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High levels of interstrand cross-link damage in mammalian cells cause chromatid breaks and radial formations recognizable by cytogenetic examination. The mechanism of radial formation observed following DNA damage has yet to be determined. Due to recent findings linking homologous recombination and non-homologous end-joining to the action of the Fanconi anemia pathway, we speculated that radials might be the result of defects in either of the pathways of DNA repair. To test this hypothesis, we have investigated the role of homologous recombination proteins RAD51 and RAD52, non-homologous end-joining proteins Ku70 and LIG4, and protein MRE11 in radial formation and cell survival following interstrand crosslink damage with mitomycin C. For the studies we used small inhibitory RNA to deplete the proteins from cells, allowing for evaluation of radial formation and cell survival. In transformed normal human fibroblasts, depletion of these proteins increased interstrand crosslink sensitivity as manifested by decreased cell survival and increased radial formation. These results demonstrate that inactivation of proteins from either of the two separate DNA repair pathways increases cellular sensitivity to interstrand crosslinks, indicating each pathway plays a role in the normal response to interstrand crosslink damage. We can also conclude that homologous recombination or non-homologous end-joining are not required for radial formation, since radials occur with depletion of these pathways. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Context.—: Genomic reports are primarily organized in a narrative and unstructured format with variations in content and format. Regulatory requirements and professional guidelines for genetic test reporting exist but provide little guidance for effective communication of information., Objective.—: To assess clinical genomic reporting practices across 5 disciplines within molecular diagnostics, including germline, somatic solid tumors, somatic hematologic malignancies, pharmacogenomics, and prenatal cell-free DNA screening., Design.—: Reporting practices were assessed by using a structured review of clinical genomic reports from multiple laboratories in 5 molecular disciplines spanning different practice settings. Report content was reviewed by the presence/absence of from 27 to 44 elements, including 23 elements required by the College of American Pathologists and/or the Clinical Laboratory Improvement Amendments of 1988 (CLIA). If present, the element's location on the report was recorded., Results.—: A total of 69 genomics reports from 31 laboratories were reviewed. Overall, the reports were compliant with regulatory requirements but showed variability in both format and content. Six of 7 required reporting elements (per CLIA, 42 CFR [Code of Federal Regulations] 493.1291) were included in 90% of the reports. However, these elements were often located in different report sections. Only patient demographics were always found in a specific report section (header)., Conclusions.—: These results show that reports are overall compliant with regulatory requirements, despite some reporting elements being less consistently reported. The lack of consistent presentation of the data elements presents an opportunity to improve the communication of molecular testing results to clinicians and patients., (© 2024 College of American Pathologists.)

