A 2-year-old boy with a family history of consanguineous marriage and with no documented genetic disease in his family. He was quite well until he showed fever and abdominal enlargement that required medical advice, and therefore, the patient was admitted to the hematology unit in our pediatric hospital for suspicion of malignancy.
On admission, the patient’s temperature was greater than 38.5 °C and hepatosplenomegaly was present (liver span 13 cm, spleen span 11 cm). There were no neurological symptoms. Laboratory studies revealed bicytopenia (hemoglobin 7 g/dL and platelet 13 × 109/L) and notable abnormal findings related to liver function tests. He had an increased level of AST (109 U/L; reference, up to 40 U/L), ALT (116 U/L; reference, up to 41 U/L), LDH (898 U/L; reference interval, 120–300 U/L), total and direct bilirubin (1.1 and 0.8 mg/dL; reference, up to 1 and 0.2 mg/dL respectively), triglyceride level (520 mg/dL; reference interval, 60–160 mg/dL), serum ferritin (> 5000 ng/mL; reference interval, 70–140 ng/mL), and low level of fibrinogen (64 mg/dL; reference interval, 200–400 mg/dL), total protein (5.6 g/dL; reference interval, 6.5–8.5 g/dL), and albumin (2.9 g/dL; reference interval, 3.5–5.2 g/dL). NK function and perforin flow cytometry were not available in our institute. HLH diagnosis was suspected in our patient as he fulfilled five out of the eight required diagnostic criteria according to the HLH-2004 guidelines, but we screened him for serological markers of Epstein-Barr virus (EBV), cytomegalovirus (CMV), hepatitis A, B, and C and all were negative and bone marrow aspiration showed hyper-cellular bone marrow with absent erythroid series without hemophagocytosis. We did not repeat bone marrow aspiration, as our patient fulfilled five out of the eight HLH diagnostic criteria according to the HLH 2004 guidelines, and its repetition would be time-consuming regarding his poor general condition. We screened the patient for the presence of mutations (the screening was not performed for his parents) in the coding exons of PRF1 gene by PCR amplification of genomic DNA, followed by direct sequencing of the PCR products in the following steps:
- 1.
We extracted genomic DNA from fresh peripheral blood using Thermo Scientific Gene JET Whole Blood Genomic DNA Purification Mini kit (Thermo Fisher Scientific Inc., Carlsbad, CA, USA).
- 2.
We used PCR to amplify the coding exons 2 and 3 of the PRF1 gene, including the exon-intron boundaries, using the following primers for exon 2: 5′ CCCTTCCATGTGCCCTGATAATC-3′ and 5′ AAGCAGCCTCCAAGTTTGATTG-39; and exon 3: 5′-CCAGTCC TAGTTCTGCCCACTTAC-3′ and 5′-GAACCCCTTCAGTCCAAG CATAC-3′.
- 3.
We performed amplification of 500 ng of DNA in a 50-μL assay of 25 μL Dream Taq Green PCR Master Mix) (Thermo Fisher Scientific Inc., Carlsbad, CA, USA) 0.4 mmol/L of each primer, and to the rest of volume water.
- 4.
Reaction conditions were 3 min at 95 °C followed by 30 cycles of 45 s at 95 °C, 30 s at 60 °C, 1 min 45 s at 72 °C, and then 10 min at 72 °C.
- 5.
Detection of the amplification product by Agarose gel Electrophoresis.
- 6.
Primers used for sequencing were the same as those for amplification.
- 7.
We performed cycle sequencing using the Big Dye terminator cycle sequencing reaction kit (version 2; Applied Biosystems, Foster City, CA, USA) and separated the DNA fragments on an ABI Prism 3700 DNA Analyzer (Applied Biosystems).
- 8.
We compared sequences with the published PRF1 gene sequence (GenBank accession no. M28393) using MEGA software (Molecular Evolutionary Genetic Analysis) which is a powerful sequence analysis software package, Basic Local Alignment Search Tool (BLAST) on The National Center for Biotechnology Information (NCBI), and Chromas software.
Results revealed a novel homozygous frameshift mutation in PRF1 gene (M28393: exon 2: c.536delAinsCG p.F178fs) with the production of stop codon as shown in Figs. 1, 2, and 3.
We treated our patient medically with the HLH-2004 protocol including etoposide, dexamethasone, and cyclosporine A. He showed dramatic responses with a resolution of fever, decrement in size of hepatosplenomegaly, and correction of hepatitis and cytopenia. We plan for hematopoietic stem cell transplantation (HSCT), and he is on the waiting list as HSCT is not available in our institute.