Purine Nucleoside Phosphorylase Deficiency Research Articles

Purine nucleoside phosphorylase (PNP) deficiency: across-the-board severe combined immunodeficiency

BackgroundPurine nucleoside phosphorylase (PNP) deficiency is a rare, autosomal recessive, inborn error of immunity. It is characterized by progressive immune abnormalities ranging from severe combined immunodeficiency (SCID) to combined immunodeficiency less profound than SCID, neurological abnormalities and autoimmunity. Early detection and diagnosis before the development of life-threatening complications are crucial.MethodsImmune cell subsets were assessed by flow cytometry, serum immunoglobulins and uric acid levels were evaluated, and genetic testing was performed for all patients.ResultsHerein, we present six Egyptian PNP deficiency patients from four different families. We describe the patients’ clinical phenotypes, their immunological profile as well as their genetic results. Sequence analysis results detected 4 different variants in the PNP gene; 1 likely pathogenic frameshift deletion c.452del; p.Asn151MetfsTer20 was found in one family, 1 pathogenic nonsense variant c.172C > T; p.Arg58Ter, and 2 likely pathogenic missense variants c.682G > C; p.Ala228Pro and c.722T > C; pIle2241Thr.ConclusionIn conclusion, PNP deficiency is a variable immunodeficiency and should be considered in various clinical contexts, with or without neurological manifestations. Hematopoietic stem cell transplantation offers a good treatment option, with excellent clinical outcomes, when performed in a timely manner.

Expanded Newborn Screening for Inborn Errors of Immunity: The Experience of Tuscany

Inborn errors of immunity (IEIs) include 485 inherited disorders characterized by an increased susceptibility to life threatening infectious diseases, autoimmunity and malignant diseases with a high mortality rate in the first years of life. Severe Combined Immunodeficiency is the most severe of the IEIs and its detection should be a primary goal in a newborn screening (NBS) program. The term "actionable" has recently been used for all IEIs with outcomes that can be demonstrably improved through early specialized intervention. to evaluate the results of the expanded NBS strategy for IEIs in Tuscany Region (Italy), based on TREC (T-cell Receptor Excision Circles), KREC (Kappa Recombining Excision Circles) and Tandem Mass-based assays. This is a retrospective study collecting data from all infants born in Tuscany from October 10, 2018, to October 10, 2022. Tandem mass assay to identify Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency, together with TREC and KREC molecular analysis were conducted on dried blood spot (DBS) from the newborns' Guthrie Cards. A new DBS and evaluation by an immunologist were carried out when the results of the first test were outside the diagnostic cut-offs. 94,319 newborns were evaluated. Referral rates for TREC (0.031%) and KREC (0.074%) in this study are in line with the data available in literature. The results from the expanded NBS strategy revealed an incidence rate of 1/9,431 affected newborns. This work represents the first description of a sustainable and real-life based expanded NBS program for IEIs with a high diagnostic incidence facilitating prompt management of identified patients.

Abstract 5865: Characterization and resistance mechanisms of PNP inhibitor forodesin-resistant cell lines

Abstract Background: The enzyme purine nucleoside phosphorylase (PNP) is involved in the purine salvage pathway and is present at high levels in lymphoid tissues. Children born with PNP deficiency are known to have significantly reduced T-cell counts but relatively normal B-cell immunity, suggesting PNP as one of the most promising therapeutic targets for T-cell malignancies. Forodesine (For, BCX-1777), a novel PNP inhibitor, was approved in Japan for the treatment of relapsed/refractory PTCL. To understand the cytotoxic effect of For and its mechanism of action, we established a For-resistant (For-R) leukemia cell line and explored the mechanism of resistance. Methods: We established two For-R T-lymphoblastic leukemia cell lines (CCRF-CEM and MOLT4) through repeated escalating exposure to For and 2’-deoxyguanosined(dGuo). Gene expression analysis was performed to identify the mechanisms of resistance. Results: For-R cells showed very high resistance compared to parental cells (IC50 value, CEM/CEM-R 4.2nM/>5x105nM, MOLT4/MOLT4-R 8nM/>5x105nM). When cross-resistance was examined in these For-R cells showed mild resistance to cytarabine. The expression of hENT1, a nucleoside transporter, was examined and found to be upregulated in both CEM-R and MOLT4-R cells, while the expression level of deoxycytidine kinase (dCK) was unchanged in both cells. Next, high performance liquid chromatography was used to assess the intracellular dGTP in each parental and its For-R cells. In the parental cells, intracellular dGTP levels increased promptly 4-6 hours after fosteredin administration, whereas in resistant cells, intracellular dGTP levels were higher than before fosteredin administration, and there was little change in dGTP levels after administration. Conclusion: The established For-R cell line acquired a high resistance to For and showed high intracellular dGTP levels. Imbalance in intracellular GTP levels may be involved in the induction of cell death by For. IC50 value of each cell lines Citation Format: Naoko Hosono, Kana Oiwa, Toshiki Tasaki, Rie Nishi, Luigi Scott, Owen A. O’Connor, Takahiro Yamauchi. Characterization and resistance mechanisms of PNP inhibitor forodesin-resistant cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5865.

