This document provides an overview of Fanconi anemia, including its pathogenesis, diagnosis, and management. Fanconi anemia is a genetic disorder caused by defects in DNA repair pathways that leads to bone marrow failure and cancer predisposition. It is diagnosed through tests showing chromosomal fragility when blood or skin cells are exposed to DNA damaging agents. Management involves treating bone marrow failure through blood product transfusions or hematopoietic stem cell transplant, as well as cancer screening due to the increased risk of malignancy.

1. PATHOGENESIS, DIAGNOSIS AMD MANAGEMENT OF
FANCONI ANAEMIA

2. OVERVIEW
• Introduction
• History
• Pathogenesis
• Diagnosis
• Treatment

3. Introduction
• Fanconi anaemia is a genetic instability disorder
• Chromosomal fragility
• Defective DNA repair
• BM failure
• Peripheral blood cytopenia
• Developmental anomalies
• Increased risk of haematological or solid neoplasm
• 98% inherited as Autosomal Recessive, rarely as XLR/ AD

4. History
• In 1917 Guido Fanconi-
• Observation of three brothers with a lethal hyperchromic anaemia
• Anisocytosis and poikilocytosis
• No splenomegaly
• Possibility of familial pernicious anaemia
• Few days later he discarded his diagnosis because of the lack of the
typical bone marrow findings at autopsy
Lobitz, S., Velleuer, E. Guido Fanconi (1892–1979): a jack of all trades. Nat Rev Cancer 6, 893–898 (2006)

5. He mentioned
“Die Knochenmarksdysfunktion, die zum
perniziösem Blutbild führt, ist
wahrscheinlich auch nur ein Zeichen einer
ererbten Minderwertigkeit”
The bone marrow dysfunction that leads to
the pernicious blood film is probably only a
symptom of an inherited deficiency

6. • In 1929 Erwin Uehlinger
• Constitutional anaemia
• Often associated with pancytopenia and
• Congenital malformations
• In 1931 Otto Naegeli
• Introduced the name ‘Fanconi's Anaemia'

7. Pathogenesis
• FA genes are involved in DNA repair
• 22 genetic groups (A, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, Q, R, S, T,
U, V, W) of FA genes have been proposed
• FANC C was the 1st discovered FA gene in 1992 in Toronto
• The most commonly mutated genes are FANCA (∼61%), FANCC
(∼16%), and FANCG (∼10%)
Che R, Zhang J, Nepal M, Han B, Fei P. Multifaceted Fanconi Anemia Signaling. Trends in Genetics. 2018
Mar 1;34(3):171–83

8. Major function of FA genes
• Repair interstrand DNA crosslinks
• Three steps in the FA DNA damage
response pathway
• Core complex
• ID2 complex
• Downstream effector complex

9. Core complex-
• Recruited to fork-like DNA structures via FAAP24 and FANCM
• Nine FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG,
FANCL, FANCM, and FANCT) and
• FA associated proteins (FAAP20, FAAP24, and FAAP100)
• Form a single large nuclear protein “core complex”
• Functions as a ubiquitin ligase

10. ID2 complex-
• Heterodimer of FANCI and FANCD2 is
called the ID2 complex
• Binds to an arrested replication fork
at interstrand DNA crosslinks
• Activated core complex converts the
heterodimer
• From unubiquitinated isoforms to
monoubiquitinated isoforms

11. Downstream effector
• ID2 complex forms binding interface for DNA
and downstream effector complexes
• The scaffold protein FANCP/SLX4 binds first
• Then endonucleases cleave DNA interstrand
crosslinks
• This results in DNA adducts and dsDNA breaks
• DNA adducts are resolved by translesion
synthesis complex.
• dsDNA breaks are repaired by homologous
recombination

12. • Three DNA repair processes are associated with FA genes
• Nucleotide excision repair
• Translesion synthesis
• Homologous recombination
• Nucleotide excision repair-
• Excises one DNA strand at the interstrand crosslink
• Via interaction between FANCP/SLX4
• Translesion synthesis
• One strand incised around the interstrand crosslink
• ICL unhooked
• Uncut strand extended
• Recruitment of translesion polymerase

13. •Homologous recombination
• Initiated after incisions by
endonucleases (FANCQ/ ERCC4)
• dsDNA break defect generated
• Repaired using the homologous
sequence from the sister chromatid
•Other DNA-repair proteins
• MRE11-RAD50-NBS1, PCNA
• Involved in the later stages of the DNA
repair response

