Fanconi Anemia: Guidelines For Diagnosis And Management

IntroductionGeneral ConsiderationsAs was true of earlier occasions, the Consensus Conference was guided bythe follow ing general considerations that form the underlying basis for morespecific recommendations.FA is a very rare genetic disorder. Accuracy in diagnosis is crucial and requires sophisticated expertise. The mode of inheritance is important for fur ther genetic testing ofsiblings; finding matched donors; identification of genotype for purposeof predicting onset of symptoms and conse quences; family planning(including PGD); selection of appropriate persons for FA gene therapytrials; and genetic counseling to the family. Expertise in FA treatment is highly specialized and to date is heavilyconcentrated in a few, criti cally important centers. Many persons withFA do not have access to such expertise locally, but the use of referralnetworks and provider cooperation should help provide adequate care.FA is a complex and chronic disorder. Well-orchestrated multi-disciplinary care across several medical andsurgical specialties is typically required for adequate monitoring andtreatment. Clinical trials or at least the collection of longitudinal data are required toinform treatment choices for patients with FA in the future. The transition from pediatric to adult care, and from parent monitoringto self-care, presents particularly important challenges which requirethoughtful management.FA must be considered a multi-system disease. The name of the disorder, Fanconi anemia, may disserve both doctors andpatients because the hematologic manifesta tions of FA are not the sole (oroften even the most important) problem for persons with FA. The FA phenotype is quite variable and leads to misdiagnosis and failureof diagnosis. Monitoring must be multi-disciplinary and include hearingevaluation, assessment of endocrine system and GI tract issues, and longterm cancer surveillance. For the majority of persons with FA, hematopoietic stem celltransplantation is the ultimate therapy for marrow dysfunction.3

Fanconi Anemia: Guidelines for Diagnosis and ManagementConsequently, early involvement with a major transplant centerexperienced in FA transplants and with a multi-disciplinary consultationteam is optimal.FA is a cancer-prone disorder. Close monitoring, especially for the high incidence of SCC, is a specialconsideration throughout the FA person’s lifetime, even post-transplant. The intrinsic genetic instability of FA cells means that exposure to ionizingradiation, envi ronmental carcinogens, and chemotherapeutic agents likelyposes special risks to persons with FA. Consequently, diagnostic x-rayexposure and some otherwise routine medical tests or agents may needto be limited, or used with great caution. Thus, lifestyle choices such astobacco and alcohol use may well have serious adverse consequences, evenbeyond those encountered in the general population.FA is a psychosocially demanding disorder. The pressures on patients, parents, and siblings over an extended timecan be over whelming, particularly where there are multiple affectedfamily members. Persons with FA, their families, and providers must be sensi tive to issuesof expense, the sophistication and availability of medical and familycounseling, and the significant and continuing emotional trauma resultingfrom this diagnosis. FA adults experience quite distinct issues, and their psychosocial concernsare emerging as a distinct field of inquiry.The underlying diagnosis and the many drugs often necessary fortreatment may put FA patients at particular risk for hazardouspharmaceutical cross-reactions. The family and primary physician must continu ously coordinate andmonitor both prescribed and over-the-counter medications taken bya patient.The authors recognize that a significant proportion of affected families seekout and utilize “alternative” medicine. We accept this approach, but at the same time ask families to be open withtheir providers in discussing what alternative practices they are using.Effective therapies may emerge and need to be shared. However, we alsocaution that unforeseen toxicities and drug interactions need to be identified.4

IntroductionWe commend these guidelines in the profound hope that they will betterserve the lives of patients who have this serious and life-threatening disorder.We welcome comments that may inform future improvements in care andtreatment.On behalf of the Fanconi Anemia Research Fund, we extend profoundthanks to the many authors and editors who contributed to this work. Ourspecial gratitude goes to those persons with FA, and their families. Thetoll of this affliction inspires our efforts, and their fervent hope for a curemotivates the urgency of our collective work. Finally, the remarkableprogress in understanding FA biology buoys our optimism for ever-improvingclinical outcomes.Dave Frohnmayer, JDPresident Emeritus and Professor of LawUniversity of OregonCo-founder, Fanconi Anemia Research Fund, Inc.Advisor to the Board of DirectorsEva Guinan, MDModerator, Consensus ConferenceDirector of Translational Research, Dept. of Radiation OncologyDana-Farber Cancer InstituteDirector, Reactor Program, Harvard CatalystProfessor of Radiation OncologyHarvard Medical SchoolJeffrey M. Lipton, MD, PhDChief, Pediatric Hematology/Oncology and Stem Cell TransplantationSteven and Alexandra Cohen Children’s Medical Center of New YorkProfessor of Pediatrics and Molecular MedicineHofstra North Shore-LIJ School of MedicineInvestigator and Professor, Elmezzi GraduateSchool of Molecular MedicineThe Feinstein Institute for Medical Research5