Neurological involvement in patients with primary immunodeficiency.

Primary immunodeficiency diseases (PID) are defined by recurrent infections, allergies, autoimmunity, and malignancies. Neurologic symptoms are one of the major components of some immunodeficiency syndromes, such as Ataxia-Telangiectasia (AT), Nijmegen breakage syndrome (NBS), and Purine Nucleoside Phosphorylase (PNP) deficiency, which are considered as the primary involvement. Various pathological mechanisms, DNA repair disorders, metabolic abnormalities, and autoimmune phenomena have also been linked with neurological conditions. We retrospectively assessed the neurological involvement in 108 patients out of 6000 with PID in this study. The female/male ratio of the cases was 49/59, and the median age was 13 years (min = 1; max = 60). Neurological problems were detected at a median age of 7 years (min = 0.5; max = 30). Di George Syndrome (DGS) and CVID (common variable immunodeficiency) were the most common diseases in our cohort (n = 31, 30% and n = 30, 27%, respectively). The most frequent outcomes were cognitive delay (n = 63, 58%), epilepsy (n = 25, 23%), and ataxia (n = 20, 18%). Central nervous system involvement was found in 99% of the patients (n = 107), and peripheral nervous system complication was found in only one patient with CVID and chronic inflammatory demyelinating polyneuropathy (CDIP). Cranial MRI was found to be abnormal in 74% (n = 80) of the patients. MRI findings included cerebellar atrophy (n = 33, 34%), white matter lesion (n = 27, 28.4%), cerebral atrophy (n = 21, 22.3%), gray matter lesion (n = 6, 6.3%), hydrocephalus (n = 5, 5,3%), and pituitary gland lesion (n = 3, 3.2%), intracranial hemorrhage (n = 3, 3%), intracranial vasculitis (n = 3, 2.7%), and arterio-venous malformation (n = 1, 0,9%). Primary involvement (a component of the disease) was 60% (n = 65), and secondary (infection or autoimmunity) and tertiary involvements (structural or incidental lesions) contributed 20% (n = 20) each in the patients. In this study, we describe the various neurologic findings of patients with PID. The neurologic presentation may represent the initial manifestation of certain types of PID. Early diagnosis and treatment are essential to prevent or reduce further neurologic damages.

Purine Nucleoside Phosphorylase Deficiency in Two Unrelated Patients with Autoimmune Hemolytic Anemia and Eosinophilia: Two Novel Mutations.

Two Iranian patients with purine nucleoside phosphorylase (PNP) deficiency are described in terms of their clinical and molecular evaluations. PNP deficiency is a rare form of combined immunodeficiency with a profound cellular defect. Patients with PNP deficiency suffer from variable recurrent infections, hypouricemia, and neurological manifestations. Furthermore, patient 1 developed mild cortical atrophy, and patient 2 presented developmental delay, general muscular hypotonia, and food allergy. The two unrelated patients with developed autoimmune hemolytic anemia and T cells lymphopenia and eosinophilia were referred to Immunology, Asthma and Allergy Research Institute (IAARI) in 2019. After taking blood and DNA extraction, genetic analysis of patient 1 was performed by PCR and direct sequencing and whole exome sequencing was applied for patient 2 and the result was confirmed by direct sequencing in the patient and his parents. The genetic result showed two novel variants in exon 3 (c.246_285+9del) and exon 5 (c.569G>T) PNP (NM_000270.4) in the patients, respectively. These variants are considered likely pathogenic based on the American College of Medical Genetics and Genomics (ACMG) guideline. PNP deficiency has a poor prognosis; therefore, early diagnosis would be vital to receive hematopoietic stem cell transplantation (HSCT) as a prominent and successful treatment.

