Publications by authors named "Arleen D Auerbach"

61 Publications

Comparison of the clinical phenotype and haematological course of siblings with Fanconi anaemia.

Br J Haematol 2020 Aug 31. Epub 2020 Aug 31.

MSK Kids - Memorial Sloan Kettering, Stem Cell Transplantation and Cellular Therapies, New York, NY, USA.

Fanconi anaemia (FA) is a genetic disorder due to mutations in any of the 22 FANC genes (FANCA-FANCW) and has high phenotypic variation. Siblings may have similar clinical outcome because they share the same variants; however, such association has not been reported. We present the detailed phenotype and clinical course of 25 sibling sets with FA from two institutions. Haematological progression significantly correlated between siblings, which was confirmed in an additional 55 sibling pairs from the International Fanconi Anemia Registry. Constitutional abnormalities were not concordant, except for a moderate degree of concordance in kidney abnormalities and microcephaly.
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http://dx.doi.org/10.1111/bjh.17061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914271PMC
August 2020

Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand cross-links.

Genes Dev 2020 06 30;34(11-12):832-846. Epub 2020 Apr 30.

Laboratory of Genome Maintenance, The Rockefeller University, New York, New York 10065, USA.

DNA interstrand cross-links (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene when monoallelic, predispose to breast and ovarian cancer, and when biallelic, result in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of programmed DNA double-strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of DNA-binding domain (DBD) mutants (c.8488-1G>A and c.8524C>T) discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for the protection of DNA at ICLs. Cells carrying DBD mutations are sensitive to ICL-inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease that is implicated in the resection of HU-induced stalled replication forks. Our results also indicate that unlike the processing at HU-induced stalled forks, the function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.
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http://dx.doi.org/10.1101/gad.336446.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263144PMC
June 2020

Association of clinical severity with FANCB variant type in Fanconi anemia.

Blood 2020 04;135(18):1588-1602

Cancer Genomics Unit, Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD.

Fanconi anemia (FA) is the most common genetic cause of bone marrow failure and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry. Those with FANCB deletion or truncation demonstrate earlier-than-average onset of bone marrow failure and more severe congenital abnormalities compared with a large series of FA individuals in published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Aberrant splicing and transcript destabilization were associated with 2 missense variants. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination in biochemical and/or cell-based assays tended to show earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our study reveals a genotype-phenotype correlation within the FA-B complementation group of FA, where severity is associated with level of residual FANCD2 monoubiquitination.
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http://dx.doi.org/10.1182/blood.2019003249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193183PMC
April 2020

A founder variant in the South Asian population leads to a high prevalence of FANCL Fanconi anemia cases in India.

Hum Mutat 2020 01 26;41(1):122-128. Epub 2019 Sep 26.

Department of Cytogenetics, National Institute of Immunohaematology (ICMR), Mumbai, Maharashtra, India.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, predisposition to cancer, and congenital abnormalities. FA is caused by pathogenic variants in any of 22 genes involved in the DNA repair pathway responsible for removing interstrand crosslinks. FANCL, an E3 ubiquitin ligase, is an integral component of the pathway, but patients affected by disease-causing FANCL variants are rare, with only nine cases reported worldwide. We report here a FANCL founder variant, anticipated to be synonymous, c.1092G>A;p.K364=, but demonstrated to induce aberrant splicing, c.1021_1092del;p.W341_K364del, that accounts for the onset of FA in 13 cases from South Asia, 12 from India and one from Pakistan. We comprehensively illustrate the pathogenic nature of the variant, provide evidence for a founder effect, and propose including this variant in genetic screening of suspected FA patients in India and Pakistan, as well as those with ancestry from these regions of South Asia.
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http://dx.doi.org/10.1002/humu.23914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362330PMC
January 2020

Somatic mosaicism of an intragenic FANCB duplication in both fibroblast and peripheral blood cells observed in a Fanconi anemia patient leads to milder phenotype.

Mol Genet Genomic Med 2018 01 30;6(1):77-91. Epub 2017 Nov 30.

Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA.

