Autoinflammatory encephalopathy due to PTPN1 haploinsufficiency: a case series

Abstract

Background: Through the agnostic screening of patients with uncharacterised disease phenotypes for an upregulation of type I interferon (IFN) signalling, we identified a cohort of individuals heterozygous for mutations in PTPN1, encoding the protein-tyrosine phosphatase 1B (PTP1B). We aimed to describe the clinical phenotype and molecular and cellular pathology of this new disease.

Methods: In this case series, we identified patients and collected clinical and neuroradiological data through collaboration with paediatric neurology and clinical genetics colleagues across Europe (Czechia, France, Germany, Italy, Slovenia, and the UK) and Israel. Variants in PTPN1 were identified by exome and directed Sanger sequencing. The expression of IFN-stimulated genes was determined by quantitative (q) PCR or NanoString technology. Experiments to assess RNA and protein expression and to investigate type 1 IFN signalling were undertaken in patient fibroblasts, hTERT-immortalised BJ-5ta fibroblasts, and RPE-1 cells using CRISPR-Cas9 editing and standard cell biology techniques.

Findings: Between Dec 20, 2013, and Jan 11, 2023, we identified 12 patients from 11 families who were heterozygous for mutations in PTPN1. We found ten novel or very rare variants in PTPN1 (frequency on gnomAD version 4.1.0 of <1·25 × 10:sup>-6). Six variants were predicted as STOP mutations, two involved canonical splice-site nucleotides, and two were missense substitutions. In three patients, the variant occurred de novo, whereas in nine affected individuals, the variant was inherited from an asymptomatic parent. The clinical phenotype was characterised by the subacute onset (age range 1-8 years) of loss of motor and language skills in the absence of seizures after initially normal development, leading to spastic dystonia and bulbar involvement. Neuroimaging variably demonstrated cerebral atrophy (sometimes unilateral initially) or high T2 white matter signal. Neopterin in CSF was elevated in all ten patients who were tested, and all probands demonstrated an upregulation of IFN-stimulated genes in whole blood. Although clinical stabilisation and neuroradiological improvement was seen in both treated and untreated patients, in six of eight treated patients, high-dose corticosteroids were judged clinically to result in an improvement in neurological status. Of the four asymptomatic parents tested, IFN signalling in blood was normal (three patients) or minimally elevated (one patient). Analysis of patient blood and fibroblasts showed that tested PTPN1 variants led to reduced levels of PTPN1 mRNA and PTP1B protein, and in-vitro assays demonstrated that loss of PTP1B function was associated with impaired negative regulation of type 1 IFN signalling.

Interpretation: PTPN1 haploinsufficiency causes a type 1 IFN-driven autoinflammatory encephalopathy. Notably, some patients demonstrated stabilisation, and even recovery, of neurological function in the absence of treatment, whereas in others, the disease appeared to be responsive to immune suppression. Prospective studies are needed to investigate the safety and efficacy of specific immune suppression approaches in this disease population.

Funding: The UK Medical Research Council, the European Research Council, and the Agence Nationale de la Recherche.

Figure 1. Genetic data

(A) Family pedigrees where an affected individual carries a novel or very rare heterozygous non-synonymous substitution in PTPN1. Circles and squares indicate female and males respectively. Dark and light colouring denote, respectively, affected and clinically asymptomatic mutation-positive individuals. NT = not tested; WT = wild-type. (B) Cartoon of PTPN1 locus with perpendicular blue lines indicating exons numbered above. Numbers below (1, 22, 52, etc) indicate the first amino acid (AA) position in the respective exon. The two splicing variants are depicted on the top with red arrows showing the relative position next to the exon. (C) Cartoon of the protein domains of PTP1B, with amino acid numbering below. STOP mutations and non-synonymous missense substitutions are indicated. N = N terminal domain; CD = catalytic domain; PRD = proline-rich domain; ERT = ER-targeting domain.

Figure 2. Representative axial T2 cerebral MRIs in affected patients.

(A) AGS1036 at age 5 years, 5 months post symptom-onset, showing generalised volume loss more evident in the right cerebral hemisphere. (B) AGS1036 at age 8 years, 35 months post symptom-onset and 23 months post initiation of treatment, showing almost complete normalisation of the changes seen initially. (C) AGS2942 at age 9 years showing significant cortico-subcortical atrophy with passive enlargement of the ventricles and cerebral sulci, and some hypersignal of the deep white matter frontally. (D) AGS3542 at age 2 years 2 months showing right-sided hemi-atrophy and non-specific hypersignal of the deep white matter. (E) AGS3542 at age 3 years 8 months showing bilateral atrophy. (F) AGS3542 at age 7 years 8 months showing normalised brain volume without significant asymmetry and normalised white matter.

Figure 3. Interferon scores in patients and asymptomatic parents

Interferon (IFN) scores (median fold expression of a panel of IFN stimulated genes (ISGs)) recorded in symptomatic patients and their asymptomatic parents carrying a mutation in PTPN1. In families AGS492 – AGS1421 IFN scores were derived using a six ISG panel measured by qPCR (A), while in families AGS2942 – AGS3561 a 24 ISG panel was generated using NanoString technology (B). The upper range of normal is calculated as +2 SD above the mean of the control group, as indicated by the dotted lines: qPCR = 2.466; NanoString = 2.758. Red lines indicate the median values for the respective groups.

Figure 4. Studies of patient-derived primary fibroblasts and PTPN1 deficient cells

(A) qPCR analysis of PTPN1 mRNA expression, and (B) representative immunoblots of PTP1B protein expression, in patient-derived primary dermal fibroblasts carrying the indicated heterozygous variants. HDF = control human dermal fibroblasts. For qPCR, n=5-7 experiments and one-way ANOVA with Fisher’s LSD test was used to compare the expression levels of PTPN1 in healthy control cells and patient cells. (C) Representative immunoblot of phospho-STAT1 (p-STAT1), STAT1 and loading control vinculin in lysates of primary fibroblasts stimulated for 15 min with IFNα2b, and quantification of phospho-STAT1 signal over vinculin signal, averaging control HDF lines. N=3 experiments and mixed model with Dunnett’s multiple comparison test was performed. (D-F) qPCR analysis of the expression of five representative interferon (IFN) stimulated genes (ISGs) (IFI27, IFI44L, MX1, OAS1 and RSAD2), with an ISG score calculated as the median of the expression of these genes in BJ-5ta human fibroblast cell clones wild-type (WT), knock-out (KO) (D), WT/KO (E) or WT/knock-in (KI) for the c.63+1G>C nucleotide substitution (F). Each circle or triangle (D-F) represents the average ISG score from independent clones of the same genotype (WT, two to four clones; WT/KO, four clones; WT/KI, one clone) in one experiment. ISG score is calculated as above. Unpaired t test (D-F) was used to compare the baseline ISG expression levels in WT and mutant BJ-5ta clones. (G) qPCR of baseline ISG expression on a PTPN1 WT or KO background in hTERT RPE-1 cells, and either WT or single KO of IFNAR1 or STING1. (H) qPCR of baseline ISG expression on a PTPN1 WT or KO background in hTERT RPE-1 cells, and cells WT or double KO for IFNAR1 and STING1. ISG score is calculated as the median of the expression of four ISGs, IFI27, IFI16, MX1 and IRF7. N=3-4 experiments and one-way ANOVA (G-H) was used to compare the levels of baseline ISG expression in the indicated genotypes.