1. NGS panel - Genetic testing for Fanconi anemia

Fanconi anemia

April 25, 2017

Clinical features

Fanconi Anemia (FA) is a rare inherited chromosome breakage syndrome characterized by physical abnormalities, bone marrow failure, and an increased risk of development of various malignancies. FA is the most common genetic cause of aplastic anemia and is one of the most common genetic causes of hematologic malignancy, with an estimated prevalence of 1:100,000 live births1. However, in some populations (Ashkenazi Jewish, Spanish Gypsy, and black South African) the carrier frequency of FA is estimated to be around 1:1001, 2.

Fanconi anemia (FA) is suspected in individuals with the following clinical features:

  • Physical abnormalities, present in approximately 75% of FA-affected include the following1 :

    • Abnormal skin pigmentation with café au lait spots is present in about 40% of all FA-affected cases, commonly associated with generalized hypopigmentation1
    • Malformations of the skeletal system are commonly present (in ~35% of cases1) in form of absent thumbs, absent or hypoplastic radii followed by weak pulse, congenital hip dislocation and syndactyly (in about 5% of FA-cases)1
    • Short stature, pre- or postnatal accompanied by low birth weight in a majority of the affected
    • Microcephaly or CNS-anomalies in 20% of FA-cases1
    • Ocular anomalies (in 20% affected) including cataracts, microphthalmia, strabismus, astigmatism, ptosis, and others.
    • Gastrointestinal and urinary system anomalies, including renal anomalies, hypospadias and cryptorchidism in men and small ovaries in women.

  • Progressive bone marrow failure manifested as thrombocytopenia, leukopenia, anemia, macrocytosis and/or increased fetal hemoglobin levels. These features are easily detected in blood sample laboratory analysis.
  • Increased risk of malignancy, including the following:

    • Adult-onset aplastic anemia.
    • Myelodysplastic syndrome (MDS).
    • Acute myelogenous leukemia (AML): the relative risk of AML is ~500 times higher than in non-affected people 3, 4
    • Solid tumors (early-onset) that may be the first manifestation of disease, including squamous cell carcinomas of the head and neck (most common form, with an incidence >700 times higher compared to normal population 1, 5), esophagus, and vulva; cervical cancer, and liver tumors.

Many different genes, primarily involved in DNA repair, underlie Fanconi anemia (FA) and these account for different phenotypic complementation groups. The Fanconi anemia complementation group currently includes the FANCA, FANCB, FANCC, FANCD1 (BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP1), FANCL, FANCM and FANCN (PALB2) genes (Table 1). FA-associated proteins (namely: FANCA, FANCB, FANCE, FANCF, FANCG, and FANCL) are assembled in a nuclear “FA core” complex, a multi-subunit ubiquitin ligase complex (see Figure 1). Additional FA proteins (FANCD2 and FANCI) are associated with the FA core complex, and after ubiquitination by ubiquitin ligase FANCL they are translocated to nuclear foci that include the “DNA repair” proteins BRCA2, RAD51, NBS1, and others (Figure 1). The latest proteins actively participate in DNA repair and therefore, when they carry a pathogenic variant, the DNA repair function is missing and as a result the DNA is no longer stable and tumors can develop.

Figure 1:
Schematic presentation of the Fanconi anemia DNA repair pathway: DNA damage activates Fanconi anemia complex which includes the Fanconi anemia-associated proteins FANCA, FANCB, FANCE, FANCF, FANCG and subsequently FANCL. FANCL is a ubiquitin ligase and it ubiquitinate FANCD2 and FANCI. This FANCD2/FANCI ubiquitinated/activated complex further activates “DNA repair proteins” associated with Fanconi anemia: BRCA2, RAD51, BRCA1, NBS1 and PCNA, subsequently leading to DNA repair.

CENTOGENE´s Fanconi anemia panel includes the 18 most relevant genes associated with FA: BRCA2, BRIP1, ERCC4, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, PALB2, RAD51C, SLX4, UBE2T, XRCC2 (Table 1).

The pathogenic variants of FANCA are numerous and highly variable among affected families1. A small percentage of families share the pathogenic variants p.Phe1263del and p.Val372AlafsTer42. The latter is found in affected individuals of northern European ancestry1, 15. Also, 3 common pathogenic variants in the FANCC gene have been identified in several affected families (c.456+4A>T, c.1642C>T, and c.67delG)6. Pathogenic variants in FANCG are highly variable, but more common variant alleles have been described in specific populations: c.307+1G>C (Korean/Japanese), c.925-2A>G (Brazilian), c.1480+1G>C (French Canadian), and p.Gly395TrpfsTer5 7 and p.Trp599ProfsTer49 8 (northern European). Also, pathogenic variants in the RAD51C gene have been recently identified in a few FA-affected consanguineous families, as well as in families with breast and ovarian cancer susceptibility16. Among the FA-associated genes is gene encoding BRCA2 which is required for homology-directed repair of chromosomal breaks. Thus, BRCA2 has a clear role in regulating homologous recombination by controlling the activity of RAD51. A rare genetic subtype of FA (FA-D1) is caused by mutations in the BRCA2 gene. According to some genetic studies, mutations in BRCA2 (FANCD1) account for ~3% of all FA cases1, 17. Recently, a new gene associated with FA was reported: another RAD51 paralog gene, XRCC2/FANCU, encoding protein involved in homologous recombination repair of DNA damage18. So far 13 mutations have been reported in the XRCC2 gene, 12 associated with breast cancer and only 1 associated with Fanconi anemia (truncated mutation c.643C>T; p.R215*)19.

