Delineation of an unknown significance FANCA genetic variant in a recurrent breast cancer patient

Abstract

Fanconi anaemia is a heterogeneous condition associated with mutations in the Fanconi anaemia complementation group (FANC). The FANC group has also been extensively associated with tumourigenesis due to its intricate association with the cellular repair mechanism. In this case report, we are drawing initial associations between a previously unreported FANC-A gene point mutation (P1222L) and familial breast cancer, by examining the presentation and management of a 65-year-old female patient with history of bilateral breast cancer of two different histological categories (ductal and in situ lobular). Here, we present a further genetic analysis beyond the common clinical practice to understand the patient’s genetic predisposition and improve their long-term management.

Background

Breast cancer (BC) is the most common malignancy in women. This disease is the second-leading cause of cancer-related deaths among women in most industrialised countries. There are a lot of risk factors for development of BC with the strongest being family history. The overall risk of developing BC is 1.9–3.9 times higher in women with an affected mother or sister.¹ The mentioned risk increases with increasing number of relatives affected with BC and the decreasing age at which it was first diagnosed. Therefore, the evaluation of the pedigree is necessary to assess the likelihood of presenting of a predisposing gene within a family.² While, 10%–30% of BC cases are attributed to hereditary factors, only 5%–10% of BC cases are identified with a strong inherited component and a small fraction of these cases (4%–5%) is explained by mutations in high penetrant genes, such as BRCA1 and BRCA2, transmitted in an autosomal dominant manner.³ A further proportion is caused by a number of moderate penetrance genes.^4–6 These genes appear to interact, although this has yet to be completely elucidated.⁷

Fanconi anaemia (FA) is an autosomal recessive syndrome characterised by congenital abnormalities, progressive bone marrow failure and Fanconi anaemia complementation group A (FANCA).^{8 9} While BRCA1 or BRCA2 mutations are the most common causes of hereditary BC, other genes have been found to be implicated such as ATM, TP53, PTEN, CDH1, STK11 and PALB2. FANCA mutations and their implications in familial BC cases have not been as extensively studied and to the best of our knowledge it is large gene deletions and promoter sequence alterations that have been associated with BC development and progression.⁹ Consequently, they are very rarely screened potentially leading clinicians to believe that their patients’ tumour is driven by somatic rather than germline mutations. With this case report, we aim to highlight the association of the FANCA variant of unknown significance, P1222L with predisposition to BC, as well as to offer a hypothesis of how the P1222L FANCA may derange the FA/BRCA DNA repair mechanism.

Case presentation

A 65-year-old G0P0 woman presented with vaginal dryness and dyspareunia. Her medical history revealed bilateral BC of two different histological types. At the age of 53, she was diagnosed with locally advanced, invasive ductal carcinoma with middle grade differentiation, ER-/PR- and pronounced positivity to CerbB2 in the context of fibrocystic mastopathy. Given the genogram of the patient (figure 1),¹⁰ she was advised to undergo BRCA1 and 2 genetic testing which was negative for known carcinogenic mutations. The patient received three neoadjuvant cycles of chemotherapy, one every 21 days with docetaxel and trastuzumab. The patient did not agree to doxorubicin addition to her regimen given that she expressed concerns for the drug cardiotoxicity profile. The patient had a good response to treatment with an apparent decrease in tumour size. In May 2008, she had a left breast mastectomy, with sentinel lymph node biopsy, which was free of metastasis. Following the operation, the patient received five cycles of adjuvant chemotherapy with docetaxel and trastuzumab. Following the completion of the five adjuvant cycles, the patient was further treated only with trastuzumab 330 mg every 21 days for a total of 12 months. During the treatments, she was radiologically monitored every 3 months with a chest, upper and lower abdomen/pelvis CT and Echocardiogram. Unfortunately, at the 12-month follow-up, she was diagnosed with in situ lobular carcinoma of her right breast. The patient underwent right mastectomy. Her family history revealed the likelihood of presenting of a predisposing moderate or low penetrance gene within a family (figure 1) as her maternal aunts were diagnosed with BC and an uncle with pancreatic cancer diagnosed in young age.

Figure 1.

Patient genogram created with progeny online platform.

