Telomere-to-telomere assembly of a complete human X chromosome.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

First insight into the somatic mutation burden of neurofibromatosis type 2-associated grade I and grade II meningiomas: a case report comprehensive genomic study of two cranial meningiomas with vastly different clinical presentation.

BACKGROUND: Neurofibromatosis type 2 (NF2) is a rare autosomal dominant nervous system tumor predisposition disorder caused by constitutive inactivation of one of the two copies of NF2. Meningiomas affect about one half of NF2 patients, and are associated with a higher disease burden. Currently, the somatic mutation landscape in NF2-associated meningiomas remains largely unexamined. CASE PRESENTATION: Here, we present an in-depth genomic study of benign and atypical meningiomas, both from a single NF2 patient. While the grade I tumor was asymptomatic, the grade II tumor exhibited an unusually high growth rate: expanding to 335 times its initial volume within one year. The genomes of both tumors were examined by whole-exome sequencing (WES) complemented with spectral karyotyping (SKY) and SNP-array copy-number analyses. To better understand the clonal composition of the atypical meningioma, the tumor was divided in four sections and each section was investigated independently. Both tumors had second copy inactivation of NF2, confirming the central role of the gene in meningioma formation. The genome of the benign tumor closely resembled that of a normal diploid cell and had only one other deleterious mutation (EPHB3). In contrast, the chromosomal architecture of the grade II tumor was highly re-arranged, yet uniform among all analyzed fragments, implying that this large and fast growing tumor was composed of relatively few clones. Besides multiple gains and losses, the grade II meningioma harbored numerous chromosomal translocations. WES analysis of the atypical tumor identified deleterious mutations in two genes: ADAMTSL3 and CAPN5 in all fragments, indicating that the mutations were present in the cell undergoing fast clonal expansion CONCLUSIONS: This is the first WES study of NF2-associated meningiomas. Besides second NF2 copy inactivation, we found low somatic burden in both tumors and high level of genomic instability in the atypical meningioma. Genomic instability resulting in altered gene dosage and compromised structural integrity of multiple genes may be the primary reason of the high growth rate for the grade II tumor. Further study of ADAMTSL3 and CAPN5 may lead to elucidation of their molecular implications in meningioma pathogenesis.

A new glucocerebrosidase-deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease.

Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1 Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1 To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba(-/-) mice and the control littermate (gba(+/+)) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba(-/-) neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba(+/+) neurons. This null allele gba(-/-) mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies.

Juvenile myelomonocytic leukemia due to a germline CBL Y371C mutation: 35-year follow-up of a large family.

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that arises from malignant transformation of the stem cell compartment and results in increased production of myeloid cells. Somatic and germline variants in CBL (Casitas B-lineage lymphoma proto-oncogene) have been associated with JMML. We report an incompletely penetrant CBL Y371C mutation discovered by whole-exome sequencing in three individuals with JMML in a large pedigree with 35 years of follow-up. The Y371 residue is highly evolutionarily conserved among CBL orthologs and paralogs. In silico bioinformatics prediction programs suggested that the Y371C mutation is highly deleterious. Protein structural modeling revealed that the Y371C mutation abrogated the ability of the CBL protein to adopt a conformation that is required for ubiquitination. Clinically, the three mutation-positive JMML individuals exhibited variable clinical courses; in two out of three, primary hematologic abnormalities persisted into adulthood with minimal clinical symptoms. The penetrance of the CBL Y371C mutation was 30% for JMML and 40% for all leukemia. Of the 8 mutation carriers in the family with available photographs, only one had significant dysmorphic features; we found no evidence of a clinical phenotype consistent with a "CBL syndrome". Although CBL Y371C has been previously reported in familial JMML, we are the first group to follow a complete pedigree harboring this mutation for an extended period, revealing additional information about this variant's penetrance, function and natural history.

Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) support the growth and differentiation of normal hematopoietic stem cells (HSCs). Here we studied the ability of MSCs to support the growth and survival of leukemic stem cells (LSCs) in vitro. Primary leukemic blasts isolated from the peripheral blood of 8 patients with acute myeloid leukemia (AML) were co-cultured with equal numbers of irradiated MSCs derived from unrelated donor bone marrow, with or without cytokines for up to 6weeks. Four samples showed CD34(+)CD38(-) predominance, and four were predominantly CD34(+)CD38(+). CD34(+) CD38(-) predominant leukemia cells maintained the CD34(+) CD38(-) phenotype and were viable for 6weeks when co-cultured with MSCs compared to co-cultures with cytokines or medium only, which showed rapid differentiation and loss of the LSC phenotype. In contrast, CD34(+) CD38(+) predominant leukemic cells maintained the CD34(+)CD38(+) phenotype when co-cultured with MSCs alone, but no culture conditions supported survival beyond 4weeks. Cell cycle analysis showed that MSCs maintained a higher proportion of CD34(+) blasts in G0 than leukemic cells cultured with cytokines. AML blasts maintained in culture with MSCs for up to 6weeks engrafted NSG mice with the same efficiency as their non-cultured counterparts, and the original karyotype persisted after co-culture. Chemosensitivity and transwell assays suggest that MSCs provide pro-survival benefits to leukemic blasts through cell-cell contact. We conclude that MSCs support long-term maintenance of LSCs in vitro. This simple and inexpensive approach will facilitate basic investigation of LSCs and enable screening of novel therapeutic agents targeting LSCs.

Cellular calibrators to quantitate T-cell receptor excision circles (TRECs) in clinical samples.

T-cell receptor excision circles (TRECs) are circular DNA molecules formed during rearrangement of the T-cell receptor (TCR) genes during lymphocyte development. Copy number of the junctional portion of the δRec-ψJα TREC, assessed by quantitative PCR (qPCR) using DNA from dried blood spots (DBS), is a biomarker for newly formed T cells and absent or low numbers of TRECs indicate SCID (severe combined immunodeficiency) or T lymphocytopenia. No quantitation standard for TRECs exists. To permit comparison of TREC qPCR results with a reliable method for counting TRECs across different laboratories, we sought to construct a stable cell line containing a normal human chromosomal constitution and a single copy of the TREC junction sequence. A human EBV (Epstein Barr virus)-transformed B-cell line was transduced with a lentivirus encoding mCherry fluorescence, puromycin resistance and the δRec-ψJα TREC sequence. A TREC-EBV cell line, with each cell carrying a single lentiviral insertion was established, expanded and shown to have one TREC copy per diploid genome. Graded numbers of TREC-EBV cells added to aliquots of T lymphocyte depleted blood showed TREC copy number proportional to TREC-EBV cell number. TREC-EBV cells, therefore, constitute a reproducible cellular calibrator for TREC assays, useful for both population-based screening for severe combined immunodeficiency and evaluation of naïve T-cell production in clinical settings.

Transcription factor zinc finger and BTB domain 1 is essential for lymphocyte development.

Absent T lymphocytes were unexpectedly found in homozygotes of a transgenic mouse from an unrelated project. T cell development did not progress beyond double-negative stage 1 thymocytes, resulting in a hypocellular, vestigial thymus. B cells were present, but NK cell number and B cell isotype switching were reduced. Transplantation of wild-type hematopoietic cells corrected the defect, which was traced to a deletion involving five contiguous genes at the transgene insertion site on chromosome 12C3. Complementation using bacterial artificial chromosome transgenesis implicated zinc finger BTB-POZ domain protein 1 (Zbtb1) in the immunodeficiency, confirming its role in T cell development and suggesting involvement in B and NK cell differentiation. Targeted disruption of Zbtb1 recapitulated the T(-)B(+)NK(-) SCID phenotype of the original transgenic animal. Knockouts for Zbtb1 had expanded populations of bone marrow hematopoietic stem cells and also multipotent and early lymphoid lineages, suggesting a differentiation bottleneck for common lymphoid progenitors. Expression of mRNA encoding Zbtb1, a predicted transcription repressor, was greatest in hematopoietic stem cells, thymocytes, and pre-B cells, highlighting its essential role in lymphoid development.

