Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

2.1. Materials

Genomic DNA (from e.g., leukocytes or fibroblasts derived from the proband or the parents) is adequate for most mutation screening assays. Screening on cDNA is more efficient but has several drawbacks: for high-quality cDNA, growing cells (stimulated leukocytes, lymphoblastoid cell lines, or fibroblasts) are necessary. In addition, common alternative splice variants will hamper the evaluation of DNA sequences. Therefore, screening on gDNA is the preferred method for mutation screening. However, during the diagnostic process, growing cells from the proband will be helpful in a couple of situations. Growing cells are indispensable for studying the effect of unclassified variants on splicing or to verify the disease gene by functional complementation of the cellular phenotype with a construct expressing a wild type copy of the suspected gene [35–37]. Finally, if no mutations can be de detected, growing cells can be used to reconfirm the diagnosis FA by checking MMC sensitivity in cell growth or G2-arrest assays [38, 39].

2.2. Mutation Screening Strategy

3.1. Mutation Screening in Mosaic Patients

If an available lymphoblastoid cell line from an FA patient is phenotypically normal due to genetic reversion at the disease locus, mutation screening is still possible in the reverted cell line, since at least one mutation will be present [49–51]. The second mutation may be identified through investigating the parents.

3.2. Unclassified Variants

Missense mutations or in-frame deletions or insertions should be judged using in silico prediction algorithms (SIFT, POLYPHEN2, Align GVGD). Alternatively, they can be tested for pathogenicity in a cellular transfection assay to check the ability of the variant gene product to complement the cellular FA defect in a deficient cell line (see e.g., [10, 35, 52]). Generally, these tests are only feasible in a setting where a diagnostic laboratory is equipped with a research laboratory with all necessary technology.

3.3. Functional Assignment to Genetic Subtypes

Retroviral constructs have been used to identify the FA subtype by functional complementation, as an intermediate step before a mutation screen is undertaken [36]. Although knowing the disease gene facilitates mutation screening, retroviral transduction has some drawbacks in comparison to direct mutation screening: (i) growing, MMC-sensitive cells either from a cell line or fresh blood sample are required, which are not always easy to obtain; (ii) overexpression of some FA proteins (e.g., FANCM and FANCP) may be toxic for cells; (iii) novel genetic subtypes that emerge after all known groups have been excluded and cannot be readily distinguished from false negatives, that is, transductions that for some unknown reason have failed to cause complementation; (iv) the method requires relatively advanced laboratory facilities and technology. However, functional assignment of complementation group can rapidly be provided by laboratories with capability for this type of analysis [37], which has greatly facilitated reliable genotyping for over 95% of FA patients for which viral constructs are available.

3.4. Genetic Counseling

All patients with a diagnosis of FA confirmed by mutation analysis should be referred for genetic counselling, together with their parents and siblings. Mutation testing should be performed in all sibs regardless of any clinical symptoms. A complete pedigree, including a cancer history anamnesis, should be prepared. Mutation carriers might be at increased cancer risk (see Section 3.7) whose aspect should be included in the counseling (see Section 3.7).

FA patients themselves usually have decreased fertility. Women usually have late menarche, irregular menses, and early menopause. However, pregnancies in women with FA have been described, and therefore women should be adequately informed about the risks for their offspring, which is mainly related to an increase in pregnancy-related complications [53].

Sibs of the parents of an FA patient often request carrier screening to assess their risk of getting a child with FA. If a sib appears to be carrier, this risk is still minimal because of the very low carrier frequency in the population. In the US the carrier frequency has been estimated to be about 1 in 181 [54]. The risk of a proven carrier to get a child with FA is therefore about 1 in 724. However, in small communities or in consanguineous couples this risk is much higher, and mutation screening in spouses of proven carriers may be indicated.

