METHODS Patients A cohort of 50 North American families with children diagnosed with primary HLH were studied with approval by the internal review board of the Cincinnati Children’s Hospital Medical Center. The majority of children were diagnosed and treated at institutions throughout the US and had blood samples sent to Cincinnati Children’s Hospital for NK function testing, and perforin and granzyme B expression studies using flow cytometry. Most patients presented with hepatomegaly, splenomegaly, neutropenia, and hypofibrinogenaemia. Lymphopenia, hypertriglyceridaemia, central nervous system involvement, fever, anaemia, and thrombocytopenia were also widely reported. Diagnosis was later confirmed through contact with the referring institution if the diagnosis at the time of testing was uncertain. Patients with viral associated HLH were not included in this study unless a positive familial history (multiple affected children or consanguinity) could be demonstrated. DNA samples were retained from 43 of the 50 families and later sequenced for mutations in PRF1.

PRF1 gene sequencing Patients were screened for the presence of mutations in the coding exons and exon–intron boundaries of PRF1 by PCR amplification of genomic DNA followed by direct sequencing of the PCR products. Genomic DNA was extracted from either fresh peripheral blood or from Epstein-Barr virus transformed B cell lines using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN, USA). PCR was used to amplify the coding exons 2 and 3 of the PRF1 gene, including the exon–intron boundaries, using the following primers for exon 2: 59-CCCTTCCATGTGCCCTGATAATC-39 and 59-AAGCAGCCTCCAAGTTTGATTG-39; and exon 3: 59-CCAGTCC TAGTTCTGCCCACTTAC-39 and 59-GAACCCCTTCAGTCCAAG CATAC-39. Amplification of 500 ng of DNA was performed in a 50 ml assay of 16PCR buffer, 1.5 mmol/l MgCl2, 0.2 mmol/l dNTP, 0.4 mmol/l of each primer, and 2.5 U Taq DNA polymerase (Life Technologies Inc., Rockville, MD, USA). Reaction conditions were 3 minutes at 95 C followed by 30