Neurologic Status of Patients with Purine Nucleoside Phosphorylase Deficiency Before and After Hematopoetic Stem Cell Transplantation.

Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive combined immunodeficiency. The phenotype is profound T cell deficiency with variable B and NK cell functions and results in recurrent and persistent infections that typically begin in the first year of life. Neurologic findings occur in approximately two-thirds of patients. The mechanism of neurologic abnormalities is unclear. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for PNP deficiency. We report here six patients from five unrelated families with PNP deficiency treated in two centers in Turkey. We evaluated the neurological status of patients and compared to post-transplantation period if available. Then, we performed PubMed, Google Scholar, and Researchgate searches using the terms "PNP" and "hematopoietic stem cell transplantation" to find all reported cases of PNP transplantation and compared to our cohort. Six patients were treated in two centers in Turkey. One patient died from post-transplant complications. The other four patients underwent successful HSCT with good immune reconstitution after transplantation (follow-up 21-48months) and good neurological outcomes. The other patient with a new mutation is still waiting for a matching HLA donor. In PNP deficiency, clinical manifestations are variable, and this disease should be considered in the presence of many different clinical findings. Despite the comorbidities that occurred before transplantation, HSCT currently appears to be the only treatment option for this disease. HSCT not only cures immunologic disorders, but probably also improves or at least stabilizes the neurologic status of patients.

Immunodeficiency and autoimmunity: companions not opposites.

Autoimmunity has long been regarded as the polar opposite of immunodeficiency, but clinical and experimental evidence refute this notion. Indeed, numerous inborn or acquired immunodeficiency syndromes are characterized by the development of autoimmune complications in the setting of deficient immune defenses against microbial pathogens. Appreciation that much of the daily business of a healthy immune system is the avoidance of potentially harmful responses to innocuous environmental antigens or components of the host organism helps provide a context for these observations. In this issue of the JCI, Abt and colleagues report on purine nucleoside phosphorylase (PNP) deficiency, exploring the basis for the autoimmune complications that develop in this particular form of T cell immune deficiency and assigning a key role for overactivation of TLR7.

Purine nucleoside phosphorylase deficiency induces p53-mediated intrinsic apoptosis in human induced pluripotent stem cell-derived neurons

Purine nucleoside phosphorylase (PNP) is an important enzyme in the purine degradation and salvage pathway. PNP deficiency results in marked T lineage lymphopenia and severe immunodeficiency. Additionally, PNP-deficient patients and mice suffer from diverse non-infectious neurological abnormalities of unknown etiology. To further investigate the cause for these neurologic abnormalities, induced pluripotent stem cells (iPSC) from two PNP-deficient patients were differentiated into neurons. The iPSC-derived PNP-deficient neurons had significantly reduced soma and nuclei volumes. The PNP-deficient neurons demonstrated increased spontaneous and staurosporine-induced apoptosis, measured by cleaved caspase-3 expression, together with decreased mitochondrial membrane potential and increased cleaved caspase-9 expression, indicative of enhanced intrinsic apoptosis. Greater expression of tumor protein p53 was also observed in these neurons, and inhibition of p53 using pifithrin-α prevented the apoptosis. Importantly, treatment of the iPSC-derived PNP-deficient neurons with exogenous PNP enzyme alleviated the apoptosis. Inhibition of ribonucleotide reductase (RNR) in iPSC derived from PNP-proficient neurons with hydroxyurea or with nicotinamide and trichostatin A increased the intrinsic neuronal apoptosis, implicating RNR dysfunction as the potential mechanism for the damage caused by PNP deficiency. The findings presented here establish a potential mechanism for the neurological defects observed in PNP-deficient patients and reinforce the critical role that PNP has for neuronal viability.