3.5. Prenatal Diagnosis

Prenatal diagnosis of FA is relatively straightforward after the pathogenic mutations in a given family have been identified. Fetal cells can be obtained by chorionic villus sampling (CVS) during weeks 10–12 of the pregnancy or by amniocentesis, which is performed between weeks 14 and 16. However, CVS may be preferred as the diagnosis will be known at an earlier stage. If the mutation is not known, a chromosomal breakage test on fetal material may be performed [55], but this test may be considered less reliable than screening for mutations in the fetal material. Alternatively, flow cytometric testing of MMC sensitivity in amniotic cell cultures might be an option; however this technique is only available in a limited number of specialized laboratories [56]. Occasionally, FA may be suspected by fetal ultrasound imaging and confirmed by parental carrier testing when the family is not yet known to carry a risk for FA [57].

3.6. Genotype-Phenotype Correlation

FA is considered as one disease, and the question may be raised whether all fifteen genetic subtypes equally conform to the clinical FA phenotype. Genotype-phenotype correlation studies comparing the 3 most common groups A, C, and G indicated modest phenotypic differences, which were rather correlated with the relative severity of the mutations [23]. However, bias due to the ethnic distribution of the studied population is very well possible. Other studies reported significant differences between FA-A/G versus FA-C [58]. Cases in group FA-D1 (mutated in BRCA2) and FA-N (mutated in PALB2) present with a distinct, relatively severe, phenotype that is characterized by the development of leukemia at very young age (median 2.2 years) and by pediatric cancers such as nephroblastoma (Wilms tumor) or medulloblastoma [40–44]. The observations that one of the pathogenic mutations in BRCA2 in FA-D1 patients is hypomorphic and that mice with biallelic null alleles in Brca2 are embryonic lethals suggest that the BRCA2 protein serves a function that is essential for survival.

Different mutations in the same gene may be associated with divergent phenotypes, as illustrated by the two FANCC mutations, c.711+4A>T and c.67delG. The former (splice-site) mutation is associated with a relatively severe phenotype in Ashkenazi Jewish people [19] although the associated phenotype was reportedly less severe in patients of Japanese ancestry [20]. The carrier frequency for this mutation in the Ashkenazi population is relatively high (1 in 87), which has led to the recommendation of carrier detection to prevent disease [59]. In the Netherlands more than 50% of FA cases are homozygous for the FANCC frameshift mutation c.67delG. The phenotype associated with this mutation, like other exon 1 mutations, seems relatively mild, as these patients rarely have skeletal abnormalities and show a relatively late age of onset of their marrow failure [24]. Awareness of such genetically determined phenotypic differences may help in clinical decision making, including the counselling of patients and families.

3.7. Cancer Risk in Heterozygous Mutation Carriers

An important issue is whether FA mutation carriers are at increased risk to develop cancer or other types of disease. Overall, there is no increased risk for cancer among FA heterozygotes [60, 61]. However, the situation is different in some of the less prevalent FA subtypes. The FA-D1 subtype is caused by mutations in BRCA2 [62] which is a well-known breast and ovarian cancer predisposition gene [63]. In FA-D1 one of the mutations will be hypomorphic because biallelic “severe” mutations are supposed to be lethal [26]. Therefore, one of the parents of a FA-D1 patient will be a heterozygous carrier of a “severe” inactivating BRCA2 mutation and may thus have an increased risk for breast cancer and other BRCA2-associated cancers. Whether the parent with the hypomorphic mutation is also at increased risk is unknown: in breast cancer families these hypomorphic mutations are considered as variants with unknown clinical significance. Two other genes involved in FA and related to breast or ovarian cancer predisposition are PALB2/FANCN [64, 65] and RAD51C/FANCO [66]. Although cancer patients have been identified with germ-line mutations in these genes, an accurate estimate of the relative cancer risk for mutation carriers is still lacking.

Another special case is represented by female FANCB mutation carriers, who are supposed to consist of 50% FA-like cells due to silenced expression of the wild type FANCB allele by the random process of X inactivation that occurs during early embryonic development. Nevertheless, in the few female FANCB mutation carriers studied so far, inactivation appeared strongly skewed towards the mutated allele [67]. This suggests that FA cells have a poor chance to survive next to unaffected cells in the same tissue, and these FA cells may therefore not give an increased cancer risk. However, the data are scarce at present so that no firm conclusions can be drawn regarding the cancer risk of female FANCB mutation carriers [60].