Here we describe a neonate exhibiting hypotonia, macrocephaly, renal cysts, and respiratory failure requiring tracheostomy and ventilator support. Genetic analysis via rapid genome sequencing (rGS) identified a loss on chromosome 4 encompassing polycystin-2 (PKD2) and a loss on chromosome 22 encompassing SH3 and Multiple Ankyrin Repeat Domains 3 (SHANK3), indicative of Phelan-McDermid syndrome. Further analysis via traditional karyotyping, Optical Genome Mapping (OGM), and PacBio long-read sequencing revealed a more complex landscape of chromosomal rearrangements in this individual, including a balanced 3;12 translocation, and an unbalanced 17;22 translocation. The proband's phenotypic presentation is thought to be the result of Phelan-McDermid syndrome and represents an expansion of the described phenotypes to include significant respiratory failure. This study underscores the challenges and importance of comprehensive genetic testing in elucidating complex presentations and highlights the need for complementary testing methods to overcome limitations in resolution., (© 2024 The Author(s). American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Background: Chromosomal microarray (CMA) is commonly utilized in the obstetrics setting. CMA is recommended when one or more fetal structural abnormalities is identified. CMA is also commonly used to determine genetic etiologies for miscarriages, fetal demise, and confirming positive prenatal cell-free DNA screening results., Methods: In this study, we retrospectively examined 523 prenatal and 319 products-of-conception (POC) CMA cases tested at Nationwide Children's Hospital from 2011 to 2020. We reviewed the referral indications, the diagnostic yield, and the reported copy number variants (CNV) findings., Results: In our cohort, the diagnostic yield of clinically significant CNV findings for prenatal testing was 7.8% (n = 41/523) compared to POC testing (16.3%, n = 52/319). Abnormal ultrasound findings were the most common indication present in 81% of prenatal samples. Intrauterine fetal demise was the common indication identified in POC samples. The most common pathogenic finding observed in all samples was isolated trisomy 21, detected in seven samples., Conclusion: Our CMA study supports the clinical utility of prenatal CMA for clinical management and identifying genetic etiology in POC arrays. In addition, it provides insight to the spectrum of prenatal and POC CMA results as detected in an academic hospital clinical laboratory setting that serves as a reference laboratory., (© 2023 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Background: Throughout history, the field of cytogenetics has witnessed significant changes due to the constant evolution of technologies used to assess chromosome number and structure. Similar to the evolution of single nucleotide variant detection from Sanger sequencing to next-generation sequencing, the identification of chromosome alterations has progressed from banding to fluorescence in situ hybridization (FISH) to chromosomal microarrays. More recently, emerging technologies such as optical genome mapping and genome sequencing have made noteworthy contributions to clinical laboratory testing in the field of cytogenetics., Content: In this review, we journey through some of the most pivotal discoveries that have shaped the development of clinical cytogenetics testing. We also explore the current test offerings, their uses and limitations, and future directions in technology advancements., Summary: Cytogenetics methods, including banding and targeted assessments like FISH, continue to hold crucial roles in cytogenetic testing. These methods offer a rapid turnaround time, especially for conditions with a known etiology involving recognized cytogenetic aberrations. Additionally, laboratories have the flexibility to now employ higher-throughput methodologies to enhance resolution for cases with greater complexity., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Somatic mosaicism is a known cause of neurological disorders, including developmental brain malformations and epilepsy. Brain mosaicism is traditionally attributed to post-zygotic genetic alterations arising in fetal development. Here we describe post-zygotic rescue of meiotic errors as an alternate origin of brain mosaicism in patients with focal epilepsy who have mosaic chromosome 1q copy number gains. Genomic analysis showed evidence of an extra parentally derived chromosome 1q allele in the resected brain tissue from five of six patients. This copy number gain is observed only in patient brain tissue, but not in blood or buccal cells, and is strongly enriched in astrocytes. Astrocytes carrying chromosome 1q gains exhibit distinct gene expression signatures and hyaline inclusions, supporting a novel genetic association for astrocytic inclusions in epilepsy. Further, these data demonstrate an alternate mechanism of brain chromosomal mosaicism, with parentally derived copy number gain isolated to brain, reflecting rescue in other tissues during development., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Detection of hallmark genomic aberrations in acute myeloid leukemia (AML) is essential for diagnostic subtyping, prognosis, and patient management. However, cytogenetic/cytogenomic techniques used to identify those aberrations, such as karyotyping, fluorescence in situ hybridization (FISH), or chromosomal microarray analysis (CMA), are limited by the need for skilled personnel as well as significant time, cost, and labor. Optical genome mapping (OGM) provides a single, cost-effective assay with a significantly higher resolution than karyotyping and with a comprehensive genome-wide analysis comparable with CMA and the added unique ability to detect balanced structural variants (SVs). Here, we report in a real-world setting the performance of OGM in a cohort of 100 AML cases that were previously characterized by karyotype alone or karyotype and FISH or CMA. OGM identified all clinically relevant SVs and copy number variants (CNVs) reported by these standard cytogenetic methods when representative clones were present in >5% allelic fraction. Importantly, OGM identified clinically relevant information in 13% of cases that had been missed by the routine methods. Three cases reported with normal karyotypes were shown to have cryptic translocations involving gene fusions. In 4% of cases, OGM findings would have altered recommended clinical management, and in an additional 8% of cases, OGM would have rendered the cases potentially eligible for clinical trials. The results from this multi-institutional study indicate that OGM effectively recovers clinically relevant SVs and CNVs found by standard-of-care methods and reveals additional SVs that are not reported. Furthermore, OGM minimizes the need for labor-intensive multiple cytogenetic tests while concomitantly maximizing diagnostic detection through a standardized workflow., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Purpose: Workforce shortages are observed in many sectors of the economy, including clinical genomics laboratories. Although medical technologists are essential for the primary functions of laboratory operations and many institutions in the United States have reported acute staff shortages, we are unaware of any recent studies that provide concrete data detailing workforce needs. In this report, we summarize the results of a technologist-based survey sent to clinical laboratory directors across the United States., Methods: The survey was designed to provide detailed and objective evidence on the current landscape of the technologist workforce in clinical cytogenetics, molecular genetics, and laboratories that have combined both disciplines. Survey questions included demographics, salaries across career stages, retention trends, and hiring requirements and challenges., Results: Analysis of the survey data from 70 US-based submissions showed that cytogenetics laboratories had higher proportion of unfilled technologist positions, whereas molecular laboratories had more applications for each open positions. The technologist retention rate in molecular laboratories was higher than that in cytogenetics. The lack of adequately trained applicants and competitive salary offers by other laboratories were cited as top barriers for filling technologist positions., Conclusion: The results from this survey will serve as normative data in generating solutions to address acute workforce needs in the United States., Competing Interests: Y.A. and M.L.L. serve as directors in a clinical laboratory that performs an extensive menu of genetic and genomic testing on a fee-for-service basis. R.G.B. is affiliated with LabCorp. S.D. declares no conflicts of interest., (© 2023 The Authors.)