Combined immunodeficiency due to purine nucleoside phosphorylase deficiency: Outcome of three patients

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway. PNP deficiency, caused by the autosomal recessive mutations in the PNP gene, can lead to severe combined immunodeficiency (SCID).PNP deficiency patients typically have profound T-cell deficiency with variable B and NK cell functions. They present clinically with recurrent infections, failure to thrive, various neurological disorders, malignancies, and autoimmune diseases. Hematopoietic stem cell transplantation (HSCT) is the only available cure for patients with PNP deficiency.We present three patients, two of whom were successfully treated with HSCT. One patient died prior to HSCT due to EBV-associated lymphoma. Over the course of post-HSCT, there was no further aggravation of the patients’ neurological symptoms. Although both of the patients still had mild developmental delay, new developmental milestones were achieved.

Quantitation of Purine in Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Inborn errors of purine metabolism, either deficiencies of synthesis or catabolism pathways, lead to a wide spectrum of clinical presentations: urolithiasis (adenine phosphoribosyltransferase), primary immune deficiency (adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency), severe intellectual disability, and other neurological symptoms (Lesch-Nyhan disease, adenylosuccinase deficiency, and molybdenum cofactor deficiency). A rapid quantitative purine assay was developed using UPLC-MS/MS to determine purine nucleoside and base concentrations in urine. Taking advantages of ultra-performance liquid chromatography, we achieved satisfactory analyte separation and recovery with a polar T3 column in a short run time with no requirement of time-consuming sample preparation or derivatization. This targeted assay is intended for diagnosis and management of purine diseases, newborn screening follow-up of SCID, and evaluation of autism spectrum disorders.

Investigating the Variation of TREC/KREC in Combined Immunodeficiencies

T-cell receptor excision circles (TREC)/Kappa-deleting recombination excision circles (KREC) assay has been recently recognized for detecting patients with primary (T- and/or B-cell) immunodeficiency (PID). We aimed to investigate the alterations of these biomarkers in some combined immunodeficiency patients compared to the healthy controls in different age groups. TREC and KREC were assessed in a total of 82 PID patients, most of them with exact genetic diagnosis (3 months to 42 years); using quantitative real-time-polymerase chain reaction (PCR). Patients had a final diagnosis of common variable immunodeficiency (n=23), ataxia-telangiectasia (AT) (n=17), hyper-IgE syndrome (HIES) (7 with DOCK8 deficiency, 4 with signal transducer and activator of transcription 3 (STAT3) deficiency, and 8 children with unknown genetic defects), Wiskott-Aldrich syndrome (WAS) (n=20), purine nucleoside phosphorylase (PNP)deficiency(n=1), dedicator of cytokinesis2 (DOCK2) deficiency (n=1), recombinase activating gene1 (RAG1) deficiency (n=1). Very low to zero amounts of TREC and/or KREC were detected in 14 out of 23 cases of common variable immunodeficiency (CVID), 14 out of 17 cases of AT, 8 out of 20 cases of WAS, 6 out of 7 cases of DOCK8-deficiency patients, 4 out of 8 cases of HIES with unknown genetic defects and all patients with defects in DOCK2, PNP, and RAG1. STAT3-deficient patients were normal for both biomarkers. All patients showed a significant difference in both markers compared to age-matched healthy controls. Our findings highlight that apart from severe types of T/B cell defects, this assay can also be used for early diagnosis the patients with late-onset of disease and even PIDs without a positive family history.

Antileukemic Activity and Pharmacodynamics of Intravenous Forodesine (BCX-1777), a Novel Purine Nucleoside Phosphorylase (PNP) Inhibitor, in Phase I/II Trials in Patients with Advanced T-Cell Malignancies.