Background: Fanconi anemia (FA) is a rare disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer. Patients harboring X-linked FANCB pathogenic variants usually present with severe congenital malformations resembling VACTERL syndrome with hydrocephalus.

Methods: We employed the diepoxybutane (DEB) test for FA diagnosis, arrayCGH for detection of duplication, targeted capture and next-gen sequencing for defining the duplication breakpoint, PacBio sequencing of full-length FANCB aberrant transcript, FANCD2 ubiquitination and foci formation assays for the evaluation of FANCB protein function by viral transduction of FANCB-null cells with lentiviral FANCB WT and mutant expression constructs, and droplet digital PCR for quantitation of the duplication in the genomic DNA and cDNA.

Results: We describe here an FA-B patient with a mild phenotype. The DEB diagnostic test for FA revealed somatic mosaicism. We identified a 9154 bp intragenic duplication in FANCB, covering the first coding exon 3 and the flanking regions. A four bp homology (GTAG) present at both ends of the breakpoint is consistent with microhomology-mediated duplication mechanism. The duplicated allele gives rise to an aberrant transcript containing exon 3 duplication, predicted to introduce a stop codon in FANCB protein (p.A319*). Duplication levels in the peripheral blood DNA declined from 93% to 7.9% in the span of eleven years. Moreover, the patient fibroblasts have shown 8% of wild-type (WT) allele and his carrier mother showed higher than expected levels of WT allele (79% vs. 50%) in peripheral blood, suggesting that the duplication was highly unstable.

Conclusion: Unlike sequence point variants, intragenic duplications are difficult to precisely define, accurately quantify, and may be very unstable, challenging the proper diagnosis. The reversion of genomic duplication to the WT allele results in somatic mosaicism and may explain the relatively milder phenotype displayed by the FA-B patient described here.
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http://dx.doi.org/10.1002/mgg3.350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823675PMC
January 2018

A comprehensive approach to identification of pathogenic FANCA variants in Fanconi anemia patients and their families.

Hum Mutat 2018 02 22;39(2):237-254. Epub 2017 Nov 22.

Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, Bethesda, Maryland.

Fanconi anemia (FA) is a rare recessive DNA repair deficiency resulting from mutations in one of at least 22 genes. Two-thirds of FA families harbor mutations in FANCA. To genotype patients in the International Fanconi Anemia Registry (IFAR) we employed multiple methodologies, screening 216 families for FANCA mutations. We describe identification of 57 large deletions and 261 sequence variants, in 159 families. All but seven families harbored distinct combinations of two mutations demonstrating high heterogeneity. Pathogenicity of the 18 novel missense variants was analyzed functionally by determining the ability of the mutant cDNA to improve the survival of a FANCA-null cell line when treated with MMC. Overexpressed pathogenic missense variants were found to reside in the cytoplasm, and nonpathogenic in the nucleus. RNA analysis demonstrated that two variants (c.522G > C and c.1565A > G), predicted to encode missense variants, which were determined to be nonpathogenic by a functional assay, caused skipping of exons 5 and 16, respectively, and are most likely pathogenic. We report 48 novel FANCA sequence variants. Defining both variants in a large patient cohort is a major step toward cataloging all FANCA variants, and permitting studies of genotype-phenotype correlations.
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http://dx.doi.org/10.1002/humu.23366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762269PMC
February 2018

Paternal or Maternal Uniparental Disomy of Chromosome 16 Resulting in Homozygosity of a Mutant Allele Causes Fanconi Anemia.

Hum Mutat 2016 May 23;37(5):465-8. Epub 2016 Feb 23.

Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland.

Fanconi anemia (FA) is a rare inherited disorder caused by pathogenic variants in one of 19 FANC genes. FA patients display congenital abnormalities, and develop bone marrow failure, and cancer susceptibility. We identified homozygous mutations in four FA patients and, in each case, only one parent carried the obligate mutant allele. FANCA and FANCP/SLX4 genes, both located on chromosome 16, were the affected recessive FA genes in three and one family respectively. Genotyping with short tandem repeat markers and SNP arrays revealed uniparental disomy (UPD) of the entire mutation-carrying chromosome 16 in all four patients. One FANCA patient had paternal UPD, whereas FA in the other three patients resulted from maternal UPD. These are the first reported cases of UPD as a cause of FA. UPD indicates a reduced risk of having another child with FA in the family and has implications in prenatal diagnosis.
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http://dx.doi.org/10.1002/humu.22962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833600PMC
May 2016

Natural history and management of Fanconi anemia patients with head and neck cancer: A 10-year follow-up.

