Telomeric Side Research Articles

A complex network of interactions governs DNA methylation at telomeric regions.

DNA methylation modulates telomere function. In Arabidopsis thaliana, telomeric regions have a bimodal chromatin organization with unmethylated telomeres and methylated subtelomeres. To gain insight into this organization we have generated TAIR10-Tel, a modified version of the Arabidopsis reference genome with additional sequences at most chromosome ends. TAIR10-Tel has allowed us to analyse DNA methylation at nucleotide resolution level in telomeric regions. We have analysed the wild-type strain and mutants that encode inactive versions of all currently known relevant methyltransferases involved in cytosine methylation. These analyses have revealed that subtelomeric DNA methylation extends 1 to 2 kbp from Interstitial Telomeric Sequences (ITSs) that abut or are very near to telomeres. However, DNA methylation drops at the telomeric side of the telomere-subtelomere boundaries and disappears at the inner part of telomeres. We present a comprehensive and integrative model for subtelomeric DNA methylation that should help to decipher the mechanisms that govern the epigenetic regulation of telomeres. This model involves a complex network of interactions between methyltransferases and subtelomeric DNA sequences.

Human NORs, comprising rDNA arrays and functionally conserved distal elements, are located within dynamic chromosomal regions.

Human nucleolar organizer regions (NORs), containing ribosomal gene (rDNA) arrays, are located on the p-arms of acrocentric chromosomes (HSA13-15, 21, and 22). Absence of these p-arms from genome references has hampered research on nucleolar formation. Previously, we assembled a distal junction (DJ) DNA sequence contig that abuts rDNA arrays on their telomeric side, revealing that it is shared among the acrocentrics and impacts nucleolar organization. To facilitate inclusion into genome references, we describe sequencing the DJ from all acrocentrics, including three versions of HSA21, ∼3 Mb of novel sequence. This was achieved by exploiting monochromosomal somatic cell hybrids containing single human acrocentric chromosomes with NORs that retain functional potential. Analyses revealed remarkable DJ sequence and functional conservation among human acrocentrics. Exploring chimpanzee acrocentrics, we show that "DJ-like" sequences and abutting rDNA arrays are inverted as a unit in comparison to humans. Thus, rDNA arrays and linked DJs represent a conserved functional locus. We provide direct evidence for exchanges between heterologous human acrocentric p-arms, and uncover extensive structural variation between chromosomes and among individuals. These findings lead us to revaluate the molecular definition of NORs, identify novel genomic structural variation, and provide a rationale for the distinctive chromosomal organization of NORs.

Mother–child transmission of epigenetic information by tunable polymorphic imprinting

Genomic imprinting mediated by DNA methylation restricts gene expression to a single allele determined by parental origin and is not generally considered to be under genetic or environmental influence. Here, we focused on a differentially methylated region (DMR) of approximately 1.9 kb that includes a 101-bp noncoding RNA gene (nc886/VTRNA2-1), which is maternally imprinted in ∼75% of humans. This is unlike other imprinted genes, which demonstrate monoallelic methylation in 100% of individuals. The DMR includes a CTCF binding site on the centromeric side defining the DMR boundary and is flanked by a CTCF binding site on the telomeric side. The centromeric CTCF binding site contains an A/C polymorphism (rs2346018); the C allele is associated with less imprinting. The frequency of imprinting of the nc886 DMR in infants was linked to at least two nongenetic factors, maternal age at delivery and season of conception. In a separate cohort, nc886 imprinting was associated with lower body mass index in children at 5 y of age. Thus, we propose that the imprinting status of the nc886 DMR is "tunable" in that it is associated with maternal haplotype and prenatal environment. This provides a potential mechanism for transmitting information, with phenotypic consequences, from mother to child.

The Highly Prevalent Deletions in F8 Intron 13 Found in French Mild Haemophilia a Patients Result of Both Founder Effect and Recurrent De Novo Events