Fanconi Anemia: Guidelines for Diagnosis and ManagementChapter 1: Diagnosis andEvaluationNoteThis chapter is divided into two sections. The Scientific Background sectionuses technical language to effectively describe the genetics and biochemistry ofFA to scientists and clinicians. The Diagnosis section is geared towards cliniciansand families.IntroductionFanconi anemia (FA) is primarily inherited as an autosomal recessive disorder,though about 2% of all cases (1 of 16 known genotypes) are inherited as anX-linked recessive condition.This chapter will explore the underlying molecular and genetic processes bywhich FA contributes to conditions such as bone marrow failure, leukemia,squamous cell carcinoma, endocrine abnormalities, and mild-to-severe birthdefects (1-3). In general, these conditions arise from genetic mutations that causechromosome instability and reduce the cell’s ability to repair damage to DNA.At publication, FA-associated mutations have been identified in 16 genes. Afew patients with FA do not have mutations in the known genes; thus, moregenes likely await discovery.The oft-cited estimate of an FA carrier frequency of 1 in 300 was recentlyrevised to be 1 in 181 for North America and 1 in 93 for Israel. Specificpopulations have founder mutations with increased carrier frequencies (lessthan 1 per 100), including Ashkenazi Jews (FANCC, BRCA2/FANCD1),northern Europeans (FANCC), Afrikaners (FANCA), sub-Saharan Blacks(FANCG), and Spanish Gypsies (FANCA) and others as detailed inChapter 17 (4).Scientific BackgroundFA genes and the DNA damage response pathwayThe products of at least 16 FA-associated genes interact in a unified responsethat unfolds in the cell after exposure to DNA damage, i.e., a “DNA damage6

Chapter 1: Diagnosis and Evaluationresponse pathway” (Figure 1 and Table 1) (5-7). As this pathway includes the twomain breast cancer-associated genes, BRCA1 and BRCA2/FANCD1, it will bereferred to here as the FA/BRCA pathway.Helpful Words and PhrasesGenotype refers to a specific set of variations in genes or the genetic makeup. Itcan also be used to describe a particular cancer.An autosomal recessive disorder shows up clinically when a person inheritstwo copies of an abnormal gene: one copy from the mother and another from thefather. It’s recessive because the person must inherit both copies to develop thecondition. The affected gene is located on one of the chromosomes numbered1-22, which are known as autosomes.An X-linked recessive condition means that females must inherit two copies ofan abnormal gene for the disease to develop, whereas males need only inherit onecopy. That is because males have one X chromosome; females have two.The carrier frequency is the proportion of individuals who carry in their DNAa single copy of an abnormal gene for an autosomal recessive disorder. Carriersusually do not develop the disorder, but can pass a copy of the abnormal gene ontotheir children.A founder mutation is a genetic change that is present in a population overseveral generations.Biallelic mutations are genetic changes found in both copies (alleles) of thesame gene.Hypomorphic mutations are changes that cause the gene product to only losepartial function.Because each patient generally has just one FA gene containing biallelicmutations (1), patients can be assigned to complementation groups FA-A toFA-Q. These groups are defined by the absence of a normal gene product in thecells, even if the specific mutation(s) in that gene is/are not known. In 2013,the first patient (female) was reported with biallelic mutations in the BRCA1gene, which plays an important role in DNA repair and has been heavilyassociated with breast and ovarian cancer susceptibility. This patient had onehypomorphic missense mutation (known as p.Val1736Ala), and exhibited short1 With one notable exception: There has been a single published report of a FANCM patient whoalso has two germ-line mutations in FANCA (8).7

