Haemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory condition in
which absent or markedly reduced T cell and natural killer (NK) cell cytotoxicity results
in uncontrolled proliferation of T cells, activation of macrophages, hypercytokinaemia,
pancytopaenia, and hepatosplenomegaly. Familial HLH (FHL), an autosomal recessive
disorder affecting young infants and children, is fatal unless treated with chemotherapy
and allogeneic haemopoietic stem cell transplantation. Mutations in the gene encoding
perforin, a lytic protein involved in cell cytotoxicity, account for 30% of cases of FHL
(FHL II). Mutations in genes encoding hMunc 13-4, syntaxin 11, and syntaxin binding
protein 2, all essential for perforin release and T/NK cell cytotoxicity, have also been
identified in FHL (FHL III IV and V respectively). A previous report form our group
showed that in the UK, in consanguineous families of Pakistani descent, FHL (designated
FHL I) maps to 9q21.3-22 in an area spanning 5Mb and containing 14 genes. The aim of
my project was to identify the gene responsible for FHL I and to study the function of the
corresponding protein. Based on the premise that similar to other types of FHL, the
protein product of FHL I candidate gene would function in the perforin-dependent
cytotoxic pathway, 4 out of the 14 genes emerged as strong functional candidates: KIF27,
RASEF, UBQLN1 and FRMD3.
My mutation screening strategy aimed at amplifying and sequencing all exons and intronexon
boundaries of the candidate genes, followed by screening the remaining genes of the
FHL I locus. The material used was either genomic DNA extracted from 4 parental and 1
patient B cell line, or cDNA from the latter. In 2 newly identified patients, genomic DNA
from granulocytes was used. PCR amplification of genomic DNA and direct sequencing
or cloning and sequencing of individual clones or in some cases direct sequencing of the
full length cDNA from the patient B cell line did not reveal any mutations in any of the
known genes in the FHL I locus. For KIF27, mutations were excluded in gene regulatory
areas by amplification and direct sequencing of the whole promoter region, up to 1kb
upstream of the transcription start site. KIF27 protein expression was also studied by
immunoblotting and immunostaining followed by confocal microscopy but no
abnormality was identified at protein level. Promoter area mutation was also excluded in UBQLN1, the other strong candidate. The gene responsible for FHL I remains
unidentified. Further refinement of the locus with high-density SNP arrays in newly
diagnosed patients demonstrating linkage to the FHL I locus might help identify the gene
responsible for FHL I.