Overview of the genes in CENTOGENE´s Fanconi anemia panel

Gene Complementation group OMIM Chromosomal locus Frequency of mutations
FANCA FA-A 607139 16q24.3 60%-70% 1, 9
FANCB FA-B 300515 Xp22.2 2%1, 9
FANCC FA-C 613899 9q22.3 14%1, 9
FA-D1 600185 13q13.1 3%1, 9
FANCD2 FA-D2 613984 3q25.3 3%1, 9
FANCE FA-E 613976 6p21.31 3%1, 9
FANCF FA-F 613897 11p15 2%1, 9
FANCG FA-G 602956 9p13 10%1, 9
FANCI FA-I 611360 15q26.1 1%1, 9
FA-J 605882 17q23.2 2%1, 9
FANCL FA-L 608111 2p16.1 13 variants reported 10
FANCM FA-M 609644 14q21.3 1 family reported 11
FA-N 610355 16p12.1 14 variants reported 12
FA-N 602774 17q22 1 family 13
FA-P 613278 16p13.3 Rare 14
FA-Q 133520 16p13.12 Rare 1
FA-U 179617 15q15.1 Rare 1
FA-T 510538 1q32.1 Rare 1

Allogeneic hematopoietic stem cell transplant is the only curative treatment for patients with Fanconi anemia20. However, alternative treatments are beneficial and include administration of oral androgens (improvement of blood counts in 50% of FA-patients), administration of G-CSF (granulocyte-colony stimulating factor), and others20.

CENTOGENE offers the Fanconi anemia panel, including full gene sequencing (genes: BRCA2, BRIP1, ERCC4, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, PALB2, RAD51C, SLX4, UBE2T, XRCC2) and deletion/duplication analysis of selected genes (FANCD2, BRIP1, FANCB, PALB2, BRCA2, FANCA, RAD51C). In addition, any of the genes in the Fanconi anemia panel can also be ordered individually, for full gene sequencing and deletion/duplication analysis.

Differential diagnosis

The differential diagnosis of Fanconi anemia- related disorders – depending on the major symptoms in the initial case – includes the following diseases

  • Chromosome breakage syndromes, such as Bloom syndrome or ataxia-telangiectasia
  • Nijmegen breakage syndrome (NBS)
  • Seckel syndrome
  • Neurofibromatosis 1 (NF1)
  • TAR syndrome (thrombocytopenia with absent radii)
  • VACTERL association

Testing strategy

CENTOGENE offers advanced, fast and cost-effective strategy to test large NGS panels and diagnose complex phenotypes based on the PCR-free Whole Genome Sequencing and NGS technology. This approach offers an unparalleled advantage by reducing amplification/capture biases and provides sequencing of entire gene at a more uniform coverage.

To confirm/establish the diagnosis, CENTOGENE offers the following testing strategy for Fanconi anemia using NGS Panel Genomic targeted towards this specific phenotype:

Step 1: Whole genome sequencing from a single filter card. The sequencing covers the entire genic region (coding region, exon/intron boundaries, intronic and promoter) for all the genes included in the Fanconi anemia panel. Copy Number Variants analysis derived from NGS data is also included.

Step 2: If no mutation is identified after analysis of the Fanconi anemia panel, based on the approval and consent, we further recommend to continue the bioinformatics analysis of the data obtained by whole genome sequencing to cover genes that are either implicated in an overlapping phenotype or could be involved in a similar pathway but not strongly clinically implicated based on the current information in literature.

Referral reasons

The following individuals are candidates for the Fanconi anemia panel testing:

  • Individuals with a family history of Fanconi anemia and presentation of the most common symptoms
  • Individuals without a positive family history, but with symptoms resembling Fanconi anemia
  • Individuals with a negative but suspected family history, in order to perform proper genetic counseling (prenatal analyses are recommended in families with affected individuals).

Test utility

Sequencing, deletion/duplication of Fanconi anemia-related genes should be performed in all individuals suspected for Fanconi anemia. In parallel, other genes reported to be related with this clinical phenotype should also be analyzed for the presence of mutations, due to the overlap in many clinical features caused by those particular genes.

Confirmation of a clinical diagnosis of Fanconi anemia through genetic testing can allow for genetic counseling and may direct medical management.

Genetic counseling can provide a patient and/or family with the natural history of Fanconi anemia, to identify at-risk family members, provide reproductive risks as well as possible preventive therapy or preconception/prenatal options, and allow for appropriate referral for patient support and/or resources.

More information on CENTOGENE´s Fanconi anemia panel can be found in our genetic test catalogue.