Fanconi Anemia: Guidelines for Diagnosis and Managementstature, microcephaly (an abnormally small head), developmental delay, earlyonset ovarian cancer, and significant toxicity from chemotherapy. Nonetheless,though BRCA1 is an essential part of the FA/BRCA pathway, it is currently notconsidered to be a true FA gene (9).A simplified model for the roles of the FA proteins in the DNA damageresponse to interstrand cross-links at stalled replication forks is shown inFigure 1 (for reviews, see 5, 10-12). After FANCM and the FA-associated proteinFAAP24 detect the DNA damage, the proteins produced from eight FAgenes (FANCA/B/C/E/F/G/L/M) form the FA core complex, which facilitatesactivation of the pathway by monoubiquitination of the FANCD2 and FANCIproteins. These two activated proteins bind to form a dimer (ID2), whichstabilizes the stalled replication fork and then in turn interacts in nuclear repairfoci with the downstream FA gene products in the FA/BRCA DNA damagerepair pathway. Damage repair is then achieved by the late FA proteins incooperation with proteins from other DNA repair pathways (not shown inFigure 1). The FA/BRCA pathway has been elucidated almost in its entiretythrough the study of FA genetics and by biochemical studies in FA cells. Inaddition, germ-line (heritable) mutations in at least six of the downstream FAgenes, FANCD1/BRCA2, FANCJ/BRIP1, FANCN/PALB2, FANCO/RAD51C,FANCP/SLX4, and FANCQ/XPF, have been associated with breast/ovarian,pancreatic, and other cancers in heterozygote individuals. In these individuals,loss of the second, wild-type allele occurs during their lifetime in a somatic(nonreproductive) cell and subsequently leads to malignant (cancerous)transformation (13-15).Good to KnowDNA forms a double-helix structure that looks like a twisted rope ladder. Whenthis ladder is unwound so that it can be copied to make additional ladders, it formsa Y-shaped area called a replication fork.The replication process can be interrupted by cross-links, which occur whenanother molecule binds to two positions on the same side of the ladder(intrastrand cross-links) or on opposite sides of the ladder (interstrandcross-links).Two cross-linking chemicals used in screening tests for FA are mitomycin C(MMC) and diepoxybutane (DEB).8

Chapter 1: Diagnosis and EvaluationFigure 1. DNA damage response pathway, linking the FA and BRCA pathways.Complementation GroupsHistorically, a complementation group is defined by a “reference cell line”(i.e., a lineage of laboratory-grown cells with a well-studied genotype) that is notfunctionally corrected by fusion with other cells of the same complementationgroup, because both cells have defects in the same recessive gene(s) and thereforecannot “complement” (i.e., correct) each other.Cells from patients with FA can be classified into sub-categories known ascomplementation groups without knowing which defective genes or DNAmutations the patient carries. This can be done by fusing the patient’s cells withthe reference cells for a particular complementation group, or by expressing anormal (“wild-type”) cDNA for the specific gene that defines the complementationgroup. This is usually performed in a laboratory using viral or plasmid vectors.Each complementation group is defined by the defect(s) in both alleles, or copies,of a particular FA gene. That is, individuals who belong to FA complementationgroup A (known as FA-A) have at least one loss-of-function mutation in eachallele of the FANCA gene, whereas individuals who belong to complementationgroup B (FA-B) have a mutation in the X-chromosomal FANCB gene. With theexception of FANCB, patients belonging to the same complementation group aretherefore said to have “biallelic recessive mutations” in the same FA gene.9

Fanconi Anemia: Guidelines for Diagnosis and ManagementTable 1. Fanconi anemia genes and gene products.GeneLocusGenomicDNA kBcDNAkBNo. ofExonsProteinkDAminoAcidsApproximate% q25-26734.53815013281ARFANCJ (BRIP1)17q22.31804.52015012493ARFANCL (PHF9/POG)2p16.1821.71443375 1ARFANCM (Hef)14q21.32506.5222502014 1ARFANCN(PALB2)16p12.1383.5131301186 1ARFANCO(RAD51C)17q25.1421.3942376 1ARFANCP (SLX4)16p13.326.65.5152001834 1ARFANCQ (XPF/ERCC4)16p13.1239.26.811104916 1ARFANCI(KIAA1794)10