Chromosomal aberrations are among the most important prognostic parameters in AML, and conventional cytogenetic analysis remains essential for risk stratification. In this report, we describe an adult male patient with a high percentage of circulating blasts, pathologically confirmed as AML with maturation. Cytogenetic analysis of a bone marrow sample revealed heptasomy 21 and trisomy 13 within a complex karyotype of 52,XY,der(2)t(2;13)(q33.3;q32.1),+13,+21,+21,+21,+21,+21 in all 20 cells examined, which was confirmed by metaphase FISH. Chromosomal microarray analysis (CMA) revealed complete loss of heterozygosity (LOH) of chromosome 21, supporting a common origin. In addition, LOH of chromosome 1p, trisomy 13, and partial tetrasomy of 13q and partial monosomy of 2q as a result of an unbalanced translocation between chromosomes 2 and 13 were observed. Molecular analysis identified two pathogenic missense variants: RUNX1 p.D198Y and SRSF2 p.P95R. The clonal allele ratio of RUNX1 p.D198Y was consistent with all copies of chromosome 21 in the leukemic clone carrying the mutation. Within the medical literature, there are no reports of heptasomy 21 for comparison; however, there are reports of AML with either polysomy 21 or trisomy 13. Our results suggest that even relatively 'common' AML aneuploidies may be associated with much more complex genomic changes, including loss of heterozygosity, which impact prognosis., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The upcoming 5th edition of the World Health Organization (WHO) Classification of Haematolymphoid Tumours is part of an effort to hierarchically catalogue human cancers arising in various organ systems within a single relational database. This paper summarizes the new WHO classification scheme for myeloid and histiocytic/dendritic neoplasms and provides an overview of the principles and rationale underpinning changes from the prior edition. The definition and diagnosis of disease types continues to be based on multiple clinicopathologic parameters, but with refinement of diagnostic criteria and emphasis on therapeutically and/or prognostically actionable biomarkers. While a genetic basis for defining diseases is sought where possible, the classification strives to keep practical worldwide applicability in perspective. The result is an enhanced, contemporary, evidence-based classification of myeloid and histiocytic/dendritic neoplasms, rooted in molecular biology and an organizational structure that permits future scalability as new discoveries continue to inexorably inform future editions., (© 2022. The Author(s).)