PNP represents an attractive target for drug development based on the profound, selective impairment of T-cell function seen in individuals with an inherited deficiency of PNP. Forodesine, a potent, specific transition-state analog inhibitor of PNP, is currently being investigated for safety and efficacy in T-cell and other malignancies. In a phase I/II single-center, intrapatient dose-escalation study, forodesine (starting dose: 40 mg/m2) was given to 5 patients with relapsed/refractory, aggressive T-cell leukemia/ lymphoma via a 30-min IV infusion, followed 24 hrs later by doses administered every 12 hrs for a total of 9 doses; courses could be repeated every 3–4 weeks, as required. Two patients with refractory T-cell prolymphocytic leukemia (T-PLL) and two patients with T-cell acute lymphoblastic leukemia (T-ALL) showed a decrease in leukemic or prolymphocytic counts in peripheral blood and/or bone marrow. All responders had increases in plasma 2′-deoxyguanosine (dGuo) (Cmax: 2.9 to 34 μM; baseline concentration: <.004 μM) and 4/5 responders showed elevated intracellular dGTP levels (10- to 60-fold). Forodesine was generally safe and well tolerated at all dose levels with no dose-limiting toxicities and only two adverse events (ie, acute renal failure and neutropenic infection) that were possibly drug-related. These promising results led to a phase II multi-center, open-label, repeat-dose trial in patients with advanced T-cell malignancies, in which forodesine (40 mg/m2) is infused for 30 mins for 5 days followed by at least 2 non-treatment days for up to 5 additional cycles. Out of 3 patients enrolled to-date, 2 T-ALL patients completed 6 courses of therapy with a dramatic reduction in WBC count (Fig 1A and 1B); 1 splenic T-cell lymphoma patient discontinued due to disease progression. Forodesine resulted in striking elevations in plasma dGuo. Peripheral blast cells in Patient 01 were undetectable at the start of the second course and remained undetectable after 6 courses (Fig 1A), and the bone marrow blast cells were reduced from >90% to near-normal levels. However, the contribution of a single dose of dexamethasone (40 mg IV given on Day 3) to the reduction in WBC count and blast cells is unknown. This patient's peripheral blood hematologic profile (WBC, absolute neutrophil, platelet counts and hemoglobin level) returned to near-normal, indicating a specificity of forodesine for leukemic cell populations and supporting that forodesine is an important breakthrough in the development of less toxic ALL therapy. Additional phase II results will be presented. [Display omitted]

Purine Nucleoside Phosphorylase (PNP) Deficiency: A Case Report

Background: Purine nucleoside phosphorylase (PNP) deficiency is a rare genetic disease that results in combined immunodeficiency. Its inheritance is autosomal recessive and affects the purine metabolic pathway. There is a profound effect on T-cells and variable B-cell dysfunction. There is a strong association with neurological dysfunction in as many as two thirds of cases and autoimmunity in one third. When PNP activity is absent or greatly diminished, deoxyguanosine triphosphate (dGTP) is believed to accumulate in the mitochondria, which inhibits ribonucleotide reductase and mitochondrial DNA repair. This is very harmful to T lymphocytes and leads to DNA damage and apoptosis during thymus selection. Case: A toddler girl diagnosed with PNP) deficiency that is currently being managed using different type of modalities with good response. Results: The patient was admitted in an outside hospital when she developed fever for 4 days along with a perianal abscess. Septic shock was suspected, and she was initially started on gentamycin and tazocin then switched to vancomycin, mereponem and metrodinazole. After resolution of fever, the patient was transferred to our tertiary care center to undergo immunology work up. Upon finally receiving the patient she was stable and afebrile, however there was severe neutropenia and leukopenia. A multidisciplinary team, which included general pediatrics, hematology, Infectious diseases and immunology, handled her case. Furthermore, the patient was switched back to tazocin and gentamycin and completed 14 days with good response and full resolution of fever. Following the multidisciplinary team plan, an immunology/hematology work up was initiated. Conclusion: After 14 days of treatment, there was a good response and resolution of fever. Then, a multidisciplinary team plan, an immunology/hematology work up was initiated.

Purine Nucleoside Phosphorylase Inhibitors as Novel Immuno-Oncology Agent and Vaccine Adjuvant

Purine Nucleoside Phosphorylase (PNP) deficiency in humans causes lymphopenia and this provided the rationale for developing PNP inhibitors as immunosuppressive agents. However, carefulre-evaluation of clinical history of PNP deficient patients and clinical experience with PNP inhibitors together with new experimental data suggest inhibition of PNP may have immune activating effects through elevation of guanosine and activation of various toll-like receptors (TLRs). This paper proposes a mechanism of action for the immune activating effects of PNP inhibition.

The Broad Clinical Spectrum and Transplant Results of PNP Deficiency.

