ADA and PNP Deficiency
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Both enzymes are ubiquitous, and defective ADA or PNP proteins are unable to effectively detoxify several naturally occurring methylated adenosine compounds of the purine salvage pathway; this results in “metabolic poisoning” that manifests its most deleterious effects in dividing cells such as lymphocytes. Although these defects primarily affect lymphocyte development, viability, and function, they also impact multiple organ systems and are considered systemic metabolic disorders (Whitmore and Gaspar 2016). The primary immunodeficiency called ADA deficiency is a consequence of ADA1 enzyme deficiency. ADA has two isoenzymes, ADA1 and ADA2, of which ADA1 is largely intracellular and widely distributed in various tissues, with the highest amounts found in the thymus, brain, and gastrointestinal tract. As a result, the major source of ADA replacement therapy was bovine intestine until 2018 when recombinant bovine ADA was first introduced.
ADA2 is more abundant in serum and is secreted by the monocyte-macrophage system. The deficiency of ADA2 (DADA2) is a completely different disease entity, a new autoinflammatory disorder characterized by an early-onset vasculopathy with livedoid skin rash associated with systemic manifestations, CNS involvement, and variable severity of CVID-like immunodeficiency. This condition is secondary to autosomal-recessive mutations of the CECR1 (Cat Eye Syndrome Chromosome Region 1) gene on chromosome 22q11.1, which encodes for the enzymatic protein ADA2.
Prior to the introduction of newborn screening (NBS) for SCID, ADA deficiency was diagnosed in approximately 40% of autosomal-recessive forms and 20% of all cases of severe combined immunodeficiency (SCID); since then, the relative frequency of SCID-ADA deficiency has decreased to 10% among all variants (Fischer et al. 2015). PNP deficiency, also inherited in an autosomal-recessive fashion, is less frequent, with fewer than 100 patients identified worldwide; hence, literature is limited.
ADA deficiency presents with clinical and immunological manifestations typical of SCID or in a less severe form as “delayed/late”-onset combined immunodeficiency (CID). PNP-deficient patients exhibit T cell dysfunction with normal B-cell function and a variable degree and extent of immunodeficiency.
Age-Related Clinical Presentations of ADA Deficiency
ADA deficiency presents with a broad clinical spectrum in terms of onset and severity of immunodeficiency-related infections and impact on non-immunological organ systems. There are three major clinical phenotypes in ADA deficiency:
Clinically indistinguishable from SCID, except for bony abnormality in half of the patients. Affected patients suffer from disseminated viral (mainly herpes and respiratory tract viruses), fungal (thrush caused by Candida albicans), and opportunistic (Pneumocystis jiroveci) infections, but bacterial infections, especially sepsis, are not uncommon. All these infections are the consequences of a marked and progressive depletion of T, B, and NK lymphocytes and an absence of both humoral and cellular immune function. ADA-deficient infants exhibit failure to thrive, and maternal T cell engraftment can cause graft-versus-host disease (GVHD).
Delayed or Late Onset
Diagnosed from 3 years of age until early adulthood and presents as a combined immunodeficiency with recurrent bacterial otitis and sinopulmonary infections, often resulting in chronic pulmonary insufficiency. Septicemia, especially caused by Streptococcus pneumoniae, is not uncommon. Persistent viral warts, recurrent herpes zoster and Candida infections, asthma, allergies, and immune abnormalities such as elevated serum concentration of IgE, as well as eosinophilia and lymphopenia, are the characteristics. Autoimmunity is frequently observed. Late-onset patients may retain 2–5% of normal ADA1 activity.
Partial ADA Deficiency
A benign condition without immunodeficiency, identified upon analysis of blood relatives of affected individuals. ADA activity is very low or absent in erythrocytes but 5–80% in nucleated cells. ADA genotype has a major effect on clinical phenotype, to the extent that it determines the level of exposure to ADA substrates.
Clinical Presentation of PNP Deficiency
The most common infectious complications are recurrent bacterial and viral, upper and lower respiratory tract infections. PNP-deficient patients often suffer from opportunistic infections caused by Aspergillus fumigatus, mycobacteria, JC virus, and P. jiroveci. Disseminated varicella and persistent herpes simplex virus infections were also observed.
Noninfectious Manifestations of ADA and PNP Deficiencies
Immune dysregulation is common in delayed or late-onset phenotype. The clinical spectrum of immune dysregulation includes multiple forms of autoimmunity, such as autoimmune cytopenia (hemolytic anemia, immune thrombocytopenia), type 1 diabetes mellitus, hypothyroidism, hepatitis, and glomerulonephritis in both ADA and PNP deficiencies. Lupus and central nervous system vasculitis were only described in PNP deficiency (Sauer et al. 2012). Malignancy, especially EBV-related lymphoma, was also observed. Patients with ADA deficiency have susceptibility to a rare mesenchymal tumor of the skin known as dermatofibrosarcoma protuberans.