Chromosomal rearrangements of the NTRK genes generate kinase fusions that are targetable oncogenic drivers in diverse adult and pediatric malignancies. Despite robust clinical response to targeted NTRK inhibition, the emergence of therapeutic resistance poses a formidable clinical challenge. Here we report the characterization of an ETV6-NTRK3 fusion-driven pediatric glioma that progressed through NTRK-targeted treatments with entrectinib and selitrectinib. Genetic analysis of multifocal recurrent/resistant lesions identified a previously uncharacterized NTRK3 p.G623A and a known p.G623E resistance mutation, in addition to other alterations of potential pathogenic impact. Functional studies using heterologous reconstitution model systems and patient-derived tumor cell lines establish that NTRK3 G623A and NTRK3 G623E mutated kinases exhibit reduced sensitivity to entrectinib and selitrectinib, as well as other NTRK inhibitors tested herein. In summary, this genetic analysis of multifocal recurrent/resistant glioma driven by ETV6-NTRK3 fusion captured a cross section of resistance-associated alterations that, based on in vitro analysis, likely contributed to resistance to targeted therapy and disease progression., (© 2021 Keddy et al.; Published by Cold Spring Harbor Laboratory Press.)

Chromosomal microarray technologies, including array comparative genomic hybridization and single-nucleotide polymorphism array, are widely applied in the diagnostic evaluation for both constitutional and neoplastic disorders. In a constitutional setting, this technology is accepted as the first-tier test for the evaluation of chromosomal imbalances associated with intellectual disability, autism, and/or multiple congenital anomalies. Furthermore, chromosomal microarray analysis is recommended for patients undergoing invasive prenatal diagnosis with one or more major fetal structural abnormalities identified by ultrasonographic examination, and in the evaluation of intrauterine fetal demise or stillbirth when further cytogenetic analysis is desired. This technology also provides important genomic data in the diagnosis, prognosis, and therapy of neoplastic disorders, including both hematologic malignancies and solid tumors. To assist clinical laboratories in the validation of chromosomal microarray methodologies for constitutional and neoplastic applications, the American College of Medical Genetics and Genomics (ACMG) Laboratory Quality Assurance Committee has developed these updated technical laboratory standards, which replace the ACMG technical standards and guidelines for microarray analysis in constitutional and neoplastic disorders previously published in 2013., (© 2021. The Author(s), under exclusive licence to the American College of Medical Genetics and Genomics.)

In the current era of personalized medicine, the field of oncology is witnessing a paradigm shift in patient care that is driving a tighter integration of genomic analysis modalities in patient care decision. This is driven by the expanding category of targeted therapies that require a broader understanding of the mutational profile of patient samples to more precisely guided personalized treatment decisions. Next-generation sequencing (NGS) has proved to be of tremendous power in detecting and characterizing a broad spectrum of activating or loss-of-function mutations across many gene targets. This power of NGS also results in significant challenges related to technical expertise, bioinformatics, computing infrastructure, laboratory practices, and integration into clinical decision-making. These challenges are particularly relevant to smaller and mid-tier hospital networks that are faced with the need to modernize their clinical practices and offer their patients access to advanced genomic technologies to improve outcomes. Adoption of such personalized medicine relies on information about a patient's cancer genome and the identification of its variants. This is best achieved using NGS. However, there are challenges to the adoption of such a complex technology and workflow, especially in smaller hospital systems. This commentary summarizes key considerations and challenges related to implementation of NGS in a community hospital setting., (© 2019 Akkari et al.; Published by Cold Spring Harbor Laboratory Press.)