Background: Genetic variations that are found at a relatively high frequency can be the consequence of a founder effect or repeated de novo events. Recently, our group has identified an intronic deletion, c.2113+461_2113+473del [DEL13T], in the F8 intron 13, in two mild haemophilia A patients. This deletion removed a part of the poly(T)-tail from the right arm of antisens AluY element and led to AluY exonisation. Then, deletions in the poly(T)-tail of AluY in F8 intron 13, from 10 to 14 pb in size, were found in approximately 6% of all cases of mild haemophilia in France.Aim: In this study, we determined whether these highly prevalent deletions are the result of recurrent molecular mechanism or of a founder effect.Methods: Haplotype reconstruction was performed after analysis of F8 extragenic and intragenic polymorphic markers in 71 unrelated French mild haemophilia A patients carrying a deletion in the poly(T)-tail of AluY in F8 intron 13 (c.2113+460_2113+473del [DEL14T] n=1; DEL13T, n=62; c.2113+462_2113+473del [DEL12T], n=2; c.2113+463_2113+473del [DEL11T], n=5 and c.2113+464_2113+473del, n=1 [DEL10T]) and in 50 non-haemophilia A subjects. The ESTIAGE tool was used to estimate the age of the DEL13T. Nineteen genetically unresolved mild Haemophilia A patients from Queen's University, Canada, were also included in the study. All patients and controls gave informed consent for genetic studies.Results: Two intragenic (STR13 and STR22) and 3 extragenic (DXS8061, ST14 and DF2) microsatellites were investigated in the DNA of the 71 mild haemophilia A patients. This set of polymorphisms markers covered a genomic region of 2,779,113 nucleotides (4.17 cM).Among the 62 patients carrying the DEL13T, 60 patients had the same allele for the marker directly flanking the deletion on the centromeric side (STR13, genetic distance 0.0062 cM). Two of them differed from the others at the next centromeric maker STR22 (genetic distance 0.2047 cM) and 20 patients differed from the others at ST14 (genetic distance 3.4237 cM). On the telomeric side, only 8 patients differed from the others at DF2 (genetic distance 0.0292 cM). None of control individuals shared such haplotypes with these 60 patients. These results provided evidence that the founder effect hypothesis was very plausible for the variant DEL13T. The ESTIAGE tool estimated that the DEL13T occurred about 61 generations ago (95% CI : 51-74 generations). Assuming that a generation spanned 25 years, the French common ancestor carrying the c.2113+461_2113+473del was estimated to have lived between the 2th and the 8th century.However, two patients carrying the DEL13T and 9 patients carrying the other deletions (DEL10T, DEL11T, DEL12T and DEL14T) had a different haplotype suggesting that these deletions arose independently. In order to support the hypothesis of a recurrent molecular mechanism, we have investigated the presence of these deletions in other geographies. F8 intron 13 deletions were found in 3/19 Canadian patients included in this study (DEL13T, n=2 and DEL10T, n=1). Haplotype analysis performed in these three patients suggested a de novo mechanism for two of them.Conclusion: This study supports both a founder effect for the c.2113+461_2113+473del in the French mild haemophilia A patients and a recurrent molecular mechanism leading to deletion in the poly(T)-tail of AluY in F8 intron 13. We recommended that these deletions be specifically investigated in all mild haemophilia A patients in whom no genetic abnormality has been detected by standard genetic analysis. Finally, these results suggest that large poly(T)-tail of inverted Alu elements may be a mutational hot spot and such deletions leading to alu-exonization, could occur in other genes. DisclosuresNegrier:Sobi/Bioverativ: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Alnylam: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; LFB: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta/Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

High Order Chromatin Structure Regulates Gene Expression in Hematopoietic Stem Cell Self-Renewal and Erythroid Differentiation

High order chromatin structure is implicated in multiple developmental processes and disease. However, a global picture of chromosomal looping interaction alterations during stem cell self-renewal and differentiation is lacking. Hematopoietic stem cell (HSCs) and their differentiated progenitors (HSPCs) offer a system in which to examine this. Of the key differentiated lineages, the erythroid lineage undergoes a unique nuclear condensation process during a well-characterized differentiation process which can be induced in vitro from CD34+ HSPCs. Thus erythroid differentiation offers an ideal model system to study differentiation-associated changes in high order chromatin structure.We have thus generated the in situ Hi-C contact map for human cord blood CD34+ CD38- HSPC (CD34+) and erythroid progenitors undergoing differentiation in vitro at day 7 from CD34+ HSPCs (EryD7). In our 5kb resolution map, we identified over 2000 chromosomal loop interactions in both CD34+ and Day 7 erythroid respectively . The EryD7 sample exhibited higher random intra-chromosomal interactions in comparison with CD34+, presumably due to nuclear condensation.By comparing the chromosomal loop interactions in the 2 cell types. We identified self-renewal and erythroid differentiation-specific looping patterns in the two cell types. Strikingly, we found that a gene depleted region (GDR) 2MB upstream of the HOXA cluster forms a strong chromosome loop with the HOXA cluster exclusively in the HSPCs (Fig1A). Within this GDR site, we identified two conserved CTCF sites, which are thought to organized chromosome looping. Utilizing the CRISPR-mediated deletion of each of the two CTCF sites, we found that deletion of either site reduce the colony forming ability of CD34+, indicating a loss of stem cell self-renewal. (Fig 1B) Gene expression analysis showed that HOXA9 expression was compromised the CTCF site deletion. These data suggest that the GDR is forming a distant regulatory loop which controls the expression of HOXA9 in HSPCs. Because the GDR is implicated in controlling HOXA9 expression, a key gene in leukemogenesis, we then tested the importance of this looping site in different leukemia cell lines that are dependent on HOXA9. Of those cell lines, we found the deletion of the CTCF sites inhibit the growth of DNMT3A and NPM1 mutated OCI-AML3 and promote the apoptosis. In contrast, growth of the MLL translocation cell line MV 4:11 was not abrogated by their deletion (Fig 1C). As a control cell line which doesn't express HOXA9, HL60 cells were not sensitive to the deletion of the GDR CTCF sites. Together, these data indicate leukemic cells may adopt different strategies to activate HOXA9. MLL translocation leukemias activates HOXA9 by the direct binding of the MLL fusion protein, while the NPM1 mutated leukemia is more likely to utilize the stem cell looping to activate HOXA9 expression.Among EryD7 specific interactions, we found the β-globin locus specifically forms chromatin loops at Day7 that are not evident in the CD34+ HSPCs. Detailed examination showed that Dnase I hypersensitivity sites HS5 and 3'HS1 both contains CTCF site and form chromosomal loops. Two other loop-forming CTCF sites, both on the telomeric and centromeric side of β-globin locus were also identified. Interestingly, we found a CTCF binding site adjacent to OR52A5 gene which forms a chromosomal loop with HS5 and is not well studied. To test the role of the chromosomal looping in the regulation of hemoglobin gene expression in β-globin locus, we deleted the OR52A5-CTCF site and the 3'HS1 CTCF site in K562 and adult CD34+ HSPCs. We found the deletion of OR52A5-CTCF resulted in a decrease of HBE and increase of HBB expression in K562 cells, which suggest the OR52A5 CTCF also plays a role in regulating hemoglobin gene expression in the β-globin locus (Fig 1D). Furthermore, we found the deletion of 3'HS1 CTCF resulted in a 4-fold increase of HBG2 expression in adult CD34+ HSPC during erythroid differentiation (Fig1 E). Thus this indicating the 3'HS1 and OR52A5-CTCF CTCF sites in β-globin locus are forming loops that regulate the β-globin locus gene expression.In summary, we have mapped the higher order chromatin structure alterations during stem cell differentiation and identified the critical looping interaction essential for the self-renewal and differentiation specific functions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A genome-wide association study reveals a quantitative trait locus for days open on chromosome 2 in Japanese Black cattle.