14. Pathology of cells with mutated FA genes
Due to defect in DNA damage repair response
• Accumulation of DNA adducts,
• Failure to arrest DNA synthesis in response to DNA damage,
• Defective nonhomologous end joining,
• Abnormal induction of p53,
• G2/M cell cycle arrest,
• Increased apoptosis and
• Replication induced telomere damage- short telomeres

15. Cell Survival and Oxidative Stress
• Protein-protein interactions between FA proteins and non-FA
“binding partners”
• Important for cell survival
• Signal transduction and activators of transcription
• FANCD2 form complexes with STAT family
• Heat shock proteins
• Provide several cell survival signals
• FANCC protein facilitates the anti-apoptotic role of Hsp70

16. • Reactive oxygen species (ROS)
• Elevated in FA cells
• Increased DNA damage
• Increased hematopoietic stem cell (HSC) senescence
• Decreased HSC pool
• Leading to BM failure
• sensitive to ROS due to impaired DNA repair mechanisms
• Deficiency in superoxide dismutase
• Leads to poor cell growth at ambient oxygen
• Ex-vivo culture with anti-oxidant
• reduced DNA damage, improve HSC reconstitution ability

17. Bone Marrow Failure
• Marked reduction in both multipotent (HSC) and oligopotent cells (CMP, MEP)
• Reduced colony-forming cells in almost all patients after aplastic anemia
• But modest reduction in granulocyte-monocyte progenitors is noted
• Pathogenesis
• Increased BM cell apoptosis
• Mediated by Fas, contain death domain
• TNF-α, INF-γ, Fas ligand, and dsRNA mediated exaggerated apoptotic
responses

18. • FA patients’ BM
• IL-6 production is reduced
• TNF-α generation is markedly increased
• Transforming growth factor-β (TGF-β) signaling
• Suppressed in FA cells
• Decreased survival and proliferation of HSPCs
• Resulted in non-homologous end-joining
• Leukocyte telomere length
• Significantly shortened
• Despite increased telomerase activity
• Due to defect prone DNA repair mechanism

19. Clinical Features
Short stature, microcephaly, microphthalmia, Café au lait spot and hypopigmented area
and dangling thumbs
Alter BP et all, Hematology of Infancy and Childhood, 4th ed. Philadelphia, PA: WB Saunders, Inc, 1993: 216-316

20. Incidence of physical abnormality and cytopenia
• 30% have no physical anomalies during the time of diagnosis
• 25% may be diagnosed on physical anomalies without cytopenias
• 7% have no physical malformations or cytopenias at the time of
diagnosis

21. Physical Findings
• 70% of individuals with FA
• Growth deficiency- Prenatal/ postnatal short stature, low birth weight
• Abnormal skin pigmentation (40%). Generalized hyperpigmentation;
café au lait macules, hypopigmentation
• Skeletal malformations of upper limbs, unilateral or bilateral (35%):
• Thumbs (35%). Absent, hypoplastic, bifid, duplicated
• Radii (7%). Absent or hypoplastic (only with abnormal thumbs),
• Hands (5%). Flat thenar eminence, clinodactyly, polydactyly
• Ulna (1%). Dysplastic, short Shimamura A, Alter BP. Pathophysiology and management of inherited bone marrow failure
syndromes. Blood Rev. 2010 May;24(3):101-22

22. • Skeletal malformations of lower limbs (5%):
• Syndactyly, abnormal toes, club feet
• Congenital hip dislocation
• Microcephaly (20%)
• Ophthalmic (20%)
• Microphthalmia, cataracts, astigmatism, epicanthal folds
• Genitourinary tract anomalies:
• Renal (20%). Horseshoe, ectopic, pelvic, hypoplastic
• Males (25%). Hypospadias, micropenis, cryptorchidism
• Females (2%). Bicornuate or uterus malposition, small ovaries

23. • Endocrine disorders (50%-75%).
• Hypothyroidism (30%-60%),
• Diabetes (8%-10%)
• Insensitivity to growth hormone
• Hearing loss (10%).
• Conductive secondary to middle-ear bony anomalies
• Congenital heart defect (6%)
• PDA, ASD, VSD, CoA
• Gastrointestinal (5%).
• Esophageal, duodenal, or jejunal atresia
• Imperforate anus
• Tracheoesophageal fistula

