Fanconi Anemia via the RFWD3 Gene

Fanconi Anemia (FA) is an inherited disease associated with aberrant DNA repair, an increased risk of bone marrow failure (BMF), hematologic malignancy, and solid tumor development. Many FA patients develop acute myelogenous leukemia (AML), gynecologic or GI tract cancers, or cancers of the head and neck at an earlier age than patients in the general population (Auerbach. 2009. PubMed ID: 19622403). For example, FA patients may be up to 800 fold more susceptible to AML than patients in the general population with a median age of onset of 13 years (Rosenberg et al. 2003. PubMed ID: 12393424). FA affects all systems of the body. While cytopenias due to BMF are the most common presentation of FA, patients may or may not also have additional physical abnormalities and developmental delays, including radial ray defects (absent thumb or radius), skin pigmentation defects, short stature, microphthalmia, renal and urinary tract defects, genital defects (males in particular), gastrointestinal malformations (atresia), congenital heart disease, hearing deficits, and learning difficulties (Tischkowitz and Hodgson. 2003. PubMed ID: 12525534; Dokal. 2000. PubMed ID: 11030042). Up to 79% of FA patients have at least one physical abnormality (Fiesco-Roa et al. 2019. PubMed ID: 31351673) and about 33% of FA patients have three or more features of VACTERL-H (Vertebral, Anal, Cardiac, Tracheo-esophageal fistula, Esophageal atresia, Renal and upper Limb and Hydrocephalus) (Alter and Giri. 2016. PubMed ID: 27028275).

Diagnosis of FA can be complicated as up to one-third of FA patients have no obvious physical or developmental abnormalities and are diagnosed only after a family member is diagnosed, or after developing hematologic complications such as thromobocytopenia, leukopenia, or anemia (Giampietro et al. 1997. PubMed ID: 8986277). A hallmark of FA is hypersensitivity of chromosomes to inter cross-strand linkage (ICL) agents such as diepoxybutane (DEB) or mitomycin C (MMC) (Sasaki and Tonomura. 1973. PubMed ID: 4352739). Exposure of primary cell cultures from FA patients to DEB or MMC results in chromosomal aberrations (breaks, radials, rearrangements) due to damaged DNA repair mechanisms that require functional gene products of the 22 FA genes identified to date.

The FANCA, -B, -C, -E, -F, -G, -L, and -M proteins are part of a nuclear core complex that regulates monoubiquitination of the FANCD2 and FANCI proteins (ID complex) during S-phase and after exposure to DNA crosslinking agents (Moldovan and D'Andrea. 2009. PubMed ID: 19686080). In unaffected individuals, ubiquitination helps localize the ID complex to sites of DNA damage and facilitate repair (Grompe and van de Vrugt. 2007. PubMed ID: 17488615; Smogorzewska et al. 2007. PubMed ID: 17412408), but in FA patients, this mechanism is impaired. Several FA genes are also associated with increased risk of breast cancer including FANCS / BRCA1 (Miki et al. 1994. PubMed ID: 7545954), FANCD1 / BRCA2 (Wooster et al. 1995. PubMed ID: 8524414), FANCJ / BRIP1 (Pennington and Swisher. 2012. PubMed ID: 22264603), FANCN / PALB2 (Walsh and King. 2007. PubMed ID: 17292821), and FANCO / RAD51C (Meindl et al. 2011. PubMed ID: 21637635). FA is phenotypically diverse even among related patients that harbor the same pathogenic variants; null alleles however are reported to result in more severe phenotypes (Faivre et al. 2000. PubMed ID: 11110674). FANCB, -D1, -D2, -I, -J, and –N have been associated with at least one physical abnormality consistently among the literature (over 80% of patients in Fiesco-Roa et al. 2019. PubMed ID: 31351673). FA affects males and females roughly equally and affects all ethnic groups. The range of onset is birth to over 50 years of age, though onset occurs most frequently at younger ages with a median age of onset of 7 years (Shimamura and Alter. 2010. PubMed ID: 20417588; Fiesco-Roa et al. 2019. PubMed ID: 31351673). Given the genotypic and phenotypic heterogeneity found in FA patients, simultaneous genetic testing for a large subset of FA-related genes may provide the most efficient approach for establishing an accurate and timely diagnosis.

In the United States, the carrier frequency for Fanconi anemia is estimated at 1 in 180, and the incidence rate is estimated at 1 in 130,000 (http://www.fanconi.org/; Rosenberg et al. 2011. PubMed ID: 21739583).

FA is a genetically heterogeneous disorder. At least 22 genes have been associated with FA or FA-like disorders. Inheritance is primarily autosomal recessive, or with cases of FANCB, X-linked, though a case of heterozygous FA-like syndrome was associated with a dominant-negative variant in the RAD51 (FANCR) gene (Ameziane et al. 2015. PubMed ID: 26681308). Approximately 86% of all FA cases are attributed to variants in one of three genes: FANCA (~ 60%), FANCC (~ 16%), and FANCG (~ 10%) (Auerbach. 2009. PubMed ID: 19622403). Since variants in FANCA are the most common cause of FA, it is important to note that large deletions make up over one-third of all reported pathogenic variants in FANCA. Nearly 95% of all FA cases are attributed to variants in eight genes, FANCA, -C, -G, -D1 (aka BRCA2), -D2, -E, -F, and –L that are either part of the core complex required for ID complex ubiquitination and facilitation of DNA repair, or function directly in ICL recognition and repair (Grompe and van de Vrugt. 2007. PubMed ID: 17488615; Khincha and Savage. 2013. PubMed ID: 24246701).

To date, only one patient with RFWD3-associated FA has been reported which represents another subtype of FA defined as FANCW. This patient was born with several symptoms typical of FA including short stature, duodenal atresia, small midface, radial ray malformations, absent thumb, and other physical abnormalities. Beginning around age 10 the patient developed cytopenic bone marrow with various dysplastic features. Genetic analysis showed this patient was compound heterozygous for two pathogenic variants in RFWD3 that are located on opposite chromosomes (Knies et al. 2017. PubMed ID: 28691929).

RFWD3 encodes an E3 ubiquitin ligase that plays a key role in DNA damage repair (Liu et al. 2011. PubMed ID: 21558276). Knies et al. showed that cells from their patient were sensitive to cross-linking agents, a hallmark of FA, and demonstrated that variant RFWD3 protein disrupted homologous recombination and failed to properly localize in cells (Knies et al. 2017. PubMed ID: 28691929). Mice deficient in RFWD3 protein (Rfwde-/-) were viable with no overt phenotypes except a possible decrease in fertility. It is worth noting is that homozygous Rfwd3-/- mice were observed at a lower than expected frequency which may be explained by embryonic lethality. Skin fibroblasts from Rfwd3-/- mice were sensitive to DNA-cross linking agents and chromosomal breakage typical of FA (Knies et al. 2017. PubMed ID: 28691929).

Over 95% of all Fanconi Anemia (FA) patients harbor pathogenic variants in one of the 22 known FA or FA-like genes (www.fanconi.org). However, pathogenic variants in RFWD3 are a rare cause of FA.

This test is performed using Next-Gen sequencing with additional Sanger sequencing as necessary.

This test provides full coverage of all coding exons of the RFWD3 gene plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).