Days open (DO), which is the interval from calving to conception, is an important trait related to reproductive performance in cattle. To identify quantitative trait loci for DO in Japanese Black cattle, we conducted a genome-wide association study with 33,303 single nucleotide polymorphisms (SNPs) using 459 animals with extreme DO values selected from a larger group of 15,488 animals. We identified a SNP on bovine chromosome 2 (BTA2) that was associated with DO. After imputation using phased haplotype data inferred from 586 812 SNPs of 1041 Japanese Black cattle, six SNPs associated with DO were located in an 8.5-kb region of high linkage disequilibrium on BTA2. These SNPs were located on the telomeric side at a distance of 177 kb from the parathyroid hormone 2 receptor (PTH2R) gene. The association was replicated in a sample of 1778 animals. In the replicated population, the frequency of the reduced-DO allele (Q) was 0.63, and it accounted for 1.72% of the total genetic variance. The effect of a Q-to-q allele substitution on DO was a decrease of 3.74 days. The results suggest that the Q allele could serve as a marker in Japanese Black cattle to select animals with superior DO performance.

Molecular cloning of IGλ rearrangements using long‐distance inverse PCR (LDI‐PCR)

Malignant cells of mature B-cell origin show tumor-specific clonal immunoglobulin gene (IG) rearrangements, including V(D)J recombinations, nucleotide mutations, or translocations. Rapid molecular cloning of the breakpoint sequence by long-distance inverse PCR (LDI-PCR) has so far been applied to rearrangements targeted to IGH joining, IGH switch, and IGκ regions. We tended to apply LDI-PCR method for cloning of IGλ rearrangements. To identify which IGλ isotype segment was rearranged, we performed Southern blot analysis using isotype-specific probes. We set inverse primers on the telomeric side of each joining region and amplified rearranged bands detected by Southern blot analysis as corresponding PCR products. All germline IGλ segments were successfully amplified as expected PCR products. We determined breakpoint sequences of five chromosome translocations involving IGλ locus: three novel t(8;22)(q24;q11), one known t(3;22)(q27;q11), and one partially known t(11;22)(q13;q11). Two of the three t(8;22)(q24;q11) were involved in Jλ with a recombination signal sequence and one of three in the first exon of IGLL5, which lies upstream of Jλ1. Three 8q24 breakpoints were widespread at 132, 260 and 366kb downstream of MYC locus. The t(3;22)(q27;q11) showed a juxtaposition of Jλ2 and the first intron of BCL6, as previously reported. In t(11;22)(q13;q11), 3'UTR of cyclin D1 fused to the constant region of λ7 with nucleotide mutations. We also amplified four Vλ/Jλ recombination sequences. Our method is a useful tool for molecular analysis of genetic events in IGλ.

