# Presentation: 835535

## Fanconi Anemia

- Priya Gopalan
- April 7, 2006

- Case
- Clinical Features
- Diagnosis
- FA pathway
- Management
- Conclusions

## Case

- A 21 year old Mexican woman with a history of anemia, presented to a local clinic with a positive home pregnancy test.  After confirming her pregnancy (she was approximately 13 weeks pregnant), she had several labs drawn, including a CBC. She was found to be pancytopenic, and was told to come to BJC for admission.

## Case

- She complained of fatigue and easy bruising for many months, but no epistaxis, hematochezia, melena or hematuria.  Furthermore, her menstrual cycles had been regular and moderate until 3 months ago.

## Case

- Past Medical History:  She was first told that she was anemic 5 years ago, in Mexico.  At that time, she had a bone marrow biopsy performed.  She is not sure what it showed, but she was told to take Proverone (medroxyprogesterone) once a day, which she was unable to afford.
- Medications:  None
- Allergies:  None

## Case

- Social History:  She moved to the U.S. 2 years ago, from Mexico.  She is single and works as a housekeeper.  She denies any tobacco, alcohol or drug use.

## Case

- Family History:
- She has 3 siblings in the U.S., and 10 siblings in Mexico.  One sister in Mexico, who is now 15 years old, was just diagnosed with anemia, and had a bone marrow biopsy.  She is not sure what it showed.
- No family history of hematological disorders, cancers or early deaths.  Her grandparents and parents are alive and healthy.

## Case

- Physical examination:
- Height:  5’0”
- Significant only for bilaterally short thumbs
- No dermatologic abnormalities
- Labs:
- WBC:   2.1 (41%N, 53%L;  ANC  900)
- Hemoglobin / Hematocrit:  4.9/14.2 (MCV 111)
- Platelets:  17,000

## Case

- Other labs:
- CMP WNL (Cr 0.4)
-  Vitamin B12 and folic acid WNL
-  Reticulocyte 2.4%
- Hepatitis panel/HIV negative, ANA negative
- Peripheral smear:  normal-appearing WBC, RBC and platelets, but greatly reduced in number

## Case

- Bone Marrow Biopsy:
- Markedly hypocellular marrow, with markedly decreased trilineage hematopoiesis, consistent with aplastic anemia (cellularity <5%)
- No overt dysplastic features
- Cytogenetics were pending

## Case

- A diagnosis of Fanconi Anemia was suspected based on:
- History of “anemia” at age 16
- History of “anemia” in a sister, at age 15
- Short stature
- Shortened thumbs bilaterally
- Pancytopenia
- Bone marrow hypocellularity

## Fanconi Anemia

- Inherited recessive disease
- Usually autosomal recessive
- Recently, an X-linked form has been described (Meetei et al 2004)
- 12 different complementation groups
- Determined by somatic cell hybridization analysis
- 11 genes cloned

## FA complementation groups and FA genes

| FA complementation group | FA gene | Approx. frequency in FA patients (%) | Chromosomal Location |
| --- | --- | --- | --- |
| A | FANCA | 60 | 16q24.3 |
| B | FANCB | Rare | Xp22.31 |
| C | FANCC | 15 | 9q22.3 |
| D1 | BRCA2 | 5 | 13q12.3 |
| D2 | FANCD2 | 5 | 3p25.3 |
| E | FANCE | Rare | 6p21.3 |
| F | FANCF | Rare | 11p15 |
| G | FANCG | 10 | 9p13 |
| I | Unknown | Rare | Unknown |
| J | BRIP1 | Rare | 17q23.2 |
| L | FANCL | Rare | 2p16 |
| M | FANCM | Rare | 14q21.2 |

- from Kennedy and D’Andrea, Genes & Development, 2005

## Clinical features

- Incidence is 3/1,000,000
- Heterozygote frequency ~1/300 in U.S. and Europe
- Ashkenzai Jewish population carrier frequency 1/89
- Afrikaners carrier frequency 1/83
- Median age at diagnosis is 5-7 (Alter et al., Blood 2003)
- Median survival is 20-30 yrs. (Alter et al., Blood 2003)
- Phenotypic variability occurs even within families (Koc et al., Br J Haem 1999)

## Clinical Features

- Characterized by
- Congenital abnormalities
- Progressive bone marrow failure
- Increased risk of malignancies