Laryngoscope 2016 Apr 20;126(4):870-9. Epub 2015 Oct 20.

Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, U.S.A.

Objectives/hypothesis: To describe the management and outcomes of Fanconi anemia (FA) patients with head and neck squamous cell carcinoma.

Study Design: Cohort study.

Methods: Demographic information, prognostic factors, therapeutic management, and survival outcomes for FA patients enrolled in the International Fanconi Anemia Registry who developed head and neck squamous cell carcinoma (HNSCC) were analyzed.

Results: Thirty-five FA patients were diagnosed with HNSCC at a mean age of 32 years. The most common site of primary cancer was the oral cavity (26 of 35, 74%). Thirty patients underwent surgical resection of the cancer. Sixteen patients received radiation therapy with an average radiation dose of 5,050 cGy. The most common toxicities were high-grade mucositis (9 of 16, 56%), hematologic abnormalities (8 of 16, 50%), and dysphagia (8 of 16, 50%). Three patients received conventional chemotherapy and had significant complications, whereas three patients who received targeted chemotherapy with cetuximab had fewer toxicities. The 5-year overall survival rate was 39%, with a cause-specific survival rate of 47%.

Conclusions: Fanconi anemia patients have a high risk of developing aggressive HNSCC at an early age. Fanconi anemia patients can tolerate complex ablative and reconstructive surgeries, but careful postoperative care is required to reduce morbidity. The treatment of FA-associated HNSCC is difficult secondary to the poor tolerance of radiation and chemotherapy. However, radiation should be used for high-risk cancers due to the poor survival in these patients.

Level Of Evidence: 4.
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http://dx.doi.org/10.1002/lary.25726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803627PMC
April 2016

A Dominant Mutation in Human RAD51 Reveals Its Function in DNA Interstrand Crosslink Repair Independent of Homologous Recombination.

Mol Cell 2015 Aug;59(3):478-90

Laboratory of Genome Maintenance, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Repair of DNA interstrand crosslinks requires action of multiple DNA repair pathways, including homologous recombination. Here, we report a de novo heterozygous T131P mutation in RAD51/FANCR, the key recombinase essential for homologous recombination, in a patient with Fanconi anemia-like phenotype. In vitro, RAD51-T131P displays DNA-independent ATPase activity, no DNA pairing capacity, and a co-dominant-negative effect on RAD51 recombinase function. However, the patient cells are homologous recombination proficient due to the low ratio of mutant to wild-type RAD51 in cells. Instead, patient cells are sensitive to crosslinking agents and display hyperphosphorylation of Replication Protein A due to increased activity of DNA2 and WRN at the DNA interstrand crosslinks. Thus, proper RAD51 function is important during DNA interstrand crosslink repair outside of homologous recombination. Our study provides a molecular basis for how RAD51 and its associated factors may operate in a homologous recombination-independent manner to maintain genomic integrity.
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http://dx.doi.org/10.1016/j.molcel.2015.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529964PMC
August 2015

Deficiency of UBE2T, the E2 Ubiquitin Ligase Necessary for FANCD2 and FANCI Ubiquitination, Causes FA-T Subtype of Fanconi Anemia.

Cell Rep 2015 Jul 25;12(1):35-41. Epub 2015 Jun 25.

Laboratory of Genome Maintenance, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT.
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http://dx.doi.org/10.1016/j.celrep.2015.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497947PMC
July 2015

Diagnosis of Fanconi anemia by diepoxybutane analysis.

Curr Protoc Hum Genet 2015 Apr 1;85:8.7.1-8.7.17. Epub 2015 Apr 1.

Program in Human Genetics and Hematology, The Rockefeller University, New York, New York.