A common copy-number breakpoint of ERBB2 amplification in breast cancer colocalizes with a complex block of segmental duplications

IntroductionSegmental duplications (low-copy repeats) are the recently duplicated genomic segments in the human genome that display nearly identical (> 90%) sequences and account for about 5% of euchromatic regions. In germline, duplicated segments mediate nonallelic homologous recombination and thus cause both non-disease-causing copy-number variants and genomic disorders. To what extent duplicated segments play a role in somatic DNA rearrangements in cancer remains elusive. Duplicated segments often cluster and form genomic blocks enriched with both direct and inverted repeats (complex genomic regions). Such complex regions could be fragile and play a mechanistic role in the amplification of the ERBB2 gene in breast tumors, because repeated sequences are known to initiate gene amplification in model systems.MethodsWe conducted polymerase chain reaction (PCR)-based assays for primary breast tumors and analyzed publically available array-comparative genomic hybridization data to map a common copy-number breakpoint in ERBB2-amplified primary breast tumors. We further used molecular, bioinformatics, and population-genetics approaches to define duplication contents, structural variants, and haplotypes within the common breakpoint.ResultsWe found a large (> 300-kb) block of duplicated segments that was colocalized with a common-copy number breakpoint for ERBB2 amplification. The breakpoint that potentially initiated ERBB2 amplification localized in a region 1.5 megabases (Mb) on the telomeric side of ERBB2. The region is very complex, with extensive duplications of KRTAP genes, structural variants, and, as a result, a paucity of single-nucleotide polymorphism (SNP) markers. Duplicated segments are varied in size and degree of sequence homology, indicating that duplications have occurred recurrently during genome evolution.ConclusionsAmplification of the ERBB2 gene in breast tumors is potentially initiated by a complex region that has unusual genomic features and thus requires rigorous, labor-intensive investigation. The haplotypes we provide could be useful to identify the potential association between the complex region and ERBB2 amplification.

A new case with 10q23 interstitial deletion encompassing both PTEN and BMPR1A narrows the genetic region deleted in juvenile polyposis syndrome

We report on a patient with a contiguous interstitial germline deletion of chromosome 10q23, encompassing BMPR1A and PTEN, with clinical manifestations of juvenile polyposis and minor symptoms of Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS). The patient presented dysmorphic features as well as developmental delay at the age of 5 months. Multiple polyps along all parts of the colon were diagnosed at the age of 3 years, following an episode of a severe abdominal pain and intestinal bleeding. The high-resolution comparative genomic hybridisation revealed a 3.7-Mb deletion within the 10q23 chromosomal region: 86,329,859-90,035,024. The genotyping with four polymorphic microsatellite markers confirmed a de novo 10q deletion on the allele with a paternal origin, encompassing both PTEN and BMPR1A genes. The karyotype analysis additionally identified a balanced translocation involving chromosomes 5q and 7q, and an inversion at chromosome 2, i.e. 46,XY,t(5;7)(q13.3-q36), inv(2)(p25q34). Although many genetic defects were detected, it is most likely that the 10q23 deletion is primarily the cause for the serious phenotypic manifestations. The current clinical findings and deletion of BMPR1A indicate a diagnosis of severe juvenile polyposis, but the existing macrocephaly and PTEN deletion also point to either CS or BRRS, which cannot be ruled out at the moment because of their clinical manifestation later in life and the de novo character of the deletion. The deletion detected in our patient narrows the genetic region deleted in all reported cases with juvenile polyposis by 0.04 Mb from the telomeric side, mapping it to the region chr10:88.5-90.03Mb (GRCh37/hg19), with an overall length of 1.53 Mb.

Abstract 3117: Complex genomic regions associated with ERBB2 amplification in breast cancer

Abstract Repeated DNA segments (Low Copy Repeats, Segmental Duplications) are highly homologous sequences within the human genome. They are typically 10-300 kb in length, and bear >95% sequence identity. In germline, repeated DNA segments mediate recurrent DNA rearrangements and thus cause specific genetic disorders called genomic disorders. Repeated genomic segments could also be prone to rearrangements in somatic cells and thus could mediate recurrent rearrangements in cancer. However, the important role of repeated genomic segments in genome instability in cancer remains elusive, mainly because such regions are less accessible by current genomic technologies due to the difficulty in assembling and interrogating sequences. Gene amplification is a cellular process characterized by the production of multiple copies of a particular gene through DNA rearrangements. ERBB2, an epidermal growth factor receptor gene, is amplified in 15-20% of breast tumors. Due to excessive cell growth signaling, this recurrent amplification is associated with advanced stages and poor prognosis. Despite the clinical importance, the mechanisms giving rises to recurrent ERBB2 amplification remains unclear. We hypothesized that, similar to the recurrent rearrangements for genomic disorders, repeated genomic segments cause DNA rearrangements for ERBB2 amplification. To test the hypothesis and understand the mechanism of ERBB2 amplification, we mapped a common copy-number breakpoint for primary ERBB2-amplified (HER2-positive) breast tumors. Copy number breakpoints were examined at every 300-kilo bases (kb) from the location of ERBB2 to its telomeric region using real-time PCR based assays. The breakpoint that marked a region between normal copy number and low level amplification resided within a 300-kb complex genomic region at the 1.5 mega-bases telomeric side of ERBB2. The region contains many repeated segments from two gene families. Furthermore, the HapMap SNPs, a set of SNPs that tag haploblocks in the human genome, are less well-defined, indicating the difficulty in interrogating the genomic region by current genomic technologies. Normal copy-number variations seem common, as there is several deletion polymorphisms reported in the region. Two sub-regions, each covering 40-kb region, are particularly complex due to a number of direct and inverted repeats. The prevalence of this copy number breakpoint in HER2-positive breast tumors was also shown in a study by Sircoulomb et al.,(BMC Cancer 2010), in which common amplifications and deletions were determined for 54 HER-2 positive breast tumors. Defining (1) the copy-number breakpoint with very high resolution and (2) critical DNA sequences for ERBB2 amplification will lead to better understanding the amplification mechanism underlying this aggressive type of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3117. doi:1538-7445.AM2012-3117