Mitotic recombination of chromosome arm 17q as a cause of loss of heterozygosity of NF1 in neurofibromatosis type 1-associated glomus tumors.

Neurofibromatosis type 1 (NF1) is a common, autosomal dominant, tumor-predisposition syndrome that arises secondary to mutations in NF1. Glomus tumors are painful benign tumors that originate from the glomus body in the fingers and toes due to biallelic inactivation of NF1. We karyotyped cultures from four previously reported and one new glomus tumor and hybridized tumor (and matching germline) DNA on Illumina HumanOmni1-Quad SNP arrays (≈ 1 × 10(6) SNPs). Two tumors displayed evidence of copy-neutral loss of heterozygosity of chromosome arm 17q not observed in the germline sample, consistent with a mitotic recombination event. One of these two tumors, NF1-G12, featured extreme polyploidy (near-tetraploidy, near-hexaploidy, or near-septaploidy) across all chromosomes. In the remaining four tumors, there were few cytogenetic abnormalities observed, and copy-number analysis was consistent with diploidy in all chromosomes. This is the first study of glomus tumors cytogenetics, to our knowledge, and the first to report biallelic inactivation of NF1 secondary to mitotic recombination of chromosome arm 17q in multiple NF1-associated glomus tumors. We have observed mitotic recombination in 22% of molecularly characterized NF1-associated glomus tumors, suggesting that it is a not uncommon mechanism in the reduction to homozygosity of the NF1 germline mutation in these tumors. In tumor NF1-G12, we hypothesize that mitotic recombination also "unmasked" (reduced to homozygosity) a hypomorphic germline allele in a gene on chromosome arm 17q associated with chromosomal instability, resulting in the extreme polyploidy.

Myelodysplasia in autosomal dominant and sporadic monocytopenia immunodeficiency syndrome: diagnostic features and clinical implications.

A novel, genetic immunodeficiency syndrome has been recently described, herein termed "MonoMAC". It is characterized by severe circulating monocytopenia, NK- and B-lymphocytopenia, severe infections with M. avium complex (MAC), and risk of progression to myelodysplasia/acute myelogenous leukemia. Detailed bone marrow analyses performed on 18 patients further define this disorder. The majority of patients had hypocellular marrows with reticulin fibrosis and multilineage dysplasia affecting the myeloid (72%), erythroid (83%) and megakaryocytic (100%) lineages. Cytogenetic abnormalities were present in 10 of 17 (59%). Despite B-lymphocytopenia, plasma cells were present but were abnormal (e.g. CD56(+)) in nearly half of cases. Increased T-cell large granular lymphocyte populations were present in 28% of patients. Chromosomal breakage studies, cell cycle checkpoint functions, and sequencing of TERT and K-RAS genes revealed no abnormalities. MonoMAC appears to be a unique, inherited syndrome of bone marrow failure. We describe distinctive bone marrow features to help in its recognition and diagnosis. (Clinicaltrials.gov identifiers: NCT00018044, NCT00923364, NCT01212055).

DNA damage responses by human ELG1 in S phase are important to maintain genomic integrity.

Genomic integrity depends on DNA replication, recombination and repair, particularly in S phase. We demonstrate that a human homologue of yeast Elg1 plays an important role in S phase to preserve genomic stability. The level of ELG1 is induced during recovery from a variety of DNA damage. In response to DNA damage, ELG1 forms distinct foci at stalled DNA replication forks that are different from DNA double strand break foci. Targeted gene knockdown of ELG1 resulted in spontaneous foci formation of gamma-H2AX, 53BP1 and phosphorylated-ATM that mark chromosomal breaks. Abnormal chromosomes including fusions, inversions and hypersensitivity to DNA damaging agents were also observed in cells expressing low level of ELG1 by targeted gene knockdown. Knockdown of ELG1 by siRNA reduced homologous recombination frequency in the I-SceI induced double strand break-dependent assay. In contrast, spontaneous homologous recombination frequency and sister chromatin exchange rate were upregulated when ELG1 was silenced by shRNA. Taken together, we propose that ELG1 would be a new member of proteins involved in maintenance of genomic integrity.