Chapter 1: Diagnosis and EvaluationGood to KnowA heterozygote individual is a person who carries two different copies of a gene:one normal copy and one mutated copy. Wild-type refers to the natural, nonmutated copy of a gene.Complementation analysis by somatic cell methods refers to the study of apatient’s nonreproductive cells to determine to which complementation group(defined by a single FA gene) the patient might belong.Flow cytometry is a laboratory technique in which single cells in solution areused to diagnose blood cancers and other conditions. This technique can separate,count, and evaluate cells with distinct characteristics.A Western blot is a laboratory technique that allows identification of proteins incell extracts based on their size and movement in an electric field.Scientific techniques used for diagnosticsAlthough next-generation sequencing is currently available as a standardroutine diagnostic procedure for patients in most developed countries(discussed in detail in Chapter 2), complementation analysis by somatic cellmethods has been the mainstay for distinguishing specific genetic lesions/complementation groups and is still used in a number of countries. In thistechnique, cells from an FA patient are tested by various methods in culture toidentify the gene that corrects the FA cell’s hypersensitivity to DNA damagingagents. Initially, this was done by fusing FA cells possessing unknowndefects with reference cells from patients possessing a known FA gene defector defined complementation groups, resulting in hybrid cells in which theunknown FA cells grew normally if they were corrected (or “complemented”)with the known FA genes (5).A more modern method—the only one clinically-certified in the U.S. (albeitused both clinically and in research worldwide)—is the transduction/infectionof the unknown FA cells with retroviral vectors expressing normal wild-typeFA proteins (16). The patient cells can be either Epstein-Barr virus-transformedlymphoblast cell lines, primary or transformed skin or bone marrow fibroblasts,or primary T-cells from peripheral blood or bone marrow. Read-out of theretroviral complementation analysis can be any type of cellular or biochemicalassay. The clinically-certified approach uses flow cytometry to determinecorrection of G2/M arrest in cells treated with DNA-damaging agents (17).Each retrovirus contains only one of the FA genes. While the early FA genes11

Fanconi Anemia: Guidelines for Diagnosis and Management(FANCA, -B, -C, -E, -F, -G, and -L, though not –M) are easier to express,vectors also exist for the other genes and have been used for research purposes.Alternatively, or if correction does not occur with these vectors, a Western blotcan be performed to identify FANCD2 or mono-ubiquitinated FANCD2 proteinand thus to determine whether the mutated FA gene is 1) upstream, whichwould involve the core complex needed for the ubiquitination of FANCI andFANCD2, or which would occur if FANCD2 itself is mutated and absent; or 2)downstream, which would involve FANCD1/BRCA2, FANCJ/BRIP1, FANCN/PALB2, FANCO/RAD51C, FANCP/SLX4, or FANCQ/XPF (18). As shown inFigure 2A, mutations in the FA core complex early genes lead to a single bandof FANCD2 protein (D2-S short), while mutations in the late FA genes areassociated with normal monoubiquitination of FANCD2 and therefore haveboth the D2-L ( long) and the D2-S bands. Here, the classification of a patientas having defects in a late FA gene can be based on the hypersensitivity of cellsin the chromosomal breakage test after crosslinker exposure and the normalFANCD2 Western blot. At least one of the mutations in FANCD2 patients ishypomorphic and associated with residual protein function (19). Therefore,residual FANCD2 protein can be detected in Western blots of all FANCD2patient cells using longer exposures. Defects in FANCI are associated withreduced FANCD2 protein levels (20), albeit monoubiquitination of the residualFANCD2 protein can be detected (Figure 2B).Figure 2. FANCD2 Western blots for identifying the defect in the FA/BRCA

Fanconi Anemia: Guidelines for Diagnosis and Management, Fourth Edition, is the result of a Consensus Conference held by the Fanconi Anemia Research Fund in Herndon, Va., April 5-6, 2013. It replaces earlier editions published in 1999, 2003, and 2008. These guidelines are published for physicians who