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer, particularly hematological malignancies and solid tumors of the head and neck. The main role of FA proteins is in the repair of DNA interstrand crosslinks (ICLs). FA results from pathogenic variants in at least sixteen distinct genes, causing genomic instability. Although the highly variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, diagnosis based on a profound sensitivity to DNA-crosslinking agents can be used to identify the pre-anemia patient as well as patients with aplastic anemia or leukemia who may or may not have the physical stigmata associated with the syndrome. Diepoxybutane (DEB) analysis is the preferred test for FA because other agents have higher rates of false-positive and false-negative results.
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http://dx.doi.org/10.1002/0471142905.hg0807s85DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408609PMC
April 2015

Comprehensive analysis of pathogenic deletion variants in Fanconi anemia genes.

Hum Mutat 2014 Nov;35(11):1342-53

Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland, 20892.

Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high-resolution comparative genome hybridization arrays, single-nucleotide polymorphism arrays, and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy-five percent of the FANCA deletions are Alu-Alu mediated, predominantly by AluY elements, and appear to be caused by nonallelic homologous recombination. Individual Alu hotspots were identified. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry. Knowing the exact molecular changes that lead to the disease may be critical for a better understanding of the FA phenotype, and to gain insight into the mechanisms driving these pathogenic deletion variants.
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http://dx.doi.org/10.1002/humu.22680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407816PMC
November 2014

Telomere phenotypes in females with heterozygous mutations in the dyskeratosis congenita 1 (DKC1) gene.

Hum Mutat 2013 Nov 11;34(11):1481-5. Epub 2013 Sep 11.

Department of Oncology and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Dyskeratosis congenita (DC) is a telomere-mediated syndrome defined by mucocutaneous features. The X-linked mode of inheritance accounts for half the cases, and is thought to predominantly manifest in childhood as bone marrow failure. We identified two male probands who presented in the fifth decade with idiopathic pulmonary fibrosis and cancer. Their pedigrees displayed consecutively affected generations. Five of six females (83%) manifested mucocutaneous features of DC, and two had wound-healing complications. No mutations in autosomal dominant telomere genes were present, but exome sequencing revealed novel variants in the X-chromosome DKC1 gene that predicted missense mutations in conserved residues, p.Thr49Ser and p.Pro409Arg. Variants segregated with the telomere phenotype, and affected females were heterozygotes, showing skewed X-inactivation. Telomerase RNA levels were compromised in cells from DKC1 mutation carriers, consistent with their pathogenic role. These findings indicate that females with heterozygous DKC1 mutations may be at increased risk for developing penetrant telomere phenotypes that, at times, may be associated with clinical morbidity.
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http://dx.doi.org/10.1002/humu.22397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926107PMC
November 2013

Massively parallel sequencing, aCGH, and RNA-Seq technologies provide a comprehensive molecular diagnosis of Fanconi anemia.

Blood 2013 May 23;121(22):e138-48. Epub 2013 Apr 23.

Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Current methods for detecting mutations in Fanconi anemia (FA)-suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom molecular inversion probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at The Rockefeller University. DNA sequencing was complemented with custom array comparative genomic hybridization (aCGH) and RNA sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 different FA genes. RNA-seq analysis revealed lack of allele specific expression associated with a deletion and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq-targeted capture, aCGH, and RNA-seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2), and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA.
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http://dx.doi.org/10.1182/blood-2012-12-474585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668494PMC
May 2013

Regulation of multiple DNA repair pathways by the Fanconi anemia protein SLX4.

Blood 2013 Jan 23;121(1):54-63. Epub 2012 Oct 23.

Laboratory of Genome Maintenance, Rockefeller University, New York, NY 10065-6399, USA.