EGFRvIII expression and PTEN loss synergistically induce chromosomal instability and glial tumors.

Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor, but it is not known if these two genetic lesions act together to transform cells. To answer this question, we infected PTEN-/- neural precursor cells with a retrovirus encoding EGFRvIII, which is a constitutively activated receptor. EGFRvIII PTEN-/- cells formed highly mitotic tumors with nuclear pleomorphism, necrotic areas, and glioblastoma markers. The transformed cells showed increased cell proliferation, centrosome amplification, colony formation in soft agar, self-renewal, expression of the stem cell marker CD133, and resistance to oxidative stress and ionizing radiation. The RAS/mitogen-activated protein kinase (ERK) and phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathways were activated, and checkpoint kinase 1 (Chk1), the DNA damage regulator, was phosphorylated at S280 by Akt, suppressing Chk1 phosphorylation at S345 in response to ionizing irradiation. The PTEN-/- cells showed low levels of DNA damage in the absence of irradiation, which was increased by EGFRvIII expression. Finally, secondary changes occurred during tumor growth in mice. Cells from these tumors showed decreased tumor latencies and additional chromosomal aberrations. Most of these tumor lines showed translocations of mouse chromosome 15. Intracranial injections of one of these lines led to invasive, glial fibrillary acidic protein-positive, nestin-positive tumors. These results provide a molecular basis for the occurrence of these two genetic lesions in brain tumors and point to a role in induction of genomic instability.

Genetics of carney triad: recurrent losses at chromosome 1 but lack of germline mutations in genes associated with paragangliomas and gastrointestinal stromal tumors.

CONTEXT: Carney triad (CT) describes the association of paragangliomas (PGLs) with gastrointestinal stromal tumors (GISTs) and pulmonary chondromas. Inactivating mutations of the mitochondrial complex II succinate dehydrogenase (SDH) enzyme subunits SDHB, SDHC, and SDHD are found in PGLs, gain-of-function mutations of c-kit (KIT), and platelet-derived growth factor receptor A (PDGFRA) in GISTs. OBJECTIVE: Our objective was to investigate the possibility that patients with CT and/or their tumors may harbor mutations of the SDHB, SDHC, SDHD, KIT, and PDGFRA genes and identify any other genetic alterations in CT tumors. DESIGN: Three males and 34 females with CT were studied retrospectively. We sequenced the stated genes and performed comparative genomic hybridization on a total of 41 tumors. RESULTS: No patient had coding sequence mutations of the investigated genes. Comparative genomic hybridization revealed a number of DNA copy number changes: losses dominated among benign lesions, there were an equal number of gains and losses in malignant lesions, and the average number of alterations in malignant tumors was higher compared with benign lesions. The most frequent and greatest contiguous change was 1q12-q21 deletion, a region that harbors the SDHC gene. Another frequent change was loss of 1p. Allelic losses of 1p and 1q were confirmed by fluorescent in situ hybridization and loss-of-heterozygosity studies. CONCLUSIONS: We conclude that CT is not due to SDH-inactivating or KIT- and PDGFRA-activating mutations. GISTs and PGLs in CT are associated with chromosome 1 and other changes that appear to participate in tumor progression and point to their common genetic cause.

Chromosome 8 BAC array comparative genomic hybridization and expression analysis identify amplification and overexpression of TRMT12 in breast cancer.

Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors. To fine map such genomic changes by comparative genomic hybridization (CGH), a high resolution (100 kb) chromosome 8 array that can detect single copy changes was developed using Phi29 DNA polymerase amplified BAC (bacterial artificial chromosome) DNA. The BAC array CGH resolved the two known amplified regions (8q21 and 8q24) of a breast cancer cell line (SKBR3) into nine separate regions including six amplicons and three deleted regions, all of which were verified by Fluorescence in situ hybridization. The extent of the gain/loss for each region was validated by qPCR. CGH was performed with a total of 8 breast cancer cell lines, and common regions of genomic amplification/deletion were identified by segmentation analysis. A 1.2-Mb region (125.3-126.5 Mb) and a 1.0-Mb region (128.1-129.1 Mb) in 8q24 were amplified in 7/8 cell lines. A global expression analysis was performed to evaluate expression changes associated with genomic amplification/deletion: a novel gene, TRMT12 (at 125.5 Mb), amplified in 7/8 cell lines, showed highest expression in these cell lines. Further analysis by RT-qPCR using RNA from 30 breast tumors showed that TRMT12 was overexpressed >2 fold in 87% (26/30) of the tumors. TRMT12 is a homologue of a yeast gene encoding a tRNA methyltransferase involved in the posttranscriptional modification of tRNA(Phe), and exploring the biological consequence of its altered expression, may reveal novel pathways in tumorigenesis. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Acinar cell apoptosis in Serpini2-deficient mice models pancreatic insufficiency.

Pancreatic insufficiency (PI) when left untreated results in a state of malnutrition due to an inability to absorb nutrients. Frequently, PI is diagnosed as part of a larger clinical presentation in cystic fibrosis or Shwachman-Diamond syndrome. In this study, a mouse model for isolated exocrine PI was identified in a mouse line generated by a transgene insertion. The trait is inherited in an autosomal recessive pattern, and homozygous animals are growth retarded, have abnormal immunity, and have reduced life span. Mice with the disease locus, named pequeño (pq), exhibit progressive apoptosis of pancreatic acinar cells with severe exocrine acinar cell loss by 8 wk of age, while the islets and ductal tissue persist. The mutation in pq/pq mice results from a random transgene insertion. Molecular characterization of the transgene insertion site by fluorescent in situ hybridization and genomic deletion mapping identified an approximately 210-kb deletion on Chromosome 3, deleting two genes. One of these genes, Serpini2, encodes a protein that is a member of the serpin family of protease inhibitors. Reintroduction of only the Serpini2 gene by bacterial artificial chromosome transgenic complementation corrected the acinar cell defect as well as body weight and immune phenotypes, showing that deletion of Serpini2 causes the pequeño phenotype. Dietary supplementation of pancreatic enzymes also corrected body size, body weight, and immunodeficiency, and increased the life span of Serpini2-deficient mice, despite continued acinar cell loss. To our knowledge, this study describes the first characterized genetic animal model for isolated PI. Genetic complementation of the transgene insertion mutant demonstrates that Serpini2 deficiency directly results in the acinar cell apoptosis, malabsorption, and malnutrition observed in pq/pq mice. The rescue of growth retardation, immunodeficiency, and mortality by either Serpini2 bacterial artificial chromosome transgenic expression or by pancreatic enzyme supplementation demonstrates that these phenotypes are secondary to malnutrition in pq/pq mice.

Common genetic changes in hereditary and sporadic pituitary adenomas detected by comparative genomic hybridization.

