The face of patients with FA has particular identifiable characteristics, such as a triangular face, bilateral epicanthic folds, micrognathia and middle facial hypoplasia. Ocular findings such as microphthalmia, cataracts, astigmatism, strabismus, hypotelorism, hypertelorism and ptosis have been described (30,31). The neck may be short with low implantation of the hairline, pterygium Colli, Sprengel deformity and Klippel-Feil anomaly (2).

Cardiac malformations can be persistent arterial ducts, atrial or ventricular septal defects, coarctation of the aorta, common arterial trunk and situs inversus totalis (32). Only 5% of patients have gastrointestinal abnormalities such as tracheoesophageal fistula, esophageal, duodenal or jejunal atresia, imperforate anus, annular pancreas and intestinal malrotation (33).

Limb defects are the most suggestive for diagnosis. However, limb defects are not observed in all patients, and may involve both the upper and lower extremities unilaterally or bilaterally (34). In the upper extremities, the radius, thumbs and hands (hypoplasia of the thenar and hypothenar eminence) are the most affected regions and less frequently, the ulna (Fig. 3A; most common malformations of the thumbs and palms of patients with FA). In the lower extremities, congenital dislocation of the hip, syndactyly and talipes have been reported (30).

In 2% of patients, spina bifida, scoliosis and abnormalities of the vertebrae (hemivertebrae, rib abnormalities, coccygeal aplasia) may be observed (2). Bone abnormalities have also been reported in the middle ear, associated with conductive hearing loss or alterations in the conformation of the auricular pavilion, which may be dysplastic or absent, low-set ears, narrow or absent internal auditory canal, absent tympanic membrane, microtia and/or fused ossicles (29).

Kidney malformations, such as horseshoe kidney, ectopic, hypoplastic, dysplastic or absence of one kidney in addition to hydronephrosis or hydroureter, have been reported in patients with FA (30). Males can present with hypospadias, micropenis, cryptorchidism, oligospermia or azoospermia, and abnormal spermatogenesis is associated with infertility (26). Females may exhibit malposition of the uterus, bicornuate uterus and smaller ovaries. Up to 50% of women are infertile, and when they achieve pregnancy, they can have complications of rapid progression, such as bone marrow failure, preeclampsia and premature delivery (29).

In the endocrinological field, >60% of affected individuals may present with a short stature due to growth hormone deficiency, hypothyroidism, or glucose or insulin abnormalities. Due to the endocrinological abnormalities, affected individuals should be monitored with regard to their hormonal profile (35,36).

The alterations described in the skin are generalized hyperpigmentation, hypopigmentation and cafe-au-lait spots (Fig. 3B; Cafe-au-lait spots and hypopigmentation in patients with FA evaluated by our FA group). The areas of hyperpigmentation are primarily on the trunk, neck, groin and armpits, with a mottled pattern of large patches and diffuse boundaries (30).

Malformations such as small pituitary gland, an absent corpus callosum, pituitary stalk interruption syndrome, cerebellar hypoplasia, hydrocephalus and dilated ventricles in the central nervous system have been described. Additionally, some affected individuals may present with neurodevelopmental delays and intellectual disability (33).

The cells of patients with FA exhibit chromosomal instability generated by the presence of unrepaired damage during the S-phase; stagnation in the G2 phase or passage to mitosis without adequate DNA repair has been proposed as one of the mechanisms that induces the depletion of hematopoietic cells by cellular senescence and the presence of damage that eventually leads to bone marrow failure, myelodysplastic syndrome or acute leukemia (2,16).

The age of onset of bone marrow failure is very variable, even in the same family, and it rarely manifests in the lactation period. The average age of onset of hematological symptoms is 7 years (9). Bone marrow failure is one of the manifestations most commonly associated with FA, so in patients with mild or imperceptible congenital malformations, the diagnosis tends to be delayed until the onset of cytopenia (37).

Generally, at the onset of the disease, thrombocytopenia or leukopenia are present, followed by anemia in fewer cases (38). In several cases, macrocytosis and increased fetal hemoglobin are observed. As bone marrow failure advances, progression to pancytopenia occurs; therefore, patients with persistent and idiopathic FA cytopenia should be suspected (39). According to Kutler et al (40), individuals with FA have a 90% risk of developing a hematologic abnormality by the age of 40.