Genetic analysis and fine mapping of the Ga1-S gene region conferring cross-incompatibility in maize

Cross-incompatibility genes known as gametophyte factors (ga) are numerous in maize. Many popcorn strains carry these genes and cannot be fertilized by pollen of dent and flint maize strains although the reciprocal crosses are successful. A Chinese popcorn strain SDGa25 carries the strongest allele of Ga1 (Ga1-S) and the majority of Chinese dent and flint maize germplasm are incompatible with SDGa25. The incompatibility is due to pollen tube growth obstruction 2 h after pollination. The pollen tube is arrested in the silk segment 5.5 cm distal to the pollination area and never reaches the ovule. The Ga1-S carried by SDGa25 behaves as a single dominant gene. This gene was mapped between markers SD3 on BAC AC200747 0.827 cM apart on the telomere side and SD12 on BAC AC204382 0.709 cM apart on the centromere side. The genetic region mapped spanning the Ga1-S locus was estimated to be 1.5 cM in length and the physical distance is 2,056,343 bp on ctg156 based on the B73 RefGen_v2 sequence. Gametophyte factors influence gene flow direction and the strongest Ga1-S allele is useful for isolating one category of commercial varieties from another. The eight tightly linked markers to Ga1-S developed in this study would greatly improve marker-assisted introgression efficiency and the fine mapping would facilitate the isolation of the Ga1-S.

Inactivation of a functional HLA-A gene: A 4-kb deletion turns HLA-A*24 into a pseudogene

An unusual haplotype without a detectable human leukocyte antigen (HLA)–A allele by serologic or molecular typing methods segregates in a Caucasian family. Microsatellite analysis and fluorescence in situ hybridization implicated that the deletion encompasses a narrow region. To identify the deleted region, five different fragments in close proximity to HLA-A, known to be highly polymorphic, were amplified and sequenced. The presence of heterozygous sequences in all five fragments of the individuals carrying the haplotype with the HLA-A deletion, indicates that the fragments are not involved in the deletion. Therefore, the 5′ primer from the fragment closest to the centromeric side of HLA-A was combined with the 3′ primer closest to the telomeric side encompassing an 11-kb region. Sequencing revealed that a deletion of 4089 bp was present, located upstream of HLA-A, including exons and introns 1–3 of the HLA gene. Sequence information of the 3′ part of HLA-A, downstream the deletion, identified that the deleted allele originates from an A*24 allele. Although different repeat sequences are present in the region both inside and outside the deletion, no evidence points to a retrotransposon mechanism. The detected partial deletion of HLA-A turns this functional gene into a pseudogene.

Biological and clinical characterization of recurrent 14q deletions in CLL and other mature B‐cell neoplasms

14q-deletions have been repeatedly described in mature B-cell neoplasms, but not yet characterized in a larger cohort. Based on chromosome banding analysis, the present study identified 47 del(14q) cases in 3054 mature B-cell neoplasms (1·5%) (chronic lymphocytic leukaemia [CLL]: 1·9%; CLL/prolymphocytic leukaemia [PL]: 9·0%; others: 0·2%). Interphase fluorescence in situ hybridization was performed with probes for 14q22.1, 14q24.1, 14q32.33, and IGH@ (14q32.3). The del(14q) had heterogeneous size but showed a breakpoint cluster at the centromeric site in 14q24.1 (62% of cases). At the telomeric side, the most frequent breakpoint was within the IGH@ locus (14q32.3) between IGH@ 3'-flanking and IGHV (IgVH) probes (45%). In 16 cases (34%), breakpoints occurred within 14q24.1 and 14q32.3. Eighty-one percent of del(14q) cases showed 1-3 additional cytogenetic alterations (in 45%, +12), and 56% were IGHV-unmutated. In all cases (16/16) with breakpoints in 14q24.1 and 14q32.3, a B-CLL immunophenotype was found. Clinical follow-up in 32 del(14q) patients was compared to 383 CLL and CLL/PL patients without del(14q). While 3-year-overall survival did not differ significantly, time to treatment was significantly shorter in the del(14q) cohort (21·0 months vs. 80·1 months, P = 0·015). In conclusion, the del(14q) is a rare recurrent alteration in diverse mature B-cell neoplasms, shows variable size but distinct clustering of breakpoints, and is associated with short time to treatment.