Advances in sequencing technologies permit the analysis of a larger selection of genes for preconception carrier screening. The study was designed as a sequential carrier screen using genome sequencing to analyze 728 gene-disorder pairs for carrier and medically actionable conditions in 131 women and their partners (n = 71) who were planning a pregnancy. We report here on the clinical laboratory results from this expanded carrier screening program. Variants were filtered and classified using the latest American College of Medical Genetics and Genomics (ACMG) guideline; only pathogenic and likely pathogenic variants were confirmed by orthologous methods before being reported. Novel missense variants were classified as variants of uncertain significance. We reported 304 variants in 202 participants. Twelve carrier couples (12/71 couples tested) were identified for common conditions; eight were carriers for hereditary hemochromatosis. Although both known and novel variants were reported, 48% of all reported variants were missense. For novel splice-site variants, RNA-splicing assays were performed to aid in classification. We reported ten copy-number variants and five variants in non-coding regions. One novel variant was reported in F8, associated with hemophilia A; prenatal testing showed that the male fetus harbored this variant and the neonate suffered a life-threatening hemorrhage which was anticipated and appropriately managed. Moreover, 3% of participants had variants that were medically actionable. Compared with targeted mutation screening, genome sequencing improves the sensitivity of detecting clinically significant variants. While certain novel variant interpretation remains challenging, the ACMG guidelines are useful to classify variants in a healthy population., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

We describe a newborn female with a de novo interstitial deletion of chromosome 21q21.1-22.12 including the RUNX1 gene who had developmental delay, multiple congenital anomalies, tetralogy of Fallot, anemia, and chronic thromobocytopenia requiring frequent platelet transfusions from birth. Because of her physical and hematologic abnormalities, she was tested for Fanconi anemia (FA). Lymphocytes and fibroblasts from this patient demonstrated increased chromosome breakage with exposure to the clastogen mitomycin C, but not, in contrast to most FA patients, to diepoxybutane. Further testing by Western analysis and complementation testing did not show a defect in the function of known Fanconi proteins. Her constitutional deletion was later found to span 13.2 Mb by chromosome microarray analysis, encompassing the RUNX1 gene that has been implicated in thrombocytopenia and predisposition to acute myelogenous leukemia (AML) when in the haploinsufficient state. We compare her phenotype to other individuals with similar 21q deletions and thrombocytopenia, as well as those with FA. We suggest that deletion of RUNX1 or another critical gene within the deleted region may result in chromosomal instability similar to that seen in FA., (Copyright © 2011 Wiley-Liss, Inc.)

The rare genetic disorder Fanconi anemia, caused by a deficiency in any of at least thirteen identified genes, is characterized by cellular sensitivity to DNA interstrand crosslinks and genome instability. The excision repair cross complementing protein, ERCC1, first identified as a participant in nucleotide excision repair, appears to also act in crosslink repair, possibly in incision and at a later stage. We have investigated the relationship of ERCC1 to the Fanconi anemia pathway, using depletion of ERCC1 by siRNA in transformed normal human fibroblasts and fibroblasts from Fanconi anemia patients. We find that depletion of ERCC1 does not hinder formation of double strand breaks in crosslink repair as indexed by gammaH2AX. However, the monoubiquitination of FANCD2 protein in response to MMC treatment is decreased and the localization of FANCD2 to nuclear foci is eliminated. Arrest of DNA replication by hydroxyurea, producing double strand breaks without crosslinks, also requires ERRC1 for FANCD2 localization to nuclear foci. Our results support a role for ERCC1 after creation of a double strand break for full activation of the Fanconi anemia pathway.

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.