24. • Central nervous system (3%)
• Small pituitary
• Absent corpus callosum,
• Cerebellar hypoplasia and hydrocephalus
• Facial features (2%).
• Triangular face shape,
• Microcephaly
• Low-set ears
• Epicanthic folds
• Hypertelorism
• Micrognathia
• Developmental delay and/or intellectual disability is seen in 10%

25. VACTERL-H
• vertebral abnormalities
• anal atresia
• cardiac defects
• tracheoesophageal fistula
• esophageal atresia
• renal and radial abnormalities
• limb abnormalities
• Hydrocephalus
• Any 3 + hydrocephalus is diagnostic
• Indicates AR or XLR disease

26. Natural history and prognosis
• Most serious consequence is BM failure
• Other causes of death
• Complications of HSCT
• Progressive malignancy/ consequence of therapy
Patients with biallelic FANCD1/BRCA2 mutation
Probability of cancer is 94% within 6 years of age
AML, Wilm’s tumour, Neuroblastoma

27. Predisposition to Malignancy
• Causes
• Chromosome fragility,
• Defect in DNA repair,
• Genomic instability, and
• Oxidative stress
• Two families of cancer predisposition syndromes
• Disorders of DNA repair (like ataxia telangiectasia, xeroderma
pigmentosum, and Bloom syndrome)
• ERCC4 mutations in is the similar genetic event
• other inherited BM failure disorders like SDS and DC
• Develop AML/solid malignancy

28. • Clonal cytogenetic abnormalities-
• 67% by 30 years of age
• Recurring cytogenetic abnormalities are
• Monosomy 7,
• rearrangement or partial loss of 7q,
• rearrangements of 1p36 and 1q24-34,
• rearrangements of 11q22-25
• Partial trisomy and tetrasomy of 3q26-q29
• rapid progression to MDS or AML

29. • By the age of 40 years,
• Incidence of non-hematologic cancers is 28%
• Most frequent solid tumor
• SCC involving the head and neck (mostly tongue)
• SCC of upper and lower esophagus
• SCC of vulva or anus, cervix
• Liver tumors (benign and malignant)

30. Diagnosis
Peripheral Blood
• Cytopenia in one or more lineages in the 1st decade of life, usually
between 4 and 8 years of age
• 5% patients develop hematological changes during the first year of life
• Thrombocytopenia with RBC macrocytosis usually develops initially
• Subsequent onset of granulocytopenia and then anemia
• Increased HbF
• Elevation of RBC MCV and HbF is higher than acquired aplastic anemia
• RBC lifespan- shortened

31. Bone Marrow features
• Early stages, BM normocellular or erythroid hyperplasia
• Sometimes with dyserythropoiesis, dysplastic changes may be noted
• Megaloblastic-appearing cells, nuclear–cytoplasmic dyssynchrony,
binucleated erythroid cells and hypolobulated megakaryocytes
• As the disease progresses
• BM becomes hypocellular and fatty,
• Sometimes in a patchy manner,
• Shows a relative increase in lymphocytes, plasma cells, reticulum cells,
and mast cells
• Incidence of BM failure by 40 years of age reaches to 90%

32. Abnormal Chromosome Fragility
• Abnormal chromosome fragility is the hallmark of FA
• Recommended diagnostic test
• Metaphase preparations of peripheral blood lymphocytes or skin fibroblasts
• Cultured with phytohemagglutinin (PHA)
• Enhanced by adding a DNA interstrand cross-linking agent, such as
mitomycin C (MMC) or diepoxybutane (DEB)
• karyotype is characterized by chromatid breaks, rearrangements, gaps,
endoreduplications, and chromatid exchanges

33. • Diagnostic implications
• Increased numbers of chromosome breaks per cell
• Normal control range of 0.00 to 0.10
• Positive range of 1.06 to 23.9
• tri-radial and quadriradial figures are supportive to diagnosis
Aberrant chromosomal events considered for the quantification of chromosomal breaks

34. 100% breaks, triradials and quadrilaterals were observed in Mitomycin induced
cultures of patients sample

35. • Few patients with clinical FA
• Do not show increased chromosome breakage
• When treated with DEB or MMC
• Due to hematopoietic cell somatic mosaicism
• Genetic correction in a hematopoietic stem or progenitor cell,
• Gene conversion events,
• Back mutations, or
• Compensatory deletions or insertions.