Zebrafish gene knockdowns imply roles for human YWHAG in infantile spasms and cardiomegaly

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder presenting with an elfin-like face, supravalvular aortic stenosis, a specific cognitive-behavioral profile, and infantile hypercalcemia. We encountered two WBS patients presenting with infantile spasms, which is extremely rare in WBS. Array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) analyses revealed atypical 5.7-Mb and 4.1-Mb deletions at 7q11.23 in the two patients, including the WBS critical region and expanding into the proximal side and the telomeric side, respectively. On the proximal side, AUTS2 and CALN1 may contribute to the phenotype. On the telomeric side, there are two candidate genes HIP1 and YWHAG. Because detailed information of them was unavailable, we investigated their functions using gene knockdowns of zebrafish. When zebrafish ywhag1 was knocked down, reduced brain size and increased diameter of the heart tube were observed, indicating that the infantile spasms and cardiomegaly seen in the patient with the telomeric deletion may be derived from haploinsufficiency of YWHAG.

14q Deletions Are Recurrent Aberrations in CLL and Other Mature B-Cell Neoplasms and Show a High Coincidence with An Unmutated IgVH Status and Trisomy 12, and Are Associated with An Intermediate Overall Survival and a Shorter Time to Treatment.

Abstract 1262Poster Board I-28414q deletions are rare aberrations in malignant neoplasia but recurrent in hematologic malignancies, where they occur most frequently in CLL and other mature B-cell neoplasms. The breakpoints and the size of the 14q deletion have not been exactly characterized in a larger cohort yet. Based on chromosome banding analysis we identified 47 cases with 14q deletion out of 3054 cases (1.5%) with mature B-cell neoplasms (CLL: 1863, CLL/PL: 92, other: 1099). 32 cases with 14q deletion were classified as CLL, 5 as CLL/PL and 10 as other mature B-cell neoplasms based on immunophenotyping or cytomorphology. Therefore, the incidence of 14q deletions was 1.7% in CLL, 4.7% in CLL/PL and 0.9% in other mature B-cell neoplasms. These cases were further analyzed by a panel of FISH probes localized in 14q22.1 (BAC RP11-218E20), 14q24.1 (BACs PR11-35D12, RP11-226F19), 14q32.33 (BAC RP11-521B24) (BlueGnome, Oxford, UK) and the IgH-locus (14q32.3). The study revealed that 14q deletions are heterogeneous in size. A breakpoint cluster on the centromeric side was identified in 14q24.1 between BAC PR11-35D12 and BAC RP11-226F19 (62% of cases), whereas the most frequent breakpoint on the telomeric side was within the IgH-locus (14q32.3) between the IgH3'-flanking probe and IgHV probe (45% of cases). In 16 cases (34%) breakpoints occurred within 14q24.1 and within 14q32.3. In 19% of patients the 14q deletion was the sole abnormality whereas 30% showed one, 15% two and 36% three or more additional aberrations. Trisomy 12 (45%) was the most frequent aberration accompanying 14q deletion. In comparison to a cohort of 1579 cases with mature B-cell neoplasms without 14q deletion which were analyzed for IgVH mutation status in the same time period, patients with 14q deletions showed more often an unmutated IgVH status (39% vs. 61%, p=0.001). In the subgroup with breakpoint in 14q24.1 and 14q32.3 even 82% showed an unmutated IgVH-status. This subgroup was also associated with a CLL immunophenotype (81%). Clinical follow up data was available in 28 patients. CLL and CLL/PL with 14q deletion were compared to 390 CLL and CLL/PL cases without 14q deletion. While overall survival (OS) did not differ between both groups (OS at 3 yrs 86.4% vs. 88.7%, p=0.195), time to treatment (TTT) was significantly shorter in cases with 14q deletion (21.0 months vs. 80.1 months, p=0.015). Next, all 418 cases were assigned into 6 subgroups with known prognostic relevance: 1) 14q deletion, 2) 17p deletion, 3) 11q deletion, 4) trisomy 12, 5) 13q deletion as the sole abnormality, and 6) none of the mentioned abnormalities. OS at 3 yrs was 86.4%, 31.4%, 77.5%, 96.9%, 93.4% and 93.9%, respectively. For TTT the respective data were 21.0 months, 21.6 months, 20.9 months, not reached, 79.6 months and 147.0 months. In conclusion, 14q deletions are recurrent abnormalities in CLL and other mature B-cell neoplasms. Comparable to 13q deletions the size of 14q deletions varies. However, recurrent breakpoints in 14q24.1 and 14q32.3 were identified. Our data suggest to add FISH probes for 14q24.1 and for the IGH locus to the standard FISH panel in CLL and other mature B-cell neoplasms to pick up cases with 14q deletion, who would otherwise be assigned to “normal” FISH subgroup. In conclusion, 14q deletions are frequently accompanied by trisomy 12, an unmutated IgVH status and are associated with an intermediate overall survival and a shorter time to treatment comparable to other high risk cytogenetic aberrations (17p deletion, 11q deletion). DisclosuresReindl:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Genetics of Behçet disease inside and outside the MHC

BackgroundBehçet disease (BD) is a rare, chronic, systemic, inflammatory disorder characterised by recurrent ocular, genital and skin lesions. Although its aetiology is still uncertain, an intricate interplay between the environment...