Patients with FA have a high risk of developing myelodysplastic syndrome, which usually precedes acute myeloid leukemia; this condition is associated with chromosomal findings in the bone marrow, such as a gain of 3q, monosomy 7 or deletions in 7q (2,41).

As mentioned above, involvement of the hematopoietic system is the most common clinical feature in FA. The failure of the bone marrow occurs early in the life of those affected, and the median survival rate is 21 years if it is not treated early (9). Currently, the only curative treatment to restore the function of the hematological system is hematopoietic stem cell transplantation. However, this procedure requires the intervention of a trained team due to the risk of recurrence, graft-versus-host disease, and mortality (42). Recently, new treatment strategies, such as gene therapy have been implemented, in which the complications associated with hematopoietic stem cell transplantation may be avoided (38). One of these therapies uses lentiviral vectors to transfer a functional copy of a specific gene (the gene that is mutated) to autologous hematopoietic stem cells. These newly edited cells remove hematologic abnormalities in the patient and restore bone marrow cell function. The mobilization of the stem cells of the patient from the bone marrow into the peripheral blood has been proposed to collect CD34⁺ cells, correct the genetic alterations, and then infuse the cells into the patient (38). To date, these therapies have a good safety profile, but additional studies are required to investigate the possible long-term effects.

In patients with FA, solid tumors have an accumulative incidence of 28% by the age of 40 years old (40,43). Solid tumors commonly occurring in the anogenital area, and the head and neck are 500 and 700 times more frequent in patients with FA than in the healthy population, especially in cases with transplanted hematopoietic stem cells (44). Tumors in the brain, in the liver (secondary to androgen treatment) and in the kidney (as Wilms tumor) can appear as other tumors (40,44). With this condition in mind, the patients should be monitored throughout a patient's life.

The most frequent carcinomas associated with FA are squamous cell carcinoma of the head and neck, preferentially located in the oral cavity, with the tongue being the most commonly affected region (45). Carcinomas appear at an earlier age than in the general population (20-40 years old) and the patients may exhibit exacerbated radiosensitivity to therapy (46,47). Patients with FA have a high risk of cancer associated with human papillomavirus, which is why they should be vaccinated (48).

The defects in DNA repair in these patients makes them extremely sensitive to chemotherapy and radiotherapy; therefore, the suggested therapy is surgical resection of the tumor. However, in cases where the diagnosis is belated and large tumor sizes are encountered in surgical management, the use of radiotherapy or chemotherapy is recommended as the only scheme or in association with surgery (43,49). Currently, the development of more secure and effective therapy protocols for the treatment of squamous cell tumors of the head and neck has been prioritized. Preclinical trials are being performed with drugs already approved for the treatment of cancer, with efficient cytotoxic and cytostatic activity that is nongenotoxic for the cells of patients with FA (50).

Fig. 3 presents the images of the most common alterations of 2 patients evaluated by our FA group. Fig. 3A corresponds to a 10-month-old male patient, with low height and weight at birth, bilateral thumb hypoplasia, ectopic right kidney, renal tubular acidosis, dysgenesis of the corpus callosum and colpocephaly, hypochromic spots, very brown skin and mild pancytopenia. Fig. 3B and C correspond to a 3-year-old male patient. Low height and weight, Left preaxial polydactyly, bilateral hypoplasia of thenar eminence, café-au-lait spots, hypochromic spots, mild VSD and aplastic anemia.

The cells of patients with FA exhibit exacerbated sensitivity to cytoreduction regimens used for bone marrow transplantation and hypersensitivity to agents that cause DNA interstrand crosslinks (60). This characteristic is the basis for the chromosome breakage test, which exposes lymphocytes or fibroblasts from individuals with suspected FA to cisplatin, MMC or DEB in vitro (61,62).