Syndromic Features in ADA Deficiency
Neurological abnormalities include neurosensorial deafness, cognitive and behavioral impairments, developmental delay, hypotonia, nystagmus, and seizures. Many of these impairments are both a consequence of the metabolic disturbance in ADA deficiency and dependent on the degree of deficiency. Skeletal abnormality may occur in costochondral junctions, detectable by lateral view of chest radiograph or by physical exam (similar to “rachitic rosary”). Kidney abnormalities result in impaired renal function secondary to mesangial sclerosis. Additional abnormalities such as pyloric stenosis and adrenal cortical fibrosis might reflect the effects of infectious agents rather than primary effects due to ADA deficiency (Hirschhorn and Candotti 2006; Grunebaum et al. 2013; Flinn and Gennery 2018).
Syndromic Features in PNP Deficiency
Neurological abnormalities develop in over 50% of PNP-deficient patients. Neurodevelopmental delay often precedes the onset of immunodeficiency. Spastic paresis such as spastic diplegia or tetraparesis (may be confused with cerebral palsy), tremor, hyper- or hypotonia, ataxia, retarded motor development, behavioral difficulties, and mental retardation are observed (Hirschhorn and Candotti 2006).
Immunological abnormalities include progressive lymphopenia affecting all compartments (T, B, and natural killer (NK) cells). Since the generation of T cells and thymic function is normal, TREC assay will only decrease after the accumulation of toxic purine pathway metabolites and death of naïve lymphocytes. In this case, patients may be identified by newborn SCID screening. ADA deficiency is, however, the only form of SCID that might be missed by newborn screening if the progression of loss of lymphocytes is slower.
In the neonatal-onset form, T cells do not proliferate in response to mitogen and antigen stimulation, and immunoglobulins are undetectable or very low around 4–6 months of age, after loss of transplacental maternal immunoglobulin G (IgG).
Thymus: severe thymus atrophy, maturation defect in thymic epithelial cells including the absence of medullary thymic epithelial cells with autoimmune regulator (AIRE) expression
Marked depletion of lymphoid tissues (thymus, tonsils, lymph nodes, and spleen)
Only plasma cells can be identified in the spleen, lymph nodes, and intestines
Absent or markedly diminished CD3+ T lymphocytes, CD4/CD8 ratio often reversed, absent T cell receptor excision circles in severe neonatal forms, reduced CD4 + CD25 + FoxP3+ Treg cells
Marked T cell lymphopenia, absence of T cell receptor excision circles
Abnormal T cell function: absent to markedly reduced proliferation to mitogens (PHA), absent to trace proliferation to antigens, abnormal CD4+ T cell TCR/CD28-driven activation
Abnormal T cell function: lack of lymphocyte response to mitogenic stimulation
Absent or markedly diminished B-cell count
Abnormal B-cell function: absent or low IgG (in late onset especially IgG2), absent or very low specific antibody titers, excess autoreactive transitional and mature naïve B-cell clones, reduced B-cell receptor and Toll-like receptor functions
Variable B-cell deficiency; immunoglobulin levels might be normal, but absent vaccine-specific antibodies
The diagnosis of ADA deficiency is established in an individual with <1% of normal ADA catalytic activity in erythrocyte lysates or – if transfused – in extracts of other cells (e.g., blood mononuclear cells, fibroblasts). Increased levels of dATP and deoxyadenosine in urine can also aid the diagnosis (Hershfield 2017). In the United States, the reference laboratory for ADA functional assays including enzyme activity and measurements of toxic metabolites is at Dr. Michael Hershfield’s laboratory at Duke University School of Medicine.
The diagnosis can be supported by identification of biallelic pathogenic variants (homozygous or compound heterozygous) in the ADA gene by next-generation sequencing (NGS). In case of variants of unknown significance, functional assays for ADA catalytic activity, measurement of protein expression, and toxic metabolites are needed to confirm the association. This is especially important in the era of gene therapy when the immunodeficiency can be corrected by transduction with the normal ADA gene.
Immunological abnormalities include progressive decline in the number of T cells, while B and NK cells are often spared. T cell function, measured by stimulation of lymphocyte proliferation with antigen and mitogen, is abnormal. PNP deficiency is not frequently associated with profound B-cell defects. Immunoglobulin levels might be within normal range, but specific antibody responses are poor (Table 1). Radiochemical or spectrophotometric assays that reveal the absence of PNP activity in erythrocyte lysates can confirm the diagnosis. Low serum and urinary levels of uric acid support the diagnosis. Rarely, serum uric acid is within the pediatric normal range; therefore, PNP deficiency should not be ruled out in the absence of hypouricemia. Urine levels of inosine, deoxyinosine, guanosine, and deoxyguanosine are characteristically high. Homozygous mutation of the PNP gene by NSG can confirm the diagnosis.