Purine nucleoside phosphorylase (PNP) is a known yet rare cause of combined immunodeficiency with a heterogeneous clinical presentation. We aim to add to the expanding clinical spectrum of disease, and to summarize the available data on bone marrow transplant for this condition. Data was collected from patient files retrospectively. A review of the literature of hematopoietic stem cell transplantation (HSCT) for PNP deficiency was conducted. Four patients were treated in two centers in Israel. One patient died of EBV-related lymphoma with CNS involvement prior to transplant. The other three patients underwent successful HSCT with good immune reconstitution post-transplant (follow-up 8-108 months) and excellent neurological outcomes. PNP is a variable immunodeficiency and should be considered in various clinical contexts, with or without neurological manifestations. HSCT offers a good treatment option, with excellent clinical outcomes, when preformed in a timely manner.

An unusual presentation of purine nucleoside phosphorylase deficiency mimicking systemic juvenile idiopathic arthritis complicated by macrophage activation syndrome

BackgroundSystemic juvenile idiopathic arthritis (sJIA) is an inflammatory condition that presents with fever, rash and arthritis. At onset systemic features are predominant and the diagnosis may be a challenge. Secondary hemophagocytic lymphohistiocytosis (sHLH) forms may be associated with different disorders, including rheumatic diseases, and this form is called macrophage activation syndrome (MAS). CXCL9 levels, a chemokine induced by IFNγ, are significantly elevated in patients with sHLH or MAS and are correlated with laboratory features of disease activity. High levels of IL-18 have been reported in patients with MAS during sJIA, as well as in some patients with sHLH and IL-18 is indeed known to induce IFNγ production.FindingsWe report a patient with a clinical presentation highly suggestive for systemic juvenile idiopathic arthritis (sJIA) onset complicated by MAS, and was later diagnosed with purine nucleoside phosphorylase (PNP)-deficiency with HLH. Some unusual features appeared when HLH was controlled and further investigations provided the correct diagnosis. Serum CXCL9 and IL-18 levels were found markedly elevated at disease onset, during the active phase of MAS and decreased progressively during the course.ConclusionThe reported case underlines the potential difficulties in discriminating sJIA from other causes of systemic inflammation. Furthermore, this supports the notion that especially in young children with a sJIA-like disease other mimicking conditions should be actively sought for. CXCL9 and IL-18 levels suggested that patients with PNP-deficiency may have a subclinical activation of the IFNγ pathway and indeed they are predisposed to develop sHLH.

The First Purine Nucleoside Phosphorylase Deficiency Patient Resembling IgA Deficiency and a Review of the Literature

ABSTRACTPurine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive primary immunodeficiency disorder characterized by decreased numbers of T-cells, variable B-cell abnormalities, decreased amount of serum uric acid and PNP enzyme activity. The affected patients usually present with recurrent infections, neurological dysfunction and autoimmune phenomena. In this study, whole-exome sequencing was used to detect mutation in the case suspected of having primary immunodeficiency. We found a homozygous mutation in PNP gene in a girl who is the third case from the national Iranian registry. She had combined immunodeficiency, autoimmune hemolytic anemia and a history of recurrent infections. She developed no neurological dysfunction. She died at the age of 11 after a severe chicken pox infection. PNP deficiency should be considered in late-onset children with recurrent infections, autoimmune disorders without typical neurologic impairment.

Direct-infusion based metabolomics unveils biochemical profiles of inborn errors of metabolism in cerebrospinal fluid

BackgroundFor inborn errors of metabolism (IEM), metabolomics is performed for three main purposes: 1) development of next generation metabolic screening platforms, 2) identification of new biomarkers in predefined patient cohorts and 3) for identification of new IEM. To date, plasma, urine and dried blood spots are used. We anticipate that cerebrospinal fluid (CSF) holds additional – valuable – information, especially for IEM with neurological involvement. To expand metabolomics to CSF, we here tested whether direct-infusion high-resolution mass spectrometry (DI-HRMS) based non-quantitative metabolomics could correctly capture the biochemical profile of patients with an IEM in CSF. MethodsEleven patient samples, harboring eight different IEM, and thirty control samples were analyzed using DI-HRMS. First we assessed whether the biochemical profile of the control samples represented the expected profile in CSF. Next, each patient sample was assigned a ‘most probable diagnosis’ by an investigator blinded for the known diagnoses of the patients. Resultsthe biochemical profile identified using DI-HRMS in CSF samples resembled the known profile, with – among others – the highest median intensities for mass peaks annotated with glucose, lactic acid, citric acid and glutamine. Subsequent analysis of patient CSF profiles resulted in correct ‘most probable diagnoses’ for all eleven patients, including non-ketotic hyperglycinaemia, propionic aciduria, purine nucleoside phosphorylase deficiency, argininosuccinic aciduria, tyrosinaemia type I, hyperphenylalaninemia and hypermethioninaemia. ConclusionWe here demonstrate that DI-HRMS based non-quantitative metabolomics accurately captures the biochemical profile of this set of patients in CSF, opening new ways for using metabolomics in CSF in the metabolic diagnostic laboratory.

