A 3.5-year-old girl, Caucasian, who was a product of a consanguineous marriage (first-degree cousins, Fig. 1 (timeline of case presentation) and Fig. 2a) was registered in our department due to petechia and nose bleeding. She was born at 37 weeks and 5 days of normal vaginal delivery. Her birth weight, head circumference, and height were 2.8 kg, 32 cm, and 46 cm, respectively. Although her development was in the normal range, the growth chart revealed that her height and head circumference were below the third percentile line, and her weight was around the 5th percentile line. In addition, she took zinc supplements due to her short stature. On physical examination, short stature, poor weight gain, microcephaly (head circumference; 46 cm) and skin hyperpigmentation were detected. She had a history of two-time hospital admission due to pneumonia.
Due to petechia, complete blood count was performed and the results identified anemia (Hb: 7.2 g/dL), leukopenia (WBC: 1.5 × 103/μL with an absolute neutrophil count of 455), and thrombocytopenia (Platelet count: 9 × 103/μL). Other laboratory and imaging findings, including C3, C4, ANA, dsDNA, CH50, Immunoglobulin level, TORCH study, metabolic panel, biochemistry studies, abdomen and pelvic sonography, brain MRI, lumbosacral and both hands X-ray, and echocardiography were normal. In addition, bone marrow aspiration and trephine biopsy revealed presented megakaryocyte and moderate to severe hypocellular bone marrow.
The patient had been on irradiated packed cell and platelet transfusion approximately every one month. She was a candidate for allogeneic bone marrow transplantation, but she did not have the HLA-match donor. She was under regular follow-up and occasionally referred due to epistaxis or pallor, and received irradiated packed cell and platelet.
Next generation sequencing (NGS)
Written informed consent was obtained from the parents. Whole blood samples were collected using EDTA tubes. Genomic DNA was prepared from peripheral leukocytes of the patient using the QIAamp DNA Blood Mini Kit (Qiagen, Germany) and then NanoDrop (ND1000, USA) was used to measure DNA concentration.
NGS covering immunological and hematological disorders was carried out on Illumina NextSeq500 machine to the sequence close to 100 million reads. Bioinformatics analysis of the sequencing results was performed using BWA aligner [6], GATK [7] and annovar [8] as well as different databases and bioinformatics software such as REVEL, MCAP, ESP6500,1000G, Clinvar, CADD-Phred, SIFT, PolyPhen, GERP, PhastCons, LRT, Mutation Assessor, Mutation Taster, phyloP46way_placental, phyloP100way_vertebrate, SiPhy_29way, FATHMM_pred, RadialSVM, ExAC. Kaviar, GME, gnomAD.
Sanger sequencing
To confirm the novel identified mutation, we performed Sanger sequencing of the genomic DNA from the proband. For this test, PCR was carried out for the patient’s DNA using the following primers: F-FANCF: CGCTGGGAGATTGACATG and R-FANCF: GACCCCAGTCTGTTAGCA (PCR product: 978 bp) to amplify a mutated region of FANCF. Then, Sanger sequencing was used to sequence amplified DNA with both forward and reverse primers using ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems®, USA). The analysis of Sanger sequencing data was performed with NCBI BLAST and Codon Code Aligner software. Multiple sequence alignment analysis was performed using the SIB BLAST+ Network Service From (https://web.expasy.org/blast/) to compare the amino acid sequence of human FANCF proteins with other proteins across different Kingdoms. STRING (STRING: functional protein association networks, https://string-db.org), tool and KEGG database (KEGG: Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg/) were also used to explain the FA pathway and its protein network.
Cytogenetic examination
Owing to suspicion to inherited bone marrow failure, chromosomal study with MMC on the peripheral blood lymphocyte culture of the proband was requested. To evaluate the types and rates of breakages and rearrangements in the chromosomes of the cells in the proband, GTG banding and the chromosome breakage test were performed on the patient’s blood sample. The blood sample was then cultured and treated with different concentrations of MMC.
NGS revealed a novel, private, homozygous, frame-shift deletion mutation in the FANCF gene (FANCF-201, ENST00000327470.4, NM_022725: exon1, c. 534delG, p. G178 fs, position 22,625,277 on chromosome 11). Using Sanger sequencing, the mutation was confirmed in the proband as homozygous (Fig. 2b and c). The identified mutation has not been reported yet in any database of genomic variants including ESP6500, 1000 Genome Project, ExAC, Kaviar, GME, gnomAD, and our internal database (Bayan Gene), confirming the novelty of mutation. This is the first report of FANCF mutation in Iranian patient affected with autosomal recessive FA, complementation group F.
The comparative amino acid alignment of FANCF protein across most kingdoms was also carried out. As shown in Fig. 3, most of the residues were highly conserved during evolution, and any frame shift mutations could be deleterious.
In the cytogenetic study, 100 metaphase spreads were studied from cultures prepared by adding MMC and compared to age-related control. The chromosomal breakage scoring was performed on 5 different slides (one untreated with MMC, one with 150 nM MMC concentration, one with 300 nM MMC concentration and one normal control sample treated with these 2 concentrations of MMC). 25 metaphases were evaluated on each slide for chromosomal aberration (gaps or breaks or radial formations). The results showed about 7–8 breaks/cell on average. In comparison to normal control sample which showed 0.3–0.5 breakages/cell. There was no radial formation in the normal control sample. The study showed 46, XX with multiple breaks and radial formation (quadri and triradial), compatible with Fanconi Anemia (Fig. 4, Additional file 1).