Verlauf betroffen. In selteneren kindlichen Fällen treten die Symptome schon in der 1. Dekade auf, und die Erkrankung verläuft schwer mit besonders starker Beteiligung der Gesichtsmuskulatur. Die Genetik der FSHD ist einzigartig und bis heute nicht endgültig geklärt. Ein direkter Gendefekt ist nicht bekannt. Es gibt eine enge Assoziation zur Deletion von subtelomerischen D4Z4-Repeat-Einheiten auf Chromosom 4q. Man vermutet einen Positionseffekt dieser D4Z4-Repeats auf zentromerwärts gelegene Gene, die bei Patienten in der Muskulatur überexprimiert werden. Die molekulargenetische Diagnostik erfolgt durch den Nachweis eines verkürzten D4Z4-Fragments im Southern-Blot. Erschwerend kommt hinzu, dass 10q-Repeat-Einheiten zu D4Z4-Einheiten hochhomolog sind und beide untereinander ausgetauscht werden können. Nur verkürzte Repeatfragmente in einer speziellen 4q-Umgebung sind pathogen.

The Begain gene marks the centromeric boundary of the imprinted region on mouse chromosome 12

Although the central portion of the imprinted region on mouse chromosome 12 has been intensively analysed in the past, little is known about the neighbouring centromeric genes. A DNA sequence comparison shows that the region upstream of Dlk1 and Gtl2 is dominated by an expanded cluster of repetitive elements in the mouse. These elements separate the paternally expressed Dlk1 gene from the centromeric Begain gene. Despite the long physical distance to the IG-DMR imprinting centre, Begain is subjected to genomic imprinting. Similar to the ovine Begain gene, the homologous mouse gene encodes two different transcript variants, one of which shows a strong bias toward paternal transcription. Nevertheless, imprinting effects do not spread further centromeric to the Wdr25 gene, which is biallelically expressed as the previously studied neighbouring Wars and Yy1 genes.

Effects of cutoff thresholds for minor allele frequencies on HapMap resolution: A real dataset-based evaluation of the Chinese Han and Tibetan populations

Genomic variation is the genetic basis of phenotypic diversity among individuals, including variation in disease susceptibility and drug response. The greatest promise of the International HapMap is to provide roadmaps for identifying genetic variants predisposing to complex diseases. Single nucleotide polymorphism (SNP) is the fundamental element of the HapMap. Allele frequency of SNPs is one of the major factors affecting the resulting HapMap, being the factor upon which linkage disequilibrium (LD) is calculated, haplotypes are constructed, and tagging SNPs (tagSNPs) are selected. The cutoff thresholds for the frequency of minor alleles used in the making of the map therefore have profound effects on the resolution of that map. To date most researchers have adopted their own cutoff thresholds, and there has been little real dataset-based evaluation of the effects of different cutoff thresholds on HapMap resolution. In an attempt to assess the implications of different cutoff values, we analyzed our own data for the centromeric genes on Chromosome 15 in Chinese Han and Tibetan populations, with respect to minor allele frequency cutoff values of ≥0.01 (0.01 group), ≥0.05 (0.05 group), and ≥0.10 (0.10 group), and constructed HapMaps from each of the datasets. The resolution, study power and cost-effectiveness for each of the maps were compared. Our results show that the 0.01 threshold provides the greatest power (P = 0.019 in Han and P = 0.029 in Tibetan for 0.01 vs. 0.05 threshold) and detects most population-specific haploypes (P = 0.012 for 0.01 vs. 0.05 threshold). However, in the regions studied, the 0.05 cutoff threshold did not significantly increase power above the 0.10 threshold (P = 0.191 in Han; 1.000 in Tibetans), and did not improve resolution over the 0.10 value for populationspecific haplotypes (P = 0.592) neither. Furthermore the 0.05 and 0.10 values produced the same figures for tagging efficiency, LD block number, LD length, study power and cost-savings in the Tibetan population. These results suggest that a lower cutoff value is more appropriate for studies in which population-specific haplotypes are crucial, and that the most appropriate cutoff value may differ between populations. Due to the limited genes studied in this project more studies should be conducted to further address this important issue.