The patient was offered genetic investigation and counselling in our clinic, with ILLUMINA TRUSIGHT CANCER (MiniSeq, 2×100 bp read length) (online supplemental file 1). This panel screened for 94 genes and 284 single-nucleotide polymorphisms associated with a predisposition towards cancer and covered a multitude of biological processes as depicted in the GO_analysis (figure 2).¹¹ A 3665 Cytosine to Thymine transition in the FANCA gene was identified resulting in a non-synonymous Proline (polar, uncharged) 1222 to Leucine (Nonpolar, aliphatic) mutation in the FANCA (PDB accession number: 6LHS) protein primary structure. This mutation has not been previously reported either as pathogenic or as normal variant and consequently its significance remained dubious.

Figure 2.

GO_Biological analysis of illumina TRUSIGHT cancer gene set. ClueGO plugin V2.3.2/Cytoscape V.4 online network analysis freeware. TOR, Target of Rapamycin; UV, Ultra-violet. Asterisks signify statistical significance where no asterisk signifies p value (P) >0.05; *: P >0.05; *: P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

We employed bioinformatic analyses to predict structural changes that this mutation may infer and analysed its impact on protein stability and functionality. We reconstructed the proline site through 3 dimensional (3D) imaging and analysed the effects of this non-synonymous mutation (figure 3).¹² Free energy component analysis indicated a ΔΔG of 2.041 kcal/mol between the wild-type and the mutant, which is denoted increased protein stability. Additionally, the difference of vibrational entropy energy between the wild-type and mutant was predicted to be −4.586 kcal/mol/K, which indicated decreased protein flexibility (figure 4, online supplemental file 1). Consequently, through this analysis, we hypothesised that the decreased flexibility of the P1222L FANCA variant may alter protein–protein interactions necessary for FA/BRCA1 complex stability and functionality. Furthermore, we recreated the molecular pathway that FANCA is involved in and then assessed its tertiary functionality through its in vivo proteomic interactome. Members of the FA pathway that have been proven to associate with high affinity to FANCA are FANC-G F, L M, C and E. Nonetheless FANC-G provides the necessary docking sites enabling FANC-A interaction with BRCA1. Consequently, we analysed the 3D structure of the mutant FANC-A and -G complex (figure 5). The decreased flexibility of the mutant FANCA significantly affected the angles of a-helices of the mutant protein which resulted in aberrant interaction of the mutant FANCA with the wild-type FANCG. This aberrant FANC-A and -G interaction may result in decreased FANC-BRCA DNA repair complex functionality (figure 6).¹³ This hypothesis warrants further in vitro testing to reach biological significance.

Figure 3.

FANCA tertiary protein reconstruction and comparison of wild-type to mutant protein sequence through DynaMut online analysis platform.

Figure 4.

Δ vibrational entropy energy and visual representation of mutant FANCA. Amino acids coloured according to the vibrational entropy change on mutation. Blue represents a rigidification of the structure and red a gain in flexibility. Δ vibrational entropy energy between wild-type and mutant ΔΔSVib ENCoM is −4.586 kcal/mol/K, which leads to decrease of molecule flexibility. FANCA, Fanconi anaemia complementation group.

Figure 5.

Space cartoon representation of FANCA (orange) and FANCG (green/grey) interaction. Location of non-synonymous amino acid substitution within the FANCA/FANCG complex is shown in red. Decrease of angle between a-helices of FANC a with connector site of FANCG. Representation was created via Geneious 2020.2.3 and DynaMUT. FANCA, Fanconi anaemia complementation group A.

Outcome and follow-up

The sister of the patient has not accepted to undergo genetic testing for emotional reasons and to the best of our knowledge she is not under current medical observation. Patient is alive and undergoing annual screening (biochemical/haematological parameters and imaging) for recurrent disease. At present, she remains disease free, non-anaemic and asymptomatic.

Discussion

The characteristics of familial BC remain a controversial issue as several studies have shown inconsistent results. A number of studies have revealed that familial BC has some specific clinical features compared with sporadic cases. Indeed, some investigations have shown that women diagnosed with positive family history present an early age of onset, bilateral BC, advanced stage, lymph node involvement and negative hormone receptors with a less favourable prognosis,¹⁴ whereas others have found no significant differences in terms of distribution of age at diagnosis, histology, tumour stage, nodal involvement and hormone receptors status.¹⁵ Nevertheless, women with a strong family history of BC could inherit genetic alterations of high, moderate or low penetrance genes. Some of these rare BC predisposing genes are FANCA. Fanconi anaemia is a heterogeneous condition associated with mutations in the FANC (figure 6).

Figure 6.