Monozygotic twin sisters, with nonhematologic symptoms of Fanconi anemia (FA), were discovered to be somatic mosaics for mutations in the FANCA gene. Skin fibroblasts, but not lymphocytes or committed hematopoietic progenitors, were sensitive to DNA cross-linking agents. Molecular analysis revealed, in skin cells of both twins, a frameshift causing deletion in exon 27 (2555deltaT) and an exon 28 missense mutation (2670G>A/R880Q). The latter resulted in primarily cytoplasmic expression and reduced function of the mutant FANCA (R880Q) protein. Surprisingly, the same acquired exon 30 missense change (2927G>A/E966K) was detected in the hematopoietic cells of both sisters, but not in their fibroblasts, nor in either parent. This compensatory mutation existed in cis with the maternal exon 28 mutation, and it restored function and nuclear localization of the resulting protein. Both sisters have been free of hematologic symptoms for more than 2 decades, suggesting that this de novo mutation occurred prenatally in a single hematopoietic stem cell (HSC) in one twin and that descendants of this functionally corrected HSC, via intra-uterine circulation, repopulated the blood lineages of both sisters. This finding suggests that treating FA patients with gene therapy might require transduction of only a few hematopoietic stem cells.

We have identified three novel FANCC mutations, a truncating single base insertion in exon 4 (c.455_456dupA), a point mutation in exon 13 (c.1390C>T), and a splice site mutation leading to deletion of exon 9, in two Brazilian FA-C patients, each a compound heterozygote. Using complementation analyses, we confirmed that two of these mutations inactivate the function of the FANCC protein., (2006 Wiley-Liss, Inc.)

Repopulation by transplanted cells can result in effective therapy for several regenerative organs including blood, liver, and skin. In contrast, cell therapies for renal diseases are not currently available. Here we developed an animal model in which cells genetically resistant to a toxic intermediate of tyrosine metabolism, homogentisic acid (HGA), were able to repopulate the damaged proximal tubule epithelium of mice with fumarylacetoacetate hydrolase (Fah) deficiency. HGA resistance was achieved by two independent mechanisms. First, Fah+ transplanted bone marrow cells produced significant replacement of damaged proximal tubular epithelium (up to 50%). The majority of bone marrow-derived epithelial cells were generated by cell fusion, not transdifferentiation. In addition to regeneration by fusion-derived epithelial cells, proximal tubular repopulation was also observed by host epithelial cells, which had lost the homogentisic acid dioxygenase gene. These data demonstrate that extensive regeneration of the renal proximal tubule compartment can be achieved through genetic selection of functional cells.

Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.

Fanconi anemia (FA) is a multigenic recessive disease resulting in bone marrow failure and increased cancer susceptibility. Cells from FA patients and mouse models are sensitive to DNA interstrand crosslinks (ICLs) and FA mice are moderately sensitive to ionizing radiation (IR). Both kinds of damage induce DNA double strand breaks (DSBs). To date, nine genes in 11 complementation groups have been identified; however, the precise function of the FA pathway remains unclear. Many of the proteins form a nuclear complex necessary for the mono-ubiquitination of the downstream protein, Fancd2. To further investigate the role of the FA pathway in repair of DSBs, we generated Fancd2(-/-)/Prkdc(sc/sc) double mutant mice. Prkdc(sc/sc) mutant mice have a defect in non-homologous end joining (NHEJ) and are sensitive to IR-induced DNA damage. Double mutant animals and primary cells were more sensitive to IR than either single mutant, suggesting that Fancd2 operates in DSB repair pathway distinct from NHEJ. Fancd2(-/-)/Prkdc(sc/sc) double mutant cells were also more sensitive to DSBs generated by a restriction endonuclease. The role of Fancd2 in DSB repair may account for the moderate sensitivity of FA cells to irradiation and FA cells sensitivity to ICLs that are repaired via a DSB intermediate.

Objective: To present a prenatal diagnosis report on a case where G-banding analysis of fetal metaphase chromosomes showed populations of cells with two different Y chromosomes; one with a short block of heterochromatin (Yqh-) and one with a longer block of heterochromatin (Yqh+)., Methods: These two populations of the Y chromosome were studied using fluorescent quinacrine banding and fluorescent in situ hybridization (FISH). A chromosome paint corresponding to the euchromatic region of the Y chromosome, and probes corresponding to the SRY, DYZ1, and DYZ3 regions were used for this study., Results: Both Y chromosomes appeared to be structurally normal by these analyses. Subsequent ultrasound examination at 20 weeks' gestation revealed normal male genitalia. Follow-up with a neonatal blood sample analysis confirmed the above findings., Conclusions: This study reports a direct prenatal diagnosis case of two populations of the Y chromosome in the same individual. This apparent mosaicism may be explained by a postzygotic simple deletion or unequal crossover event between sister chromatids in the DYZ region.