Genetic and physical mapping of blast resistance gene Pi-42(t) on the short arm of rice chromosome 12

We have identified, genetically mapped and physically delimited the chromosomal location of a new blast resistance gene from a broad spectrum resistant genotype ‘DHR9’. The segregation analysis of an F2 progeny of a cross between a susceptible cv. ‘HPU741’ and the resistant genotype ‘DHR9’ suggested that the resistance was conditioned by a single dominant gene. A RAPD marker, OPA82000, linked to the resistance gene was identified by the linkage analysis of 109 F2 individuals. By chromosomal landing of the sequence of RAPD marker on the sequence of reference cv. Nipponbare, the gene was mapped onto rice chromosome 12. Further linkage analysis with two polymorphic simple sequence repeat (SSR) markers, RM2529 and RM1337 of chromosome 12, confirmed the chromosomal localization of the resistance gene. Based on linkage analysis of 521 susceptible F2 plants and comparative haplotype structure analysis of the parental genotypes with SSR and sequence tagged site (STS) markers developed from the Nipponbare PAC/BAC clones of chromosome 12, the resistance gene was delimited within a 2 cM interval defined by STS marker, STS5, on the telomeric side and SSR marker, RRS6 on the centromeric side. By aligning the sequences of linked markers on the sequence of cv. Nipponbare, a ~4.18 Mb cross-over cold region near the centromere of chromosome 12 was delineated as the region of blast resistance gene. In this region, six putatively expressed NBS-LRR genes were identified by surveying the equivalent genomic region of cv. Nipponbare in the TIGR Whole Genome Annotation Database (http://www.tigr.org). NBS-LRR locus, LOC_Os12g18374 situated in BAC clone OJ1115_G02 (Ac. No. AL772419) was short-listed as a potential candidate for the resistance gene identified from DHR9. The new gene was tentatively designated as Pi-42(t). The markers tightly linked to gene will facilitate marker-assisted gene pyramiding and cloning of the resistance gene.

Deciphering the Heterogeneity of 13q Deletions in CLL on the Cytogenetic and Molecular Level by SNP Arrays: In 49% of Cases Deletion Breakpoints Cluster in a 200 Kb Sized Region on the Centromeric Side and in a 300 Kb Sized Region on the Telomeric Side