Clinical, Immunological, and Molecular Findings in 57 Patients With Severe Combined Immunodeficiency (SCID) From India.

Severe combined immunodeficiency (SCID) represents one of the most severe forms of primary immunodeficiency (PID) disorders characterized by impaired cellular and humoral immune responses. Here, we report the clinical, immunological, and molecular findings in 57 patients diagnosed with SCID from India. Majority of our patients (89%) presented within 6 months of age. The most common clinical manifestations observed were recurrent pneumonia (66%), failure to thrive (60%), chronic diarrhea (35%), gastrointestinal infection (21%), and oral candidiasis (21%). Hematopoietic Stem Cell Transplantation (HSCT) is the only curative therapy available for treating these patients. Four patients underwent HSCT in our cohort but had a poor survival outcome. Lymphopenia (absolute lymphocyte counts/μL <2,500) was noted in 63% of the patients. Based on immunophenotypic pattern, majority of the cases were T−B− SCID (39%) followed by T−B+ SCID (28%). MHC class II deficiency accounted for 10.5% of our patient group. A total of 49 patients were molecularly characterized in this study and 32 novel variants were identified in our cohort. The spectrum of genetic defects in our cohort revealed a wide genetic heterogeneity with the major genetic cause being RAG1/2 gene defect (n = 12) followed by IL2RG (n = 9) and JAK3 defects (n = 9). Rare forms of SCID like Purine nucleoside phosphorylase (PNP) deficiency, reticular dysgenesis, DNA-Protein Kinase (DNA-PKcs) deficiency, six cases of MHC class II deficiency and two ZAP70 deficiency were also identified in our cohort. Fourteen percent of the defects still remained uncharacterized despite the application of next generation sequencing. With the exception of MHC class II deficiency and ZAP70 deficiency, all SCID patients had extremely low T cell receptor excision (TRECs) (<18 copies/μL).

Intracellular Delivery of Human Purine Nucleoside Phosphorylase by Engineered Diphtheria Toxin Rescues Function in Target Cells.

Despite a wealth of potential applications inside target cells, protein-based therapeutics are largely limited to extracellular targets due to the inability of proteins to readily cross biological membranes and enter the cytosol. Bacterial toxins, which deliver a cytotoxic enzyme into cells as part of their intoxication mechanism, hold great potential as platforms for delivering therapeutic protein cargo into cells. Diphtheria toxin (DT) has been shown to be capable of delivering an array of model proteins of varying sizes, structures, and stabilities into mammalian cells as amino-terminal fusions. Here, seeking to expand the utility of DT as a delivery vector, we asked whether an active human enzyme, purine nucleoside phosphorylase (PNP), could be delivered by DT into cells to rescue PNP deficiency. Using a series of biochemical and cellular readouts, we demonstrate that PNP is efficiently delivered into target cells in a receptor- and translocation-dependent manner. In patient-derived PNP-deficient lymphocytes and pluripotent stem cell-differentiated neurons, we show that human PNP is efficiently translocated into target cells by DT, where it is able to restore intracellular hypoxanthine levels. Further, through replacement of the native receptor-binding moiety of DT with single-chain variable fragments that were selected to bind mouse HBEGF, we show that PNP can be retargeted into mouse splenocytes from PNP-deficient mice, resulting in restoration of the proliferative capacity of T-cells. These findings highlight the versatility of the DT delivery platform and provide an attractive approach for the delivery ofPNP as well as other cytosolic enzymes implicated in disease.