SLX4, the newly identified Fanconi anemia protein, FANCP, is implicated in repairing DNA damage induced by DNA interstrand cross-linking (ICL) agents, topoisomerase I (TOP1) inhibitors, and in Holliday junction resolution. It interacts with and enhances the activity of XPF-ERCC1, MUS81-EME1, and SLX1 nucleases, but the requirement for the specific nucleases in SLX4 function is unclear. Here, by complementing a null FA-P Fanconi anemia cell line with SLX4 mutants that specifically lack the interaction with each of the nucleases, we show that the SLX4-dependent XPF-ERCC1 activity is essential for ICL repair but is dispensable for repairing TOP1 inhibitor-induced DNA lesions. Conversely, MUS81-SLX4 interaction is critical for resistance to TOP1 inhibitors but is less important for ICL repair. Mutation of SLX4 that abrogates interaction with SLX1 results in partial resistance to both cross-linking agents and TOP1 inhibitors. These results demonstrate that SLX4 modulates multiple DNA repair pathways by regulating appropriate nucleases.
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http://dx.doi.org/10.1182/blood-2012-07-441212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538331PMC
January 2013

Human Variome Project country nodes: documenting genetic information within a country.

Hum Mutat 2012 Nov 18;33(11):1513-9. Epub 2012 Jul 18.

Department of Pharmacy, School of Health Sciences, University of Patras, Patras, Greece.

The Human Variome Project (http://www.humanvariomeproject.org) is an international effort aiming to systematically collect and share information on all human genetic variation. The two main pillars of this effort are gene/disease-specific databases and a network of Human Variome Project Country Nodes. The latter are nationwide efforts to document the genomic variation reported within a specific population. The development and successful operation of the Human Variome Project Country Nodes are of utmost importance to the success of Human Variome Project's aims and goals because they not only allow the genetic burden of disease to be quantified in different countries, but also provide diagnosticians and researchers access to an up-to-date resource that will assist them in their daily clinical practice and biomedical research, respectively. Here, we report the discussions and recommendations that resulted from the inaugural meeting of the International Confederation of Countries Advisory Council, held on 12th December 2011, during the 2011 Human Variome Project Beijing Meeting. We discuss the steps necessary to maximize the impact of the Country Node effort for developing regional and country-specific clinical genetics resources and summarize a few well-coordinated genetic data collection initiatives that would serve as paradigms for similar projects.
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http://dx.doi.org/10.1002/humu.22147DOI Listing
November 2012

FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway.

Blood 2012 Apr 17;119(14):3285-94. Epub 2012 Feb 17.

Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.

Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63-linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage-induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance.
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http://dx.doi.org/10.1182/blood-2011-10-385963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321854PMC
April 2012

Sensitive quantification of mosaicism using high density SNP arrays and the cumulative distribution function.

Mol Genet Metab 2012 Apr 24;105(4):665-71. Epub 2011 Dec 24.

Office of the Clinical Director, NHGRI, National Institutes of Health, Bethesda, MD 20892-1851, USA.

Medicine is rapidly applying exome and genome sequencing to the diagnosis and management of human disease. Somatic mosaicism, however, is not readily detectable by these means, and yet it accounts for a significant portion of undiagnosed disease. We present a rapid and sensitive method, the Continuous Distribution Function as applied to single nucleotide polymorphism (SNP) array data, to quantify somatic mosaicism throughout the genome. We also demonstrate application of the method to novel diseases and mechanisms.
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http://dx.doi.org/10.1016/j.ymgme.2011.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3309164PMC
April 2012

Postoperative clinical radiosensitivity in patients with fanconi anemia and head and neck squamous cell carcinoma.

Arch Otolaryngol Head Neck Surg 2011 Sep;137(9):930-4

Department of Otolaryngology-Head and Neck Surgery, New York Presbyterian Hospital, Weill Cornell Medical Center, NY 10021, USA.

Objective: To describe the complications and adverse effects of postoperative radiotherapy in patients with Fanconi anemia (FA).

Design: Cohort study.

Setting: Patients with FA treated at community and tertiary care hospitals throughout the United States.

Patients: The study included patients with FA who were enrolled in the International FA Registry (IFAR) and who developed head and neck squamous cell carcinoma and received postoperative radiotherapy.

Main Outcome Measures: Demographics of patients with FA and adverse effects and dosages of radiotherapy.