13q deletions are the most frequent genetic abnormalities in CLL. The aim of this study was to analyze the deleted region in 73 cases using high-resolution SNP microarrays (Affymetrix SNP 6.0). All cases showed 13q deletions as sole aberration as assessed by interphase FISH and chromosome banding analysis (CBA). The total cohort was separated into 4 subgroups:24 cases with a homozygous 13q14 deletion in the majority of cells. In two cases a homozygous deletion was observed for D13S319, but no deletion for D13S25. In one case a homozygous deletion was observed for D13S25 and a heterozygous deletion for D13S319.10 cases with a heterozygously and a homozygously 13q14 deleted clone.19 cases with a heterozygous 13q14 deletion.21 cases showing a reciprocal translocation or insertion involving 13q14 as confirmed by CBA.In 13/21 cases a heterozygous 13q14 deletion was observed, the remaining 8 cases demonstrated a homozygous 13q14 deletion. By SNP analysis the median size of the heterozygous deletion in the total cohort was assessed to be 2,335 kb (mean: 6,926 kb; range: 816–41,520 kb). The centromeric breakpoints were observed between physical map positions 29,192,645 and 49,562,978, while the telomeric breakpoints were found between 50,226,807 and 86,301,155. The minimal deleted region was 664 kb in size. The median size of the homozygously deleted region was 930 kb (mean: 1,072 kb; range: 408–2,893). The centromeric breakpoints were observed between positions 47,589,986 and 49,833,185, while the telomeric breakpoints were found between 49,933,260 and 50,708,954. Therefore, the minimal deleted region was only 100 kb in size. In this region no known gene is located. The deletion size did not differ between cases with interstitial deletion and translocations. Interestingly, in all 21 cases with translocations/insertions involving 13q14, deletions were also found on the partner chromosome in the breakpoint region with sizes between 218 and 2,164 kb (median: 898 kb). The size of heterozygous deletions varies considerably, however the centromeric breakpoints cluster between positions 48.0 MB and 49.5 MB (57% of cases) and the telomeric breakpoints between 50.2 and 50.8 (65% of cases). The size of homozygously deleted regions is smaller, the centromeric breakpoint clusters between positions 49.3 MB and 49.5 MB (79% of cases) and the telomeric breakpoint between 50.3 MB and 50.6 MB (79% of cases). Therefore, in the majority of cases one allele of the following genes was lost: CYCR, RAD17P2, FNDC3a, COX7CP1, MLNR, CDAC1, CAB39L, SETDB2, PHF11, RCTB1, ARL11, EBPL, KPNA3, FAM12A. Markers identified in the region which can also be affected by loss of both copies were: microRNA16-1, microRNA15a, DLEU2, DLEU1, BCMS, DLEU7. Breakpoints on the centromeric side are located in TRIM13 (intron 3), KPNA3 (introns 1, 13, 15), C13orf1 (introns 1, 3, 4), KCNRG (exon 2). On the telomeric side the breaks occur in DLEU7 (introns 1, 2), GUCY1B2 (introns 2, 3, 7, 10), RNASEH2B (introns 1, 4, 5, 7, 10). 22 cases showed breaks in both cluster regions suggesting the possible generation of fusion genes. Recurring combined breakpoints were C13orf1 (intron 4) - RNASEH2B (intron 1). Overall, 36 (49%) of 73 cases showed breakpoints between positions 49.3 MB and 49.5 MB as well as between positions 50.3 MB and 50.6 MB. In 8 cases with homozygous deletions a large copy-neutral LOH flanking the deleted region was observed (5 cases with whole chromosome 13 UPD; 3 cases with partial UPD ranging from 13q14-13qter, 13q14-13q31.1, and 13q14-13q21). Next, we analyzed the potential clinical impact of the localization of the deletion. Out of 46 patients analyzed prior to treatment 15 showed a deletion which started centromeric of the RB gene. Time to treatment was significantly shorter in patients whose deletion did not include the RB locus (p=0.04). Also, in 13 (34%) of 38 patients analyzed at diagnosis showing a deletion which started centromeric of the RB gene, time to treatment was significantly longer than in patients whose deletion did not include the RB locus (p=0.04). In conclusion, breakpoints cluster in a 200 kb sized region on the centromeric side and in a 300 kb sized region on the telomeric side leading to the loss of one or to copies of several genes. The genomic sequences in the breakpoint cluster regions have to be further characterized to understand the mechanism leading to deletions in the breakpoint area not only on 13q14 but also on the partner chromosome involved in 13q14 translocations. In addition, the potential formation of fusion genes has to be evaluated further as well as the clinical impact of the deletion size.

First International “Institute of Myology Workshop” on Facioscapulohumeral Muscular Dystrophy, Paris, May 22, 2007

This one-day workshop was organized by Thomas Voit and assembled 13 researchers from five countries (Belgium, The Netherlands, France, Italy, USA) in order to create a collaborative interface between several international groups who are actively investigating the molecular background of facioscapulohumeral muscular dystrophy (FSHD) as well as to give an update on therapeutic projects on FSHD at the preclinical and clinical level. Such a collective effort seemed particularly timely because there is still no unifying and generally accepted patho-physiological concept that could explain the muscle degeneration in FSHD or the less frequently associated symptoms of cochlear hearing loss, Coats’s syndrome or, in rare infantile cases, mental retardation. Furthermore, opinions diverge as to which genes are possibly directly or indirectly implicated, and no animal model exists that resumes all the particular aspects of FSHD.

Empirically derived subgroups in rheumatoid arthritis: association with single-nucleotide polymorphisms on chromosome 6

Rheumatoid arthritis (RA) is a disorder with important public health implications. It is possible that there are clinically distinctive subtypes of the disorder with different genetic etiologies. We used the data provided to the participants in the Genetic Analysis Workshop 15 to evaluate and describe clinically based subgroups and their genetic associations with single-nucleotide polymorphisms (SNPs) on chromosome 6, which harbors the HLA region. Detailed two- and three-SNP haplotype analyses were conducted in the HLA region. We used demographic, clinical self-report, and biomarker data from the entire sample (n = 8477) to identify and characterize the subgroups. We did not use the RA diagnosis itself in the identification of the subgroups. Nuclear families (715 families, 1998 individuals) were used to examine the genetic association with the HLA region. We found five distinct subgroups in the data. The first comprised unaffected family members. Cluster 2 was a mix of affected and unaffected in which patients endorsed symptoms not corroborated by physicians. Clusters 3 through 5 represented a severity continuum in RA. Cluster 5 was characterized by early onset severe disease. Cluster 2 showed no association on chromosome 6. Clusters 3 through 5 showed association with 17 SNPs on chromosome 6. In the HLA region, Cluster 3 showed single-, two-, and three-SNP association with the centromeric side of the region in an area of linkage disequilibrium. Cluster 5 showed both single- and two-SNP association with the telomeric side of the region in a second area of linkage disequilibrium. It will be important to replicate the subgroup structure and the association findings in an independent sample.