The test consists of counting both spontaneous and induced ruptures in the metaphase chromosomes of the patients after exposure of the cells to the aforementioned agents, and comparing those ruptures with those of a healthy control individual with similar demographic characteristics. The number of chromosomal breaks per cell, the presence of radial figures, and the proportion of aberrant cells (one or more breaks/cell) are identified and recorded. A patient with FA will exhibit a significant increase in chromosomal breaks and radial figures compared with the control individual, although there may be variations in this value in patients with FA with somatic mosaicism (62,63). At present, cytogenetic tests are considered the gold standard in the diagnosis of FA (64). Fig. 4 shows the different fragility expression chromosomal events that must be evaluated to establish the diagnosis of FA.

In ~25% of cases of FA, patients may present with somatic mosaicism, which reduces or eliminates lymphocyte sensitivity to clastogenic agents. The presence of this condition causes difficulties in the identification of affected individuals utilizing the chromosomal breakage test (65). Somatic mosaicism in Mendelian hematopoietic disorders is the process in which one pathogenic mutation is reversed in a cell of a tissue, resulting in a group of cells with the defect corrected (66). In FA, this process occurs primarily in hematopoietic stem cells or lymphocyte progenitors. The corrected cells proliferate and clonally expand, improving the blood counts of the individual and thus reducing the incidence of bone marrow failure and hematological malignancy (67).

The cytogenetic test analyzes peripheral blood T cells; in this way, a high proportion of reversed T cells can lead to a false-negative result. Some individuals without FA may present with a proportion of T cells sensitive to DEB or MMC treatment, which could be interpreted as mosaicism, generating false positives (64). To resolve the overlap between patients with mosaic FA and non-FA patients, and to discriminate non-mosaics in patients with FA, it is proposed to use the chromosome fragility index (CFI), which calculates the quotient between the percentage of aberrant cells (cells with 1 or more breaks) and the number of breaks in multi-aberrant cells (cells with 2 or more breaks): [CFI = percentage of aberrant cells x (number of aberrations/ number of multi-aberrant cells)]; Fig. 4 shows the establishment of the number of breaks per chromosome or chromosomal event. For the Spanish population, a patient with suspected FA and a CFI >55 is considered to have FA, while within the group diagnosed with FA, when a patient has <40% aberrant cells, it is considered mosaic (64). In studies of patients lymphocytes, where they have been reported as normal or inconclusive and reversal mosaicism of their bone marrow mutation, but FA is suspected, a test of sensitivity to ICL-inducing agents in fibroblasts is recommended (62).

In ~70% of cases of FA, the cause of the disease are pathogenic variants in the FANCA gene. Although the majority of these variants are produced by point mutations, up to 44% of cases can be caused by small deletions/insertions and large intragenic deletions. These types of variants have not only been described in the FANCA gene but also in other genes related to the disease (68).

MLPA allows the detection of intragenic deletions in FANC genes and is recommended for the initial screening of patients when the proportion of large intragenic deletions amongst mutated alleles is high, as it happens for the FANCA gene (69). This test is useful to confirm or dismiss compound heterozygosis, which explains the phenotype of the patient. Additionally, the analysis for the search for large intragenic deletions can be performed by array-CGH, which allows establishing the extension of the deletions beyond the limits of any FANC gene, and the exact points of breakage and loss in the chromosome (68). The array-CGH test is important as the additional loss of other genes involved in the deletion may contribute to the phenotype of the patient (68).

With the advent of NGS, the identification of new genes associated with FA has been achieved and has allowed the analysis of several genes involved in different diseases, where clinical diagnosis is not easy. Currently, 22 genes have been confirmed to cause FA.

Within the molecular test, clinical exome sequencing or the panel of genes specifically analyzes the exons of the genes that are involved in the disease. However, despite having improved coverage and specificity, the molecular test has the disadvantage that not all panels include the same number of genes and have a low cover of intronic regions (70). Due to the above disadvantages, the specialist must ensure that the requested study contains all the genes associated with FA. For carriers or prenatal tests, the sequencing of a single gene or a specific mutation is indicated.

Once the pathogenic variant associated with FA has been identified, carriers in the family can be identified. The case of an autosomal recessive inheritance pattern will require the study of the parents to determine their carrier status and the risk to offspring. In the case of identifying a female carrier under the context of an X-linked recessive disease, the specialist should advise not only on the risks but also on the options for having healthy children, such as preimplantation diagnosis (2). In situations where the disease is considered sporadic, the patient must attend a consultation with his geneticist if they wish to have children.