Results: Twelve patients with FA (7 men and 5 women) were identified. They developed cancers at a mean age of 35.5 years (age range, 20-48 years). The sites of primary cancer were the oral cavity (n = 8), larynx (n = 2), pharynx (n = 1), and unknown (n = 1). The median radiation dose was 5590 cGy (range, 2500-7020 cGy). The most common adverse effects were mucositis (n = 9), dysphagia (n = 8), and pancytopenia (n = 6). Other complications included esophageal stenosis, laryngeal edema, and wound breakdown. Radiotherapy could not be completed in 5 cases. Overall, 8 patients died, 4 during the course of radiotherapy. The postoperative disease-free survival time ranged from 0 to 55 months.

Conclusions: Patients with FA have a high rate of complications from radiotherapy. Common adverse effects, particularly mucositis, are especially prevalent and difficult to manage in this population. Pancytopenia is common and may lead to further complications, particularly bleeding and infection. Overall survival is poor. Further study of the response to radiotherapy in patients with FA should be attempted to establish appropriate dosages to balance treating disease while limiting adverse effects.
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http://dx.doi.org/10.1001/archoto.2011.154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3343719PMC
September 2011

The clinical phenotype of children with Fanconi anemia caused by biallelic FANCD1/BRCA2 mutations.

Pediatr Blood Cancer 2012 Mar 5;58(3):462-5. Epub 2011 May 5.

Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.

Fanconi anemia (FA) is characterized by progressive marrow failure, congenital anomalies, and predisposition to malignancy. Biallelic FANCD1/BRCA2 mutations are the genetic basis of disease in a small proportion of children with FA with earlier onset and increased incidence of leukemia and solid tumors. Patients with FA have increased sensitivity to chemotherapy and radiation, and upon development of a solid tumor, require modification of these therapies. We report clinical and molecular features of three patients with FA associated with FANCD1/BRCA2 mutations, including two novel mutations, and discuss treatment of malignancy and associated side effects in this particularly vulnerable group.
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http://dx.doi.org/10.1002/pbc.23168DOI Listing
March 2012

Origin, functional role, and clinical impact of Fanconi anemia FANCA mutations.

Blood 2011 Apr 27;117(14):3759-69. Epub 2011 Jan 27.

Genome Instability and DNA Repair Group, Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Campus de Bellaterra s/n, Barcelona, Spain.

Fanconi anemia is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. To investigate the origin, functional role, and clinical impact of FANCA mutations, we determined a FANCA mutational spectrum with 130 pathogenic alleles. Some of these mutations were further characterized for their distribution in populations, mode of emergence, or functional consequences at cellular and clinical level. The world most frequent FANCA mutation is not the result of a mutational "hot-spot" but results from worldwide dissemination of an ancestral Indo-European mutation. We provide molecular evidence that total absence of FANCA in humans does not reduce embryonic viability, as the observed frequency of mutation carriers in the Gypsy population equals the expected by Hardy-Weinberg equilibrium. We also prove that long distance Alu-Alu recombination can cause Fanconi anemia by originating large interstitial deletions involving FANCA and 2 adjacent genes. Finally, we show that all missense mutations studied lead to an altered FANCA protein that is unable to relocate to the nucleus and activate the FA/BRCA pathway. This may explain the observed lack of correlation between type of FANCA mutation and cellular phenotype or clinical severity in terms of age of onset of hematologic disease or number of malformations.
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http://dx.doi.org/10.1182/blood-2010-08-299917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083295PMC
April 2011

Mutations of the SLX4 gene in Fanconi anemia.

Nat Genet 2011 Feb 16;43(2):142-6. Epub 2011 Jan 16.

Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, USA.

Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors. At the cellular level, hypersensitivity to DNA interstrand crosslinks is the defining feature in Fanconi anemia. Mutations in thirteen distinct Fanconi anemia genes have been shown to interfere with the DNA-replication-dependent repair of lesions involving crosslinked DNA at stalled replication forks. Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase, results in increased sensitivity of the cells to DNA crosslinking agents. Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.
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http://dx.doi.org/10.1038/ng.750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345287PMC
February 2011

How to catch all those mutations--the report of the third Human Variome Project Meeting, UNESCO Paris, May 2010.