36. When FA is strongly suspected
and
chromosome fragility test on peripheral blood cells is negative
skin biopsy is performed
chromosomal breakage in cultured fibroblasts with DEB or MMC

37. • Chromosomal Breakage Syndromes
• Transmitted in an AR mode of inheritance
• Exhibit elevated rates of chromosomal breakage or instability
• Chromosomal rearrangements in ionizing radaiations
• Defect in DNA repair mechanisms or genomic stability
• Increased predisposition to cancer
• Most common are
• Ataxia telangiectasia (#ATM gene)
• Bloom syndrome (#BLM gene)
• Fanconi anaemia
• Xeroderma pigmentosum (#XP group of genes)

38. Immunoblotting for FANCD2
• Used as a diagnostic test for FA
• Tool to direct specific gene testing
• FANCD2 protein is analyzed in lymphocytes or fibroblasts after
exposure to MMC or radiation by immunoblotting
• The blot can distinguish the unubiquitinated and monoubiquitinated
FANCD2

39. • If chromosomal breakage assay is positive
• FANCD2 not detected in immunoblot, biallelic null mutations in this
gene
• detection of FANCD2 without its monoubiquitinated form suggests
mutations in one of the upstream core complex genes
• detection of monoubiquitinated FANCD2 suggests mutation in one of
the downstream FA genes
• Gene sequencing had replaced this technique as more rapid and effective

40. Flowcytometry
• FA cells exposed to DNA interstrand crosslinking agents
• Arrested in the G2/M phase of the cell cycle,
• 4N DNA cellular content
• Can be detected by flow cytometry
• Hybridization with nonmutated cell normalize the cell cycle
• Used to diagnose FA
• Due to issues of reproducibility and instrumentation cost, this method
is not used clinically

41. • Serum α-fetoprotein
• Stable, elevated levels
• Independent of liver disease and androgen therapy
• Unchanged after HSCT
• Imaging Studies
• USG of the abdomen may reveal intra-abdominal anomalies in
kidneys, urogenital system, and the gastrointestinal tract
• Ionizing radiation usually avoided

42. Summary of Diagnostic Approach
Fanconi Anemia Research Fund [Internet]. www.fanconi.org. Available from: https://www.fanconi.org/clinical-care/chapter-2

43. Differential Diagnosis
• 30% of FA patients do not have physical anomalies
• Diagnosis delayed until develop aplastic anemia, MDS, AML, or macrocytic
RBCs
• Suspected in solid malignancy at lower age group
• CA cervix < 30 years
• SCC of the head and neck < 50 years of age
• Chromosomal fragility test in “idiopathic” aplastic anemia
• All patients < 40 years
• Who failed treatment with ATG and Cyclosporine

44. Treatment
• Observation for stable patients
• Surveillance for solid cancers
• Androgens
• Growth factors
• Stem cell transplant
• Surgeries
• Others

45. Initial evaluation
• Personal and family history
• Physical examination
• Complete blood counts and biochemistry
• Bone marrow aspirate and biopsy, for- cellularity, morphology,
cytogenetics, ringed sideroblast
• Chromosomal fragility
• USG to identify inta-abdominal abnormalities
• HLA typing for HSCT

46. Stable patients
• Minimal to moderate cytopenia
• Non transfusion dependent
• Screening schedule
• CH every 3 months
• BM examination every year
• More frequent observation indicated when
• BM dysplasia
• Cytopenia
• Cytogenetic abnormality

47. Surveillance Programme for Solid Malignancy
Initiated after age of 10 years or 1 year after HSCT
For solid cancer, annual surveillance for oral cavity, 6 monthly examination
recommended, as risk increases X700
SCC in head and neck
Rhinopharynoscopy

48. For female
• Increase risk of
• Vulval SCC X400 risk
• Cervical SCC X 200 risk
• Annual gynaecological screening started at 13 years of age
• 85% cases are HPV +ve
• Quadrivalent HPV vaccine recommended at 9 years of age

49. Androgens
• Treatment response rate is 50%
• Reticulocytosis usually 1st documented evidence
Hb improves by 1-2 months
WBC improvement
PLT improvement

50. • Indications of Androgens
• Hb <8 gm/dl or Symptomatic anaemia
• ANC <500
• PLT <30,000/ dl
• Oxymetholone
• Dose is 1-5 mg/kg
• Additional corticosteroids
• Stabilize vessels and
• Decrease bleeding

