Targeted high-throughput sequencing for genetic diagnostics of hemophagocytic lymphohistiocytosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rapid-onset, potentially fatal hyperinflammatory syndrome. A prompt molecular diagnosis is crucial for appropriate clinical management. Here, we validated and prospectively evaluated a targeted high-throughput sequencing approach for HLH diagnostics. METHODS: A high-throughput sequencing strategy of 12 genes linked to HLH was validated in 13 patients with previously identified HLH-associated mutations and prospectively evaluated in 58 HLH patients. Moreover, 2504 healthy individuals from the 1000 Genomes project were analyzed in silico for variants in the same genes. RESULTS: Analyses revealed a mutation detection sensitivity of 97.3%, an average coverage per gene of 98.0%, and adequate coverage over 98.6% of sites previously reported as mutated in these genes. In the prospective cohort, we achieved a diagnosis in 22 out of 58 patients (38%). Genetically undiagnosed HLH patients had a later age at onset and manifested higher frequencies of known secondary HLH triggers. Rare, putatively pathogenic monoallelic variants were identified in nine patients. However, such monoallelic variants were not enriched compared with healthy individuals. CONCLUSIONS: We have established a comprehensive high-throughput platform for genetic screening of patients with HLH. Almost all cases with reduced natural killer cell function received a diagnosis, but the majority of the prospective cases remain genetically unexplained, highlighting genetic heterogeneity and environmental impact within HLH. Moreover, in silico analyses of the genetic variation affecting HLH-related genes in the general population suggest caution with respect to interpreting causality between monoallelic mutations and HLH. A complete understanding of the genetic susceptibility to HLH thus requires further in-depth investigations, including genome sequencing and detailed immunological characterization.

Further characterization of atypical features in auriculocondylar syndrome caused by recessive PLCB4 mutations.

Auriculocondylar syndrome (ACS) is a branchial arch syndrome typically inherited in an autosomal dominant fashion. Patients with ACS display the following core symptoms with varying severity: a specific malformation of the external ear, known as a "question mark ear," micrognathia and mandibular condyle hypoplasia. Recently, phospholipase C, ß 4 (PLCB4) mutations were identified as the major cause of autosomal dominant ACS, with mutations of the PLCB4 catalytic domain predicted to have a dominant negative effect. In addition, one ACS patient born to related parents harbored a homozygous partial deletion of PLCB4, and presented with ACS plus central apnea and macropenis; these features had not been previously reported in association with ACS. His parents, each with a heterozygous partial PLCB4 deletion, were phenotypically normal, suggesting autosomal recessive inheritance of ACS, with complete loss of function of PLCB4 predicted in the patient. We herein describe two brothers with ACS caused by compound heterozygous splice site mutations in PLCB4. The patients were born to the same unrelated and healthy parents, with each parent harboring one of the mutations, indicating autosomal recessive ACS. Both patients reported here had mixed apneas, gastrointestinal transit defects and macropenis, in addition to typical craniofacial features of ACS. This is the first example of ACS caused by compound heterozygous splice site mutations in PLCB4, the second autosomal recessive case of ACS confirmed by molecular analysis, and strengthens the link between complete loss of function of PLCB4 and extra-craniofacial features.