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure and an increased susceptibility to cancer. FA is genetically heterogeneous, consisting of at least 11 complementation groups, FA-A through L, including FA-D1 (BRCA2) and D2. We have previously reported an increased incidence of epithelial tumors in Fancd2 knockout mice. To further investigate the role of the FA pathway in tumor prevention, Fancd2 mutant mice were crossed to mice with a null mutation in the tumor suppressor gene, Trp53. The tumor spectrum in Fancd2(-/-)/Trp53(+/-) mice included sarcomas expected in Trp53 heterozygotes, as well as mammary and lung adenocarcinomas that occur rarely in Trp53 heterozygotes. These tumors occurred earlier than in Fancd2(-/-) control mice. Therefore, the Fancd2(-/-)/Trp53(+/-) mice represent an improved model for the study of adenocarcinoma in FA. In addition, it was found that Fancd2(-/-) mouse embryonic fibroblasts but not Fancd2(-/-)/Trp53(-/-) mouse embryonic fibroblasts arrest following DNA damage. Therefore, Trp53 is required for the S phase checkpoint activation observed in Fancd2 mutant cells. Fancd2(-/-)/Trp53(-/-) cells showed an increase in aneuploidy and had multiple gross chromosomal rearrangements.

Liver repopulation with bone marrow-derived hepatocytes (BMHs) can cure the genetic liver disease fumarylacetoacetate hydrolase (Fah) deficiency. BMHs emerge from fusion between donor bone marrow-derived cells and host hepatocytes. To use such in vivo cell fusion efficiently for therapy requires knowing the nature of the hematopoietic cells that fuse with hepatocytes. Here we show that the transplantation into Fah(-/-) mice of hematopoietic stem cells (HSCs) from lymphocyte-deficient Rag1(-/-) mice, lineage-committed granulocyte-macrophage progenitors (GMPs) or bone marrow-derived macrophages (BMMs) results in the robust production of BMHs. These results provide direct evidence that committed myelomonocytic cells such as macrophages can produce functional epithelial cells by in vivo fusion. Because stable bone marrow engraftment or HSCs are not required for this process, macrophages or their highly proliferative progenitors provide potential for targeted and well-tolerated cell therapy aimed at organ regeneration.

In 1967, Guido Fanconi, a Swiss pediatrician, described Fanconi anemia (FA) in two siblings with similar physical anom-alies and bone marrow failure.1 A few years earlier, Schroeder and colleagues had described an increase in spontaneous chromosome breaks in blood samples from patients afflicted with what was then called familial panmyelopathy,2 now known to have been FA. Since then, the field of FA research has undergone an amazing revolution, especially in the last decade. Because of its relation-ship to DNA repair and association with a susceptibility to neo-plastic transformation, FA has gained much attention, particularly following the elucidation of some of the genes that are mutated in FA patients. In 2002, the discovery of a connection between FA, breast cancer, and other well-known DNA repair diseases sparked numerous publications on the subject. In this review, we discuss the clinical and genetic aspects of FA.

BRCA1 and BRCA2 proteins act in repair of interstrand crosslinks (ICLs) and maintenance of genome stability and are known to be part of the Fanconi anemia (FA) pathway. We have investigated the role of the BRCA1 and BRCA2 genes in genome stability following ICL damage in normal and FA cells. To circumvent cell lethality of complete disruptions in BRCA1 or BRCA2, small inhibitory RNA (siRNA) was used to transiently deplete the expression of the proteins. Using chromosomal stability after ICL damage as the end point, we find that BRCA1 functions in more than just the FA pathway for genome maintenance, whereas BRCA2 appears to act predominantly in the FA pathway. Depletion of BRCA1 causes a marked decrease, although not a complete absence of, ubiquitination of FANCD2. In contrast to BRCA1, BRCA2 is not needed for normal ubiquitination of FANCD2 after DNA damage, a requirement for the FA pathway to function. Thus, BRCA2 is epistatic to FA genes for ICL repair, but not for damage-induced modification of FANCD2 and may act downstream form FANCD2.