51. • Danazole
• Attenuated androgens
• Dose is 2-5 mg/kg
• Lower incidence of masculinizing side effect in female
• Oxandrolone
• Causes of subclinical virilization
•Nandrolone
• Injectable androgen
• Preferred in hepatic impairment
• Dose is 1-2 mg/kg/week

52. Mechanism of action
• Stimulation of haematopoiesis
• Increase erythropoietin
• Anti-inflammatory effect
• Protection from apoptosis
When response is maximum/ adequate
• Slowly tapered
• Maintained at lower dose
• Never stopped
• Ultimately become refractory and progress to BM failure

53. Complications of Androgen Therapy
• Masculinization in female
• Peliosis hepaticus
• Cystic dilation of hepatic sinusoids with blood
• Usually silent or rt. quadrant pain
• LFT- normal
• Diagnosed by USG
• Usually regress by stopping androgens
• If ruptures- life threatening

54. • Hepatocyte damage
• Cholestatic jaundice/ transaminitis
• Usually reversable by holding therapy
• If not corrected, hepatic biopsy to exclude cirrhosis
• Hepatocellular adenoma
• Benign disease
• Rapidly increasing in size
• May cause life threating bleeding if ruptures
• Reversable by holding therapy
• Surgical removal or radio-ablation may be needed
• Hepatocellular carcinoma
• Rare
• APF is normal

55. Growth Factors
• G-CSF and GM-CSF
• Both increases ANC
• Indicated in sepsis with ANC <500
• Also increases HB and PLt
• Usually started with 5ug/kg/day; upto 40 weeks
• After response, dose may be reduced to 2-3 ug/kg/wk
• Addition of EPO + Androgen shows better outcome
• BM examination should be done before start of therapy and each year
• Document dysplasia/ leukemic clone

56. Stem Cell Transplant
• Only curative therapy available
• Best donor – HLA matched sibling, when FA excluded by
• Physical examination
• Peripheral blood counts
• Chromosomal breakage studies
• Genetic testing

57. • Indication od HCST
• Cytopenia (ANC <500/ PLt <20,000/ HB<7 gm/dl) and transfusion
dependent
• High risk for MDS/AML (monosomy ch7, trisomy/tetrasomy 3q,
deteriorating counts, blast>5%)
• Overt AML
• Complications during HSCT
• >20 blood product transfusion- delayed engraftment and poor
survival
• Risk of alloimmunization

58. Conditioning regimens-
• Initially – standard myeloablative protocols with standard GVHD prophylaxis
• Later reduced dose regimens used IVO increased chemosensitivity of cell
• Radiation abandoned doe to increased susceptibility to solid cancers
• Modern protocol
Cyclophosphamide (40mg/kg) + Fludarabine (140-180mg/kg) + ATG
With
T- Cell depleted Stem Cell
With
Cyclosporin/Sirolimus + MMF/Methylprednisolone
• 5 years OS is >90%

59. Choice of Donor
• 1st choice-
• HLA matched sibling donor,
• 8/8 matched (HLA- A, B, C, DRB1)
• MUD/ Umbilical cord stem cell
• 2nd choice
• One antigen mismatch
• 3rd choice
• Two antigen mismatch
• Haploidentical transplant

60. Preimplantation diagnosis
In-vitro fertilization
HLA matched and non-biallelic FA mutated embryo selected
Implantation
Cord blood transplanted to diseased

61. Others
• Surgery
• Structural deformities- limit functional delays
• Malignancy
• Endocrine disturbance
• Growth hormone- for decreased growth velocity
• Diabetes mellitus
• Hypothyroidism
• Gene therapy
• Vector induced transduction of wild FA gens
• Induced chimerism

62. Take Home Massage
• FA is an inherited AR disease.
• So far, 22 FANC genes and associated proteins found to be associated
• Defective DNA repair mechanism is the main pathology
• Diagnosed by clinical features, physical abnormalities, bone marrow
hypoplasia and genetic testing
• Increased chromosomal breakage is the hallmark
• Transfusion support, chelation, hormonal adjustment are useful
• Androgens have response in 50% patients but hepatic complications
• AlloHSCT is only curative therapy for cytopenia
• Increased predisposition to solid malignancy and needs regular surveillance