The functional effects of all of the FANCD1/BRCA2 mutations need to be examined more closely, to determine whether those with two mutant alleles have truly Fanconi anaemia or variant Fanconi anaemia, and to provide appropriate genetic counselling, risk assessment and cancer surveillance to relatives from both sides of the family. Our analyses suggest that FA‐D1 is indeed phenotypically distinctive among the Fanconi anaemia complementation groups, despite the known heterogeneity within Fanconi anaemia itself. Focusing on the specific types of mutations in BRCA2 may be instructive with regard to how patients with FA‐D1 differ from patients with other types of Fanconi anaemia, and with regard to why biallelic mutations in a breast/ovarian cancer susceptibility gene give rise to a totally different syndrome from that seen in heterozygotes.
To tackle this question more definitively, it is important to note that two of the patients classified as FANCD1/BRCA2
actually had only one deleterious BRCA2
allele, with a benign polymorphism in the trans allele; they should be included in defining a new disorder with caution. In patient HSC230, the K3326X allele removes the last 92 amino acids at the C‐terminus, does not change the function of the gene product, is a polymorphism found in >1% of normal individuals, and is commonly found in linkage disequilibrium with a known deleterious mutation.28,31
In addition, this patient clearly belongs to the FA‐B complementation group, with a defined deleterious X‐linked mutation in FANCB
The other patient with benign polymorphism, 900/1, has N372H, which also lacks known functional consequence, and is not associated with increased cancer risk.30,31
The report of very early‐onset breast cancer in the grandmother on this side of the family, absent direct information that she carries this allele, is provocative but not informative. Unless a truly deleterious mutation is found to be in linkage disequilibrium with the N372H allele, it is possible that patient 900/1 belongs to a different complementation group than FA‐D1, or even has another disorder. However, it must be emphasised that the usual methods for sequencing BRCA2
might miss large deletions or rearrangements, which might be in cis to the benign mutations reported in these two patients.32
Both patients carrying “benign” alleles had physical features consistent with Fanconi anaemia, as well as increased chromosome breakage, but 900/1 had T cell acute lymphoblastic leukaemia (not the typical AML of Fanconi anaemia) at age 5 years, whereas HSC230 had no cancer at age 2 years.
Six patients from five families with one deleterious and one missense BRCA2
mutation, the latter of unknown clinical significance with regard to familial breast cancer, are potentially very informative. Family histories of cancer were provided for only two of the five families, and neither was enriched with BRCA2
‐related cancers. However, it is striking that the five Fanconi anaemia‐associated missense mutations clustered tightly between amino acid positions 2336 and 2729, whereas the 20 truncation/frameshift mutations were uniformly distributed across the BRCA2
gene. This “FA cluster” is located in the most highly conserved BRCA2
region in an interspecies comparison of human/mouse/chicken BRCA2
, suggesting that these residues may be functionally constrained (data not shown). BRCA2
regions which are not implicated in breast/ovarian cancer pathogenesis in heterozygotic adults, nonetheless, may be critical interaction regions for a component of the Fanconi anaemia pathway, such as downstream of ubiquitination of FANCD2
This hypothesis provides a rationale for further evaluation of interactions between the BRCA2 protein and other proteins in the Fanconi anaemia DNA repair pathway. For example, these missense mutations (hypomorphs) may reduce the amount of functional BRCA2 protein.
Alternatively, at least some of the five families with an allele of unknown significance may be truly heterozygotic for a single biologically active BRCA2 mutation; their Fanconi anaemia could be due to unrecognised mutations in a different Fanconi anaemia gene, as we have shown with HSC230, who was reported to be an example of biallelic mutations in BRCA2 but who was clearly a patient with FA‐B.
In 19 of the 27 patients, and in 13 of the 20 families, both alleles were interpreted to be deleterious or probably deleterious; patient 4 was a compound heterozygote for two different null mutations. No patient was homozygotic for the same null allele, and thus the data on live‐born children with biallelic mutations in BRCA2
are insufficient to deal with the possibility that true homozygosity for mutations in BRCA2
is incompatible with life. Survival of homozygotic or compound heterozygotic mice with BRCA2
mutations seems dependent on mutation type and mouse strain.34,35
It would be of interest to determine whether mothers of patients with FA‐D1 experience excessive fetal loss.
