Whole Exome Sequencing Identifies Novel Lyst-Missense Mutations In Incomplete Childhood Chediak-Higashi-Syndrome Presenting As Hemphagocytic Lymphohistiocytosis (HLH)

Chediak Higashi Syndrome (CHS) is caused by defective membrane targeting of components of the lysosome, which results from inactivation of the lysosomal trafficking regulator LYST. Clinically, CHS is typically characterized by partial albinism, susceptibility to infection, lymphoproliferation with acceleration to HLH. The immunodeficiency can be cured by allogeneic stem cell transplantation (HSCT), but transplanted patients can develop picture resembling spinocerebellar degeneration in early adult life. Depending on the type of mutation, CHS can vary from a most severe childhood form with null-mutations to milder adult onset forms with hypomorphic mutations. We report on a previously healthy boy, who presented at the age of 3 years with life threatening features of HLH but no clinical features of CHS. The patient was treated with HSCT from an unrelated HLA-identical stem cell donor 4 years ago and is developing normally since. Analysis of HLH candidate genes did not result in the identification of the genetic cause at that time. At the time of the next pregnancy whole exome sequencing of DNA that had been obtained before HSCT was performed to enable specific genetic counseling. The Agilent SureSelect Target Enrichment Kit was used and the captured fragments were sequenced as 100 bp paired reads using an Illumina HiSeq2000 sequencing instrument. All sequenced DNA reads were preprocessed using Trimmomatic (Lohse et al. 2012) to clip adapter contaminations and to trim reads for low quality bases. The remaining reads greater than 36bp were mapped to build hg19 of the human reference genome with Stampy (Lunter & Goodson, 2011), using default parameters. Following such preprocessing, the number of mapped reads was >95% for all samples. Single-nucleotide variants (SNVs) were called using SAMtools mpileup (Li et al. 2009). The number of exonic SNVs varied between 23,741 and 31,418 per sample. To facilitate a fast classification and identification of candidate driver mutations, all identified coding SNVs were comprehensively annotated using the ANNOVAR framework (Wang et al., Nat. Rev. Genet., 2010). To identify possible pathogenic mutations, candidate SNVs were filtered for nonsynonymous, stopgain or stoploss SNVs, requiring an SNV quality greater or equal to 100, and requiring absence of segmental duplications. Only SNVs that were not contained in dbSNP were considered for further analysis. No homozygous and 122 heterozygous SNVs meeting those requirements were identified. Only one gene, LYST, was affected by two different SNVs and was selected for further analysis because of its known relationship to HLH. Sanger sequencing confirmed the compound heterozygous genotype for the two novel LYST missense mutations Q3057K and R3785H in the patient and the heterozygous genotype for one of these mutations in the parents. We then specifically searched for typical features of CHS in the pre-HSCT diagnostic material. The typical large lysosomal granules in blood cells could not be identified. By contrast, light microscopy of the patient’s hair showed a silvery aspect and chunky dyspigmentation in the medulla. Little granular melanin was detected in the hair cortex. Electron microscopy revealed an uneven distribution of pigment and giant melanosomes in some keratinocytes, compatible with a partial albinism. We thus conclude that this patient suffers from an incomplete albeit immunologically most severe Chediak-Higashi syndrome, which led to an early accelerated phase resembling primary HLH. This report highlights the diagnostic power of whole exome sequencing, which enables an unbiased mutation analysis and the identification of unexpected causes of genetic diseases with atypical phenotypes. At the same time, this case also highlights some of the ethical challenges associated with diagnostic genomic analyses: While a specific and clinically validated diagnosis enabled specific genetic counseling, the family now has to face the unexpected uncertainty about the neurologic prognosis of incomplete Chediak-Higashi-syndrome, which may possibly progress into untreatable neurodegeneration during early adulthood despite successful allogeneic stem cell transplantation. Apart from adding to the knowledge of the genetic and phenotypic complexity of CHS, this patient also underlines the necessity of careful counseling before diagnostic genomic analyses are offered to patients and their families.