## Frequency of abnormalities in FA

|  |
| --- |

| Abnormality | Frequency (%) |
| --- | --- |
|  |  |
| Skeletal (radial ray, hip, vertebral scoliosis, rib) | 71 |
| Skin pigmentation (café au lait, hyper- and hypopigmentation) | 64 |
| Short stature (median height 5th %ile) | 63 |
| Eyes (microphthalmia) | 38 |
| Renal and urinary tract | 34 |
| Male genitalia | 20 |
| Mental retardation | 16 |
| Gastrointestinal (eg, anorectal, duodenal atresia) | 14 |
| Cardiac abnormalities | 13 |
| Hearing | 11 |
| Central nervous system (eg, hydrocephalus, septum pellucidum) | 8 |
| No abnormalities | 30 |

**Tischkowitz, M D et al. J Med Genet 2003;40:1-10 ****(taken from Dokal, 2000)**

## (A) Typical radial ray abnormalities and 

- Copyright ©2003 BMJ Publishing Group Ltd.

**Tischkowitz, M D et al. J Med Genet 2003;40:1-10**

**(A) Typical radial ray abnormalities and **
**(B) cafe au lait patches and hypopigmentation **

## Clinical Features

- Most common etiology of inherited bone marrow failure
- Others include dykeratosis congenita, amegakaryocytic thrombocytopenia, Schwachman-Diamond syndrome
- Increased risk of MDS and AML (15,000x)
- Many have  monosomy 7, or duplication of 1q (Auerbach et al., Cancer Genet Cytogenet 1991)

**Clinical Features**

## Clinical Features

- Increased risk of solid tumor formation (hepatic, esophageal, oropharyngeal, vulvar)
- Average age at diagnosis is 23*
- Cumulative incidence ~30% by age 45**

- *Shimamura et al., Gene Reviews 2002 (genetests.org)
- **Alter et al. Blood 2003

## Diagnosis

- Pts. with congenital abnormalities are often diagnosed as neonates/infants
- Others may be diagnosed when hematological problems occur
- Median age of onset of pancytopenia is 7 (Butturini et al., Blood 1994)
- Usually normal CBC at birth
- First develop macrocytosis, then thrombocytopenia, and eventually neutropenia

## Diagnosis

- Based on chromosomal hypersensitivity to cross-linking agents
- Chromosome fragility test:  Mitomycin C (MMC) or diepoxybutane (DEB) added to lymphoctyes – increases the number of chromosome breaks and radial structures
- Very specific for FA, regardless of severity of disease
- Can do chromosome breakage analysis on amniotic cells, chorionic villus cells or fetal blood

## Diagnosis

- Difficult to screen using molecular testing due to multiple complementation groups and heterogeneity of mutations
- Afrikaners:  60% of 46 pts. had deletion of exons 12-31 in FANCA (Tipping et al., PNAS 2001)
- Ashkenazi’s:  Many have mutation IVS4+4A  T (altered splice site and deletion of exon 4) in FANCC (Gillio et al., Blood 1997)
- Also used for carrier testing and prenatal diagnosis

## Niedernhofer et al., Science 2005

## FA cells were treated with mitomycin C and harvested in metaphase. Typical abnormalities include radial formation (green circle) and chromosome breaks (red arrows).

**Chromosome breakage in Fanconi Anemia cells**

## FA pathway

- During S-phase, DNA replication is mediated by DNA polymerases acting at multiple replication forks
- Alkylating agents such as MMC and DEB induce cross-links, halting replication at the forks
- When replication is halted due to cross-linking, the FA pathway is activated

## Activation of the FA pathway

- Activated during the S phase in response to DNA replication fork arrest
- Leads to local single-stranded DNA
- Single-stranded DNA binds to, among other proteins, ATR (ataxia telangiectasia and RAD3-related protein, an S-phase checkpoint kinase)
- ATR phosphorylates many DNA repair and checkpoint proteins, including CHK1 (Checkpoint Kinase 1)
- ATR and CHK1 are somehow involved in activating the FA pathway (Seckel syndrome)

- Andreassen et al., Genes and Dev 2004

## FA gene products

- Complex 1 may detect stalled replication forks
- It appears to monoubiquitinate FANCD2 (at Lys 561)
- FANCL may be the catalytic subunit
- Complex 2 may be involved in DNA repair
- FANCD2-Ub relocalizes to chromatin (dependent on BRCA1)
- It associates with/may promote binding of other DNA damage repair proteins, including FANCD1 (BRCA2), BRCA1, RAD51, PCNA, NBS1, and possibly FANCJ (BRCA1-Interacting Protein [BRIP1])
- DNA repair pathways may be involved in the pathogenesis of inherited forms of breast cancer

- Kennedy and D’Andrea, Genes and Dev 2005,
- Fei et al., Cell Cycle 2005

## Schematic interaction of the FA pathway

**Richard D. Kennedy et al. Genes Dev. 2005; 19: 2925-2940**

**Schematic interaction of the FA pathway**

- L=catalytic element?