Are there genuine genotype/phenotype associations among patients who do seem to have bona fide deleterious mutations of both BRCA2
alleles? Short stature, microcephaly, abnormal thumbs, hyperpigmentation or café‐au‐lait spots and renal and gastrointestinal (GI) anomalies were reported frequently, and short stature, microcephaly and GI anomalies were more common in the FA‐D1 group than in other patients with Fanconi anaemia.1
In the general population, the VATER association is >50 times more common than Fanconi anaemia (160 per 1
000 live births, respectively). It has been suggested that 5% of children in the VATER category may have Fanconi anaemia,36
and that 5% of patients with Fanconi anaemia have features consistent with the VATER association.24
The identification of five cases with VATER features among 27 patients (19%) in this series suggests that there may be a higher proportion of VATER association among patients with FA‐D1 than among those with Fanconi anaemia overall (p
0.01). However, the numbers are very small, and a VATER phenotype has also been reported in FA‐A, C, E, F and G. There may be specific BRCA2
mutations that are linked with VATER association in the absence of a clinical diagnosis of Fanconi anaemia.
The risk of early onset of specific cancers is clear and dramatic in the FA‐D1 group, in which the cumulative probability of leukaemia was 79% by age 10 years, that of a solid tumour was 83% by age 6.7 years, that of a brain tumour was 85% by age 9 years, that of a Wilms' tumour was 63% by age 6.7 years (data not shown) and that of any malignancy was 97% by age 5.2 years. These probabilities are much higher than that we observed in genetically unclassified patients with Fanconi anaemia, and in our literature review,26,37
in which the cumulative probability of leukaemia reached a plateau of approximately 30% by age 30 years, and that of a solid tumour 25% by age 30 years and 75% by age 45 years. Thus, patients with FA‐D1 have a distinctively higher risk of specific cancers than patients in other Fanconi anaemia complementation groups. In addition, the correlations between IVS7 with AML and both 886delGT and 6174delT with brain tumours suggest the possibility of specific genotype/cancer associations, although the numbers of cases and families reported to date are too small to draw firm conclusions.
The recent observation that all Fanconi anaemia groups, including FA‐J, but excluding FA‐D1, develop nuclear Rad51 foci after DNA damage29
underscores FA‐D1's distinctiveness. The aetiological role of BRCA2
mutations vis‐à‐vis the typical Fanconi anaemia birth defects or the specific cancers reported in these patients remains to be elucidated, although the associations with AML and brain tumours described above may be mutation‐specific. Greater attention must be paid to the biological consequences of the specific BRCA2
mutations identified in this context, particularly as, by conventional criteria, several seem to be without known functional consequences. How do alleles, the importance of which would be dismissed in hereditary breast/ovarian cancer (HBOC), act to cause the unique phenotype observed in patients with FA‐D1? Is there a protective interaction between a BRCA2 protein with a missense mutation and the normal BRCA2 protein, which is lost in patients with a truncated protein due to a deleterious mutation? Or might the recognition that some patients with FA‐D1 have one missense mutation imply that such mutations, heretofore of “unknown” significance, are, in fact, biologically important?
A larger, more systematically ascertained series of patients with FA‐D1 must be analysed to determine whether the severe clinical phenotype observed among the limited number of patients reported to date is truly characteristic of this Fanconi anaemia subgroup, or whether this represents biased ascertainment or reporting of patients with more severe disease. Most important, we must determine whether single copies of the presumably deleterious BRCA2 mutations detected in patients with FA‐D1 also predispose to the HBOC spectrum of cancer in heterozygotic family members. Counselling those individuals is particularly difficult at present, as they are ascertained as members of families with Fanconi anaemia, rather than as members of HBOC kindreds. In addition, some of them have altered BRCA2 alleles that are currently of unknown clinical significance. The converse clinical presentation is also important: should women of child‐bearing age from BRCA2‐positive HBOC families be counselled regarding the theoretical risk of Fanconi anaemia in their offspring if their partners carried unrecognised truncating or even missense BRCA2 mutations? Such events are within the realm of possibility, particularly in specific population groups that have a higher prevalence of BRCA2 mutations (eg, 1% of unselected individuals of Ashkenazi Jewish descent carry the 6174delT founder mutation). The rarity of FA‐D1 (~3% of all Fanconi anaemia) suggests that a registry targeting patients with FA‐D1 may be the most efficient means to deal with the many unanswered questions created by the unexpected nexus between the Fanconi anaemia and BRCA pathways.