Overview of the FA/BRCA pathway. ATR-Chk1 phosphorylates multiple FA and FA-associated proteins including FANCA, FANCE, FANCD2, FANCI and BRCA1. Other proteins in the FA core complex are necessary for the monoubiquitination of the ID complex. FA, Fanconi anaemia; FAAP, Fanconi anaemia-associated protein; FANCA, Fanconi anaemia complementation group A.

In this case report, we are drawing initial associations between mutations of one of the FANC group proteins, FANCA and BC predisposition. FANCA is known to encode for a DNA repair protein promoting cytogenetic stability. FANCA has been hypothesised to also be a member of the postreplication repair mechanism with having an additional cell cycle checkpoint function. The FANC complex is composed of eight proteins (FANC-A, B, C, D1, D2, E, F, G), while the FA pathway also includes FANCJ (BRIP1), BRCA1BRCA2, RAD51C and FAN1 (figure 6). The initial complex activates through the process of mono-ubiquitination of FANC-D2 and FANCI.¹⁵ These two proteins then form a complex which acts a scaffold of assembly of other DNA repair proteins (BRCA1, BRCA2, RAD51C and FAN1) further on DNA damage areas. Generally, the mutation index of FANCA gene is significantly high leading to a variety of phenotypes emerging. Mutations extend from point mutations to large intragene deletions.^{15 16} Nonetheless, our patient, as far as they were aware, did not have any ancestry association with these populations. Most FANC complex members have been associated with the development of various types of cancers, solid and haematological in nature, which comes as no surprise taking into account their intricate association with the cellular repair mechanism. These malignancies span from acute myeloid leukaemia (AML), familial pancreatic cancer, primary cervical and of course BC.⁹ While the association of FANCD1 with familial breast is well established through is interaction with BRCA1/2 and RAD 51C, FANCA mutations are still not clearly established as drivers of BCdevelopment. FANCA deletions have been associated with familial BC syndromes in Finnish BC families and sporadic AML cases.^{17 18} Intriguingly founder mutations have been identified to stem from South African and Spanish Gypsy populations.¹⁹ A more recent study has demonstrated the association of a tandem duplication in the FANCA promoter region with high penetrance familial BC.⁹

An alternative pathway to the one presented in the current report has been described in a few studies.^{20 21} It has been proposed that FANC mutations can damage mitochondrial function that may in-turn predispose normal cell conversion to the cancer cell counterparts.^{20 21} A number of studies have evaluated the effect of FANCA mutations in BC. Litim et al, identified 24 novel polymorphisms in the FANCA gene in high risk non BRCA1/2 BC individuals from the French Canadian population.²² Moreover, del Valle et al identified 35 pathogenic variants in eight genes by analysing 1021 hereditary cancer patients.²³ They demonstrated a significant association between carriers of FANCA mutations and the risk of developing BC.

The majority of affected individuals in our patients’ genealogical tree had already deceased by the time the patient came under our care, making it extremely difficult to trace the FANCA P1222L mutation across generations and consequently prove its pathogenic or synergistic effect on carcinogenesis. As FANCA is not a commonly screened gene in cases of putative familial BC, this work could significantly benefit from in vivo murine model experiments in order to prove the P1222L as a pathogenic variant and to further understand its effects on the cellular DNA repair mechanisms in an in vivo rather than an in silico environment.

Patient’s perspective.

Having an answer of what might be going wrong, what might be the cause, and that it may not be just a wrong lifestyle choice, helps you to undergo the treatments without feeling that your health problem was of your own making, that it was not your fault. This helps you focus your efforts to fight cancer, an external “enemy”. I am just grateful that my doctors searched beyond the obvious and spent time to search for a cause. Even though not for sure, that potential explanation and seeing their efforts, gave me the boost I needed to keep on fighting.

Learning points.

• Fanconi anaemia complementation group (FANCA) P1222L mutation, which is considered a variant of unknown significance, may, through destabilisation of the FANC-BRCA1 complex, increase breast cancer risk.

• FANCA P1222L mutation decrease of FANC-A flexibility which in turn may affect complex interaction.

• The diagnosis of a genetic predisposition to carcinogenesis doe not only involve and affect the patient examined, but their entire first degree family member. The implications of disseminating a positive result expand beyond the scope of medical diagnosis and should be carefully communicated not only to the patient but also to the family members that might be affected given that consent has been obtained bilaterally. This process should also involve psychological as well as genetic counselling.