Hum Mutat 2010 Dec;31(12):1374-81

Cancer Research Program, Garvan Institute of Medical Research, St. Vincent's Clinical School, University of NSW, Sydney, NSW, Australia.

The third Human Variome Project (HVP) Meeting "Integration and Implementation" was held under UNESCO Patronage in Paris, France, at the UNESCO Headquarters May 10-14, 2010. The major aims of the HVP are the collection, curation, and distribution of all human genetic variation affecting health. The HVP has drawn together disparate groups, by country, gene of interest, and expertise, who are working for the common good with the shared goal of pushing the boundaries of the human variome and collaborating to avoid unnecessary duplication. The meeting addressed the 12 key areas that form the current framework of HVP activities: Ethics; Nomenclature and Standards; Publication, Credit and Incentives; Data Collection from Clinics; Overall Data Integration and Access-Peripheral Systems/Software; Data Collection from Laboratories; Assessment of Pathogenicity; Country Specific Collection; Translation to Healthcare and Personalized Medicine; Data Transfer, Databasing, and Curation; Overall Data Integration and Access-Central Systems; and Funding Mechanisms and Sustainability. In addition, three societies that support the goals and the mission of HVP also held their own Workshops with the view to advance disease-specific variation data collection and utilization: the International Society for Gastrointestinal Hereditary Tumours, the Micronutrient Genomics Project, and the Neurogenetics Consortium.
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http://dx.doi.org/10.1002/humu.21379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3119486PMC
December 2010

Correct mRNA processing at a mutant TT splice donor in FANCC ameliorates the clinical phenotype in patients and is enhanced by delivery of suppressor U1 snRNAs.

Am J Hum Genet 2010 Oct;87(4):480-93

Institute of Virology, Heinrich-Heine-University, D-40225 Düsseldorf, Germany.

The U1 small nuclear RNA (U1 snRNA) as a component of the major U2-dependent spliceosome recognizes 5' splice sites (5'ss) containing GT as the canonical dinucleotide in the intronic positions +1 and +2. The c.165+1G>T germline mutation in the 5'ss of exon 2 of the Fanconi anemia C (FANCC) gene commonly predicted to prevent correct splicing was identified in nine FA patients from three pedigrees. RT-PCR analysis of the endogenous FANCC mRNA splicing pattern of patient-derived fibroblasts revealed aberrant mRNA processing, but surprisingly also correct splicing at the TT dinucleotide, albeit with lower efficiency. This consequently resulted in low levels of correctly spliced transcript and minute levels of normal posttranslationally processed FANCD2 protein, indicating that this naturally occurring TT splicing might contribute to the milder clinical manifestations of the disease in these patients. Functional analysis of this FANCC 5'ss within splicing reporters revealed that both the noncanonical TT dinucleotide and the genomic context of FANCC were required for the residual correct splicing at this mutant 5'ss. Finally, use of lentiviral vectors as a delivery system to introduce expression cassettes for TT-adapted U1 snRNAs into primary FANCC patient fibroblasts allowed the correction of the DNA-damage-induced G2 cell-cycle arrest in these cells, thus representing an alternative transcript-targeting approach for genetic therapy of inherited splice-site mutations.
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http://dx.doi.org/10.1016/j.ajhg.2010.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948791PMC
October 2010

FANCJ/BRIP1 recruitment and regulation of FANCD2 in DNA damage responses.

Chromosoma 2010 Dec 31;119(6):637-49. Epub 2010 Jul 31.

Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, 3333 Burnet Ave. ML S7.203, Cincinnati, OH 45229, USA.