Correlations between specific mutations, residual ADA activity, metabolite concentration, age at onset, and severity of disease appear to be present in ADA deficiency; however, other genetic and nongenetic factors (e.g., infections) can modify the phenotype in patients with identical pathogenic gene variants. Somatic mosaicism, due to de novo mutations during embryogenesis and resulting in only a proportion of cells that carry the inherited disease-causing gene variant, can significantly modify the clinical phenotype (Hershfield 2003). Because of the low numbers of PNP-deficient patients described in the literature, there is no genotype-phenotype correlation established for this disease.
If immune function is not restored, children with ADA-deficient SCID rarely survive beyond 1 to 2 years of age.
Infections are treated with specific antibiotic, antiviral, and antifungal agents. P. jiroveci prophylaxis is recommended with trimethoprim-sulfamethoxazole. Antibody deficiency requires immunoglobulin replacement therapy (IgRT) via either intravenous or subcutaneous route.
Enzyme replacement therapy (ERT) with polyethylene glycol-modified ADA (PEG-ADA) administered weekly (30 U/kg) by intramuscular injection is recommended to stabilize the metabolic status of patients until autologous or allogenic hematopoietic stem cell transplantation (HSCT) is available. This is a lifesaving but not curative treatment for patients lacking a donor or awaiting gene therapy. Dosage regimen may be individualized, based on monitoring of PEG-ADA levels in plasma and toxic metabolite levels (dATP or total dAdo nucleotides: the sum of dAMP, dADP, and dATP) in erythrocytes. Periodic testing for anti-ADA antibodies is also recommended, since in approximately 10% of cases, neutralizing antibodies impair the effect of ERT. ERT has resulted in the development of protective, although not normal, T cell immunity in 80% of treated patients, and approximately half of PEG-ADA-treated patients have been able to discontinue IgRT. No toxic or hypersensitivity reactions to PEG-ADA have been reported, but there have been examples of several manifestations of immune dysregulation, including autoimmunity, malignancies, and progression of chronic pulmonary insufficiency (Sauer et al. 2012). The need for prolonged use of PEG-ADA for several years has been reported among patients who received and failed HSCT therapy. The expenses of few years prolonged PEG-ADA replacement therapy near the cost of HSCT. Therefore, it is still recommended medically and financially that patients be offered HSCT, if needed, even repeatedly.
Restoration of a functional immune system is essential for long-term survival. The preferred treatment is HSCT from an HLA-identical relative (usually a sibling) since this has a superior outcome in terms of overall survival as well as immune recovery in comparison to transplant from matched unrelated or haploidentical donors (Hirschhorn and Candotti 2006; Flinn and Gennery 2018).
Gene therapy with retroviral vectors has been pursued on over 200 ADA-deficient patients in clinical trials. Specifically, ex vivo autologous hematopoietic stem cells (CD34+ enriched cell fraction) are transduced with retrovirus containing the ADA cDNA sequence. Although the insertion site in the genome is not directed, ADA retroviral vectors are not associated with increased risk of malignancy, unlike first-generation IL2RG gene therapy trials. The patients require reduced intensity conditioning before the transfusion of modified stem cells. The ex vivo stem cell retroviral vector ADA gene therapy (Strimvelis™) is the first genetic treatment for SCID that received approval by the European Medicines Agency in 2016. Currently, lentiviral studies are ongoing that show great promise with high transduction rate. In summary, a high level (50–90%) of gene correction in T, B, and NK cells has been detected in all variants of gene therapy, leading to an efficient systemic detoxification and immune recovery, but there is significant risk of autoimmunity post gene therapy. No leukemic or oncogenic events have been reported, indicating that ADA-SCID gene therapy has a favorable risk/benefit profile; however, long-term safety needs to be monitored.
Genetic counseling is recommended, to reduce morbidity and mortality through early diagnosis and treatment. If the pathogenic variants in the proband have been identified by molecular genetic testing, carrier testing for at-risk family members and prenatal testing for pregnancies are recommended. Alternatively, relatives at risk can be evaluated by assaying ADA catalytic activity in red blood cells (Hirschhorn and Candotti 2006).
HSCT is the treatment of choice in PNP deficiency, but overall prognosis is poor. Even when engraftment is successful, the neurological deficit does not improve (Grunebaum et al. 2013).
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