Twenty-four pituitary adenomas, both the sporadic type (n = 18) and the type arising in association with either multiple endocrine neoplasia, type 1 (MEN1; n = 2), or Carney complex (CNC, n = 4) were analyzed by comparative genomic hybridization. Twenty-one (88%) tumors displayed chromosomal alterations. The number of chromosomal aberrations in each tumor varied from 2 to greater than 10. Several recurrent chromosomal abnormalities were identified in this study. The most frequently detected losses of chromosomal material involved 1p (14 of 24, 58%), 11p (11 of 24, 46%), 17 (10 of 24, 42%), 16p (9 of 24, 38%), 4 (8 of 24, 33%), 10p (6 of 24, 25%), 12 (6 of 24, 25%), 20 (6 of 24, 25%), 22q (6 of 24, 25%), 13q (5 of 24, 21%), and 9p (4 of 24, 17%). Copy number increases were detected on 4q (7 of 24, 29%), 17 (8 of 24, 33%), 19 (7 of 24, 29%), 1p (6 of 24, 25%), 5 (6 of 24, 25%), 20 (6 of 24, 25%), 6q (5 of 24, 21%), 13q21-qter (5 of 24, 21%), and 16p (5 of 24, 21%). Chromosome 11 loss, which involved 11p in all cases, was the most significant finding and was common to tumors arising sporadically and in association with MEN1 and CNC. In addition, the majority of the tumors (18 of 24, 75% overall and 86% of all tumors with chromosomal abnormalities) showed involvement of chromosome 1. Tumors had either loss (14 of 24, 58%) or gain (6 of 24, 25%) in the 1p32-1pter region. Finally, changes on chromosome 17, either loss or gain, occurred in 71% (17) of all 24 patients. In summary, all the tumors with chromosomal rearrangements (21 of 24, 88%), whether sporadic pituitary adenomas or those associated with MEN1 or CNC, had alteration(s) of 1p32, 11p, or 17.

Differentiation of human bone marrow-derived cells into buccal epithelial cells in vivo: a molecular analytical study.

BACKGROUND: Adult bone marrow-derived (BMD) cells could be used to repair damaged organs and tissues, but the intrinsic plasticity of these cells has been questioned by results of in-vitro studies suggesting that such cells might fuse with other cells giving the appearance of differentiation. We aimed to determine whether fusion events are important in vivo. METHODS: To test whether BMD cells can colonise an epithelial tissue and differentiate there without fusion, we did in-situ hybridisation with Y and X chromosome probes labelled with 35-sulphur or digoxigenin, or labelled fluorescently. We did immunohistochemistry with anticytokeratin 13 along with fluorescence in-situ hybridisation to identify Y-chromosome positive buccal epithelial cells in cheek scrapings obtained from five females who had received either a bone-marrow transplant or an allogeneic mobilised peripheral-blood progenitor-cell transplant (enriched in CD34+ cells) from male donors. FINDINGS: When examined 4-6 years after male-to-female marrow-cell transplantation, all female recipients had Y-chromosome-positive buccal cells (0.8-12.7%). In more than 9700 cells studied, we detected only one XXXY-positive cell (0.01%) and one XXY cell (0.01%), both of which could have arisen when an XY cell fused with an XX cell. INTERPRETATION: Male BMD cells migrate into the cheek and differentiate into epithelial cells, an occurrence that does not depend on fusion of BMD cells to recipient cells. This finding might be an example of transdifferentiation of haemopoietic or stromal progenitor cells. Plasticity of BMD cells could be useful in regenerative medicine.

A genomic map of a 6-Mb region at 13q21-q22 implicated in cancer development: identification and characterization of candidate genes.

Chromosomal region 13q21-q22 harbors a putative breast cancer susceptibility gene and has been implicated as a common site for somatic deletions in a variety of malignant tumors. We have built a complete physical clone contig for a region between D13S1308 and AFM220YE9 based on 18 yeast artificial chromosome and 81 bacterial artificial chromosome (BAC) clones linked together by 22 genetic markers and 61 other sequence tagged sites. Combining data from 47 sequenced BACs (as of June 2001), we have assembled in silico an integrated 5.7-Mb genomic map with 90% sequence coverage. This area contains eight known genes, two hypothetical proteins, 24 additional Unigene clusters, and approximately 100 predicted genes and exons. We have determined the cDNA and genomic sequence, and tissue expression profiles for the KIAA1008 protein (homologous to the yeast mitotic control protein dis3+), KLF12 (AP-2 repressor), progesterone induced blocking factor 1, zinc finger transcription factor KLF5, and LIM domain only-7, and for the hypothetical proteins FLJ22624 and FLJ21869. Mutation screening of the five known genes in 19 breast cancer families has revealed numerous polymorphisms, but no deleterious mutations. These data provide a basis and resources for further analyses of this chromosomal region in the development of cancer.