FANCJ/BRIP1 encodes a helicase that has been implicated in the maintenance of genomic stability. Here, to better understand FANCJ function in DNA damage responses, we have examined the regulation of its cellular localization. FANCJ nuclear foci assemble spontaneously during S phase and are induced by various stresses. FANCJ foci colocalize with the replication fork following treatment with hydroxyurea, but not spontaneously. Using FANCJ mutants, we find that FANCJ helicase activity and the capacity to bind BRCA1 are both involved in FANCJ recruitment. Given similarities to the recruitment of another Fanconi anemia protein, FANCD2, we tested for colocalization of FANCJ and FANCD2. Importantly, these proteins show substantial colocalization, and FANCJ promotes the assembly of FANCD2 nuclear foci. This process is linked to the proper localization of FANCJ itself since both FANCJ and FANCD2 nuclear foci are compromised by FANCJ mutants that abrogate its helicase activity or interaction with BRCA1. Our results suggest that FANCJ is recruited in response to replication stress and that FANCJ/BRIP1 may serve to link FANCD2 to BRCA1.
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http://dx.doi.org/10.1007/s00412-010-0285-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4928586PMC
December 2010

Fanconi anemia and its diagnosis.

Mutat Res 2009 Jul 28;668(1-2):4-10. Epub 2009 Feb 28.

Laboratory of Human Genetics and Hematology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, United States.

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous recessive disorder characterized by diverse congenital malformations, progressive pancytopenia, and predisposition to both hematologic malignancies and solid tumors. Congenital anomalies vary from patient to patient and may affect skeletal morphogenesis as well as any of the major organ systems. Although this highly variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, laboratory study of chromosomal breakage induced by diepoxybutane (DEB) or other crosslinking agents provides a unique cellular marker for the diagnosis of the disorder either prenatally or postnatally. Diagnosis based on abnormal response to DNA crosslinking agents can be used to identify the pre-anemia patient as well as patients with aplastic anemia or leukemia who may or may not have the physical stigmata associated with the syndrome. This overview will present our current knowledge regarding the varied phenotypic manifestations of FA and procedures for diagnosis based upon abnormal DNA damage responses.
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http://dx.doi.org/10.1016/j.mrfmmm.2009.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742943PMC
July 2009

Identification and characterization of mutations in FANCL gene: a second case of Fanconi anemia belonging to FA-L complementation group.

Hum Mutat 2009 Jul;30(7):E761-70

Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.

Fanconi anemia (FA) is a rare autosomal recessive or X-linked disorder characterized by aplastic anemia, cancer susceptibility and cellular sensitivity to DNA crosslinking agents. Eight FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL and FANCM) and three non-FA proteins (FAAP100, FAAP24 and HES1) form an FA nuclear core complex, which is required for monoubiquitination of the FANCD2-FANCI dimer upon DNA damage. FANCL possesses a PHD/RING-finger domain and is a putative E3 ubiquitin ligase subunit of the core complex. In this study, we report an FA patient with an unusual presentation belonging to the FA-L complementation group. The patient lacks an obvious FA phenotype except for the presence of a café-au-lait spot, mild hypocellularity and a family history of leukemia. The molecular diagnosis and identification of the FA subgroup was achieved by FA complementation assay. We identified bi-allelic novel mutations in the FANCL gene and functionally characterized them. To the best of our knowledge, this is the second reported case belonging to the FA-L complementation group.
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http://dx.doi.org/10.1002/humu.21032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2760491PMC
July 2009

Planning the human variome project: the Spain report.

Hum Mutat 2009 Apr;30(4):496-510

Division of Personalised Nutrition and Medicine, FDA/National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.

The remarkable progress in characterizing the human genome sequence, exemplified by the Human Genome Project and the HapMap Consortium, has led to the perception that knowledge and the tools (e.g., microarrays) are sufficient for many if not most biomedical research efforts. A large amount of data from diverse studies proves this perception inaccurate at best, and at worst, an impediment for further efforts to characterize the variation in the human genome. Because variation in genotype and environment are the fundamental basis to understand phenotypic variability and heritability at the population level, identifying the range of human genetic variation is crucial to the development of personalized nutrition and medicine. The Human Variome Project (HVP; http://www.humanvariomeproject.org/) was proposed initially to systematically collect mutations that cause human disease and create a cyber infrastructure to link locus specific databases (LSDB). We report here the discussions and recommendations from the 2008 HVP planning meeting held in San Feliu de Guixols, Spain, in May 2008.
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http://dx.doi.org/10.1002/humu.20972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5879779PMC
April 2009