Additional file 2: of Screening assays for primary haemophagocytic lymphohistiocytosis in children presenting with suspected macrophage activation syndrome

Data set summary. Clinical information from 21 patients who underwent screening for suspected MAS clinical information. (DOCX 317 kb

Table_1_A rapid turnaround gene panel for severe autoinflammation: Genetic results within 48 hours.docx

There is an important unmet clinical need for fast turnaround next generation sequencing (NGS) to aid genetic diagnosis of patients with acute and sometimes catastrophic inflammatory presentations. This is imperative for patients who require precise and targeted treatment to prevent irreparable organ damage or even death. Acute and severe hyper- inflammation may be caused by primary immunodeficiency (PID) with immune dysregulation, or more typical autoinflammatory diseases in the absence of obvious immunodeficiency. Infectious triggers may be present in either immunodeficiency or autoinflammation. We compiled a list of 25 genes causing monogenetic immunological diseases that are notorious for their acute first presentation with fulminant inflammation and which may be amenable to specific treatment, including hemophagocytic lymphohistiocytosis (HLH); and autoinflammatory diseases that can present with early-onset stroke or other irreversible neurological inflammatory complications. We designed and validated a pipeline that enabled return of clinically actionable results in hours rather than weeks: the Rapid Autoinflammation Panel (RAP). We demonstrated accuracy of this new pipeline, with 100% sensitivity and 100% specificity. Return of results to clinicians was achieved within 48-hours from receiving the patient’s blood or saliva sample. This approach demonstrates the potential significant diagnostic impact of NGS in acute medicine to facilitate precision medicine and save “life or limb” in these critical situations.</p

Table_1.DOCX

<p>Cutaneous leukocytoclastic vasculitis arises from immune complex deposition and dysregulated complement activation in small blood vessels. There are many causes, including dysregulated host response to infection, drug reactions, and various autoimmune conditions. It is increasingly recognised that some monogenic autoinflammatory diseases cause vasculitis, although genetic causes of vasculitis are extremely rare. We describe a child of consanguineous parents who presented with chronic cutaneous leukocytoclastic vasculitis, recurrent upper respiratory tract infection, and hypocomplementaemia. A homozygous p.His380Arg mutation in the complement factor I (CFI) gene CFI was identified as the cause, resulting in complete absence of alternative complement pathway activity, decreased classical complement activity, and low levels of serum factor I, C3, and factor H. C4 and C2 levels were normal. The same homozygous mutation and immunological defects were also identified in an asymptomatic sibling. CFI deficiency is thus now added to the growing list of monogenic causes of vasculitis and should always be considered in vasculitis patients found to have persistently low levels of C3 with normal C4.</p

Table_2.docx

<p>Cutaneous leukocytoclastic vasculitis arises from immune complex deposition and dysregulated complement activation in small blood vessels. There are many causes, including dysregulated host response to infection, drug reactions, and various autoimmune conditions. It is increasingly recognised that some monogenic autoinflammatory diseases cause vasculitis, although genetic causes of vasculitis are extremely rare. We describe a child of consanguineous parents who presented with chronic cutaneous leukocytoclastic vasculitis, recurrent upper respiratory tract infection, and hypocomplementaemia. A homozygous p.His380Arg mutation in the complement factor I (CFI) gene CFI was identified as the cause, resulting in complete absence of alternative complement pathway activity, decreased classical complement activity, and low levels of serum factor I, C3, and factor H. C4 and C2 levels were normal. The same homozygous mutation and immunological defects were also identified in an asymptomatic sibling. CFI deficiency is thus now added to the growing list of monogenic causes of vasculitis and should always be considered in vasculitis patients found to have persistently low levels of C3 with normal C4.</p

T cell reconstitution in patients after cell therapy.

<p>P1, a child with Fanconi anaemia, underwent a second mismatched donor, CD34 selected stem cell graft after in the context of relapsed MDS. Donor HSVTK/CD34 modified T cells were infused in two dose aliquots and were detectable at low level in peripheral blood for over 12 weeks before the patient died of disease relapse. The persistence of non-modified T cells reflects the reduced intensity conditioning and absence of serotherapy. P2, an infant with RAG1 deficient SCID had no pre-existing T cell immunity and was conditioned whist infected with H1N1 influenza. Modified T cells persisted for over 12 months, with eventual recovery of thymic derived donor T cells after one year and normalisation of immunity. P3 suffered Ligase IV deficiency, a form of radiosensitive SCID. Expansion of modified donor T cells was detected within two weeks of first infusion, but the patient died from mucositis related pulmonary and gastrointestinal haemorrhage before dose escalation.</p

Production of donor HSVTK-CD34 T cells.

<p>Production of donor HSVTK-CD34 T cells.</p

GMP compliant T cell transduction procedure.

<p>GMP compliant T cell transduction procedure.</p

T cell repertoire diversity before and after modification.

<p>Complementarity determining region-3 (CDR3) T-cell receptor (TCR) spectratyping was performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077106#pone.0077106-Qasim3" target="_blank">[18]</a>. Briefly, RNA was extracted and cDNA prepared from pre- and post-transduced cells. Twenty four Vβ-specific primers were used with a fluorescent-labelled constant region (Cβ)-specific primer to RT-PCR amplify the CDR3 region of the TCR β chain. Products were run on an AB3130 Genetic Analyzer and analysed using GeneMapper v4.0 software (Applied Biosystems, Warrington, UK). Representative data for P2 is showing preservation Vβ family distributions is shown.</p

Vector configuration and study schema.

<p>1a. A gamma retroviral platform incorporating long terminal repeals (LTRs) from Myeloproliferative sarcoma virus (MPSV) and leader sequence 71 derived from Murine embryonic stem cell virus (MESV). The splice site corrected herpes simplex virus thymidine kinase suicide gene (scHSVTK) fused to a truncated (splice variant) human CD34 gene is shown. 1b. Subjects undergoing CD34 selected mismatched allografts and receiving grafts carrying <5×10⁴ T cells/kg following conditioning (but not serotherapy) were eligible. Gene modified T cells were scheduled at two cell doses, the first 5×10⁴/kg the day following the stem cell graft, and the second programmed within 28 days at a higher dose of 5×10⁵/kg. In the event of GVHD>Grade I, Ganciclovir therapy was scheduled for seven days to eliminate gene modified T cells.</p

Transfer and tracking of T cell mediated virus specific immunity.

<p>Most compelling, and beneficial, was transfer of immunity against pandemic H1N1 infulenza in P2. The haploidentical donor had been electively vaccinated against the strain before leukapheresis harvest of peripheral blood lymphocytes. The transduced and CD34 enriched populations exhibited specific IFNγ responses against HI1N1 compared to non-stimulated control cells. Samples collected 150 days after donor lymphocyte infusion from the patient showed similar H1N1 specific IFNγ responses, which coincided with clearance of persistent H1N1 respiratory infection. These responses were still detectable after 350 days.</p