- =BRIP1

- and BRCA1, RAD51,
- PCNA, NBS1

## A schematic diagram depicting how the FA pathway may function to coordinate the cellular response to replication fork arrest following DNA damage

**Richard D. Kennedy et al. Genes Dev. 2005; 19: 2925-2940**

**A schematic diagram depicting how the FA pathway may function to coordinate the cellular response to replication fork arrest following DNA damage**

## A simplified model of FA-mediated cross-link repair that involves nucleotide excision repair (NER), translesion synthesis (TLS), and homologous recombination (HR)

**Richard D. Kennedy et al. Genes Dev. 2005; 19: 2925-2940,**
**adapted from Niedzwiedz et al. Mol. Cell 2004;  15:  607-620.**

**A simplified model of FA-mediated cross-link repair that involves nucleotide excision repair (NER), translesion synthesis (TLS), and homologous recombination (HR)**

## Initial management

- Refer for genetic counseling
- Testing of siblings
- Renal ultrasound, hearing test, eye exam
- Endocrine evaluation if evidence of growth failure (check growth hormone levels, TSH)
- Referral to hand surgeon for radial ray defects
- Bone marrow biopsy

## Management

- Bone marrow failure
- Transfusions
- Androgens (e.g. oral oxymethalone) – can improve blood counts in 50% of pts.
- Side effects:  Masculinization, acne, hyperactivity, premature closure of epiphyses, liver toxicity, hepatic adenomas
- Growth factors (G-CSF, CM-CSF) – should not be used in patients with clonal cytogenetic abnormalities
- Bone marrow transplantation
- FA cells are very sensitive to radiation and alkylating agents – can use greatly reduced doses
- 2-yr. survival 70% for allo;*  20-40% for MUD**

- *Guardiola et al. Bone Marrow Transplant 1998;
- **MacMillan et al., Br J Haematol 2000

## Management

- Surveillance for solid tumors
- 42% risk of malignancy 20 yrs. after transplant (Deeg et al. Blood 1996)
- Annual gyn exams
- If malignancy is diagnosed, treatment is limited by the increased sensitivity to DNA-damaging chemotherapeutic agents

## Management - Gene therapy

- Goal is to permanently correct hematological manifestations by transducing hematopoietic progenitor cells with a vector containing the deficient gene
- Knockout mice with FANCC using retroviral vectors - phenotypic correction (Gush et al., Blood 2000)
- Clinical trial disappointing (Liu et al., Human Gene Therapy 1999)
- Knockout mice with FANCA and FANCC using lentiviral vectors – more promising (integrates into the genome) (Galimi et al. Blood 2002)

## Conclusions

- FA is characterized by congenital abnormalities, bone marrow failure and predisposition to malignancies (leukemias and solid tumors)
- Diagnosis is based on hypersensitivity to cross-linking agents such as DEB and MMC
- FA pathway is involved in repair of DNA cross-links
- FANCD1 is BRCA2, FANCJ is BRIP1 and FANCD2 associates with BRCA1 and BRIP1, suggesting a role for DNA repair pathways in the pathogenesis of inherited breast cancers
- Treatment includes bone marrow transplantation

## Case

- The patient’s FA test was negative. She does, however, have aplastic anemia, and does want to keep her baby.  She met with Dr. DiPersio this week to discuss transplantation.

## The FA pathway may function to specifically coordinate more than one pathway following cross-linking damage, whereas IR and UV damage can be repaired through other pathways

**Richard D. Kennedy et al. Genes Dev. 2005; 19: 2925-2940**

**The FA pathway may function to specifically coordinate more than one pathway following cross-linking damage, whereas IR and UV damage can be repaired through other pathways**

## Figure 1. Some Features of Fanconi's Anemia.

**Figure 1. Some Features of Fanconi's Anemia.**
**The physical and cytogenetic findings in some patients with Fanconi's anemia are shown. Panel A shows the characteristically short thumb; Panel B shows a cafe au lait spot; and Panel C shows chromosomal breaks as revealed by cytogenetic analysis (A indicates chromatid breaks, B chromatid fragments, and C a dicentric chromosome). Other common clinical manifestations include short stature, skin hyperpigmentation, pancytopenia, and renal anomalies.**