Evidence suggests that haematopoietic stem cells might have unexpected developmental plasticity, highlighting therapeutic potential. For example, bone-marrow-derived hepatocytes can repopulate the liver of mice with fumarylacetoacetate hydrolase deficiency and correct their liver disease. To determine the underlying mechanism in this murine model, we performed serial transplantation of bone-marrow-derived hepatocytes. Here we show by Southern blot analysis that the repopulating hepatocytes in the liver were heterozygous for alleles unique to the donor marrow, in contrast to the original homozygous donor cells. Furthermore, cytogenetic analysis of hepatocytes transplanted from female donor mice into male recipients demonstrated 80,XXXY (diploid to diploid fusion) and 120,XXXXYY (diploid to tetraploid fusion) karyotypes, indicative of fusion between donor and host cells. We conclude that hepatocytes derived form bone marrow arise from cell fusion and not by differentiation of haematopoietic stem cells.

Fanconi anemia (FA) is a human genetic disorder characterized by hypersensitivity to DNA crosslinking agents. Its cellular phenotypes include increased chromosome breakage and a marked cell-cycle delay with 4N DNA content after introduction of interstrand DNA crosslinks (ICL). To further understand the nature of this delay previously described as a G2/M arrest, we introduced ICL specifically during G2 and monitored the cells for passage into mitosis. Our results showed that, even at the highest doses, postreplication ICL produced neither G2/M arrest nor chromosome breakage in FA-A or FA-C cells. This suggests that, similar to wild-type cells, DNA replication is required to trigger both responses. Therefore, the 4N cell DNA content observed in FA cells after ICL treatment also represents incomplete DNA replication and arrest in late S phase. FA fibroblasts from complementation groups A and C were able to recover from the ICL-induced cell-cycle arrest, but took approximately 3 times longer than controls. These results indicate that the FA pathway is required for the efficient resolution of ICL-induced S-phase arrest., (Copyright 2001 Elsevier Science.)

Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G(2)/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G(1), S, and G(2) cells were treated with photoactivated 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G(2) did not result in a G(2)/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G(1) did not cause a G(1)/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to approximately 2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.

The existence of gamma-tubulin was first reported approximately ten years ago, and it is appropriate to review the progress that has been made in gamma-tubulin research and to discuss some of the unanswered questions about gamma-tubulin function. gamma-Tubulin is ubiquitous in eukaryotes and is generally quite conserved. Two highly divergent gamma-tubulins have been discovered, however, one in Saccharomyces cerevisiae and one in Caenorhabditis elegans. Several organisms have two gamma-tubulin genes. In Drosophila melanogaster, the two gamma-tubulins differ significantly in sequence and expression pattern. In other organisms the two gamma-tubulins are almost identical and expression patterns have not been determined. gamma-Tubulin is located at microtubule organizing centers in many organisms, and it is also frequently associated with the mitotic spindle. gamma-Tubulin is essential for the formation of functional mitotic spindles in all organisms that have been examined to date. In animal cells, complexes containing gamma-tubulin are located at microtubule organizing centers where they nucleate the assembly of microtubules. In spite of the considerable progress that has been made in gamma-tubulin research important questions remain to be answered. The exact mechanisms of microtubule nucleation by gamma-tubulin complexes remain to be resolved as do the mechanisms by which microtubule nucleation from gamma-tubulin complexes is regulated. Finally, there is evidence that gamma-tubulin has important functions in addition to microtubule nucleation, and these functions are just beginning to be investigated.