Delayed-onset adenosine deaminase deficiency with a novel synonymous mutation and a case series from China

Yue Zhang · Wei Liu · Zhou Shu · Yan Li · Fei Sun · Zhi-Gang Li · Tong-Xin Han · Hua-Wei Mao · Tian-You Wang,5

Abstract Background Adenosine deaminase (ADA) is a key enzyme in the purine salvage pathway.Genetic defects of the ADA gene can cause a subtype of severe combined immunodeficiency.To date,few Chinese cases have been reported.Methods We retrospectively reviewed the medical records of patients diagnosed with ADA deficiency in Beijing Children’s Hospital and summarized the previously published ADA deficiency cases from China in the literature.Results Nine patients were identified with two novel mutations (W272X and Q202=).Early-onset infection,thymic abnormalities and failure to thrive were the most common manifestations of Chinese ADA-deficient patients.The ADA genotype has a major effect on the clinical phenotype.Notably,a novel synonymous mutation (c.606G＞A,p.Q202=) was identified in a delayed-onset patient,which affected pre-mRNA splicing leading to a frameshift and premature truncation of the protein.Furthermore,the patient showed γδT cells expansion with an increased effect or phenotype,which may be associated with the delayed onset of disease.In addition,we reported cerebral aneurysm and intracranial artery stenosis for the first time in ADA deficiency.Five patients died with a median age of four months,while two patients received stem cell transplantation and are alive.Conclusions This study described the first case series of Chinese ADA-deficient patients.Early-onset infection,thymic abnormalities and failure to thrive were the most common manifestations in our patients.We identified a synonymous mutation that affected pre-mRNA splicing in the ADA gene,which had never been reported in ADA deficiency.Furthermore,we reported cerebral aneurysm in a delayed-onset patient for the first time.Further study is warranted to investigate the underlying mechanisms.

Keywords Adenosine deaminase deficiency · Cerebral aneurysm · Gamma delta · Severe combined immunodeficiency · Synonymous mutation

Introduction

Adenosine deaminase (ADA) is a key enzyme in the purine salvage pathway and is responsible for catalyzing the deamination of adenosine and 2’-deoxyadenosine into inosine and 2’-deoxyinosine,respectively.ADA is present in all cell types,with the highest amounts in lymphoid tissues,particularly the thymus,brain and gastrointestinal tract [1].Genetic defects in ADA can lead to a subtype of severe combined immunodeficiency (SCID),known as ADA-SCID,which accounts for approximately 10%-15% of all causes of SCID [2].The incidence of ADA deficiency worldwide is estimated to be between 1:1,000,000 and 1:200,000 live births every year [3].Similar to other genetic forms of SCIDs,the typical presentation of the patient is life-threatening opportunistic infections early in life and failure to thrive.Furthermore,the ubiquitous nature of ADA allows for the development and observation of nonimmunological symptoms,including skeletal,pulmonary,hepatic,renal,skin,auditory,cognitive and behavioral abnormalities [4].Biochemical testing is usually characterized by reduced ADA activity,a marked elevation of the metabolite dATP and reduced activity of S-adenosylhomocysteine (SAH) hydrolase in erythrocytes.The presence of biallelic pathogenic mutations in theADAgene confirms the diagnosis [5].The affected individuals generally succumb within the first or second year of life if no intervention is performed.

Since it was first described in 1972 [6],hundreds of ADA-deficient patients have been reported,and approximately 130 pathogenic mutations have been registered in the Human Genetic Mutation Database thus far,including missense,nonsense,frameshift and splicing mutations.However,reports from China are rare.No pathogenic synonymous mutation has been documented thus far.Here,we report the first Chinese case series of ADA deficiency and identified a synonymous mutation (c.606G＞A,p.Q202=) in a patient with delayed disease onset.

Methods

Patients

Patients from the Department of Immunology,Beijing Children’s Hospital,who were diagnosed with ADA deficiency from July 2020 to December 2022 were enrolled.The diagnosis was based on the following criteria: (1) biallelic pathogenic mutations in theADAgene;(2) severe lymphocytopenia affecting the T-and B-lymphocytes and natural killer (NK) cells and low serum immunoglobulins;and/or (3) biochemical testing that shows absent or greatly reduced ADA activity (＜ 1% of normal),marked elevation of the metabolite dATP or total deoxyadenosine nucleotides (the sum of dAMP,dADP and dATP) in erythrocytes,and reduced activity of SAH hydrolase in erythrocytes (＜ 5% of normal).The less severe “delayed” onset type is usually diagnosed between the ages of one and ten years,and the “late/adult onset” type is diagnosed in the second to fourth decades [5].Informed consent to participate in the study was obtained from the participants or their parent/legal guardian in the case of children under 16.

Clinical features

The clinical details of all patients,including age,sex,age at onset,time to diagnosis,clinical manifestations,imaging,laboratory results,treatment and outcomes,were collected retrospectively.

Peripheral blood lymphocyte subpopulation analysis

Ethylene diamine tetraacetic acid-anticoagulated whole blood was split into three different panels to evaluate the lymphocyte subsets: (1) the general lymphocyte population: T cells,B cells,and NK cells;(2) the T-cell subsets;and (3) the B-cell subsets.For each panel fraction,50 μL of whole blood was incubated with an antibody cocktail specific for the target cell population: the Multitest IMK Kit (BD Biosciences,USA) was placed in tube 1;anti-T-cell receptor (TCR) γ/δ,anti-Vδ2,anti-CD27,anti-CD45RA,anti-CD45,anti-CD8,anti-CD3,anti-TCRαβ,and anti-CD4 (BD Biosciences,USA) were placed in tube 2;and anti-CD38,anti-CD24,anti-immunoglobulin D,anti-CD19,anti-CD27,and anti-CD45 (BD Biosciences,USA) were placed in tube 3.After staining for 20 minutes at room temperature in the dark,the erythrocytes in the samples were lysed by incubation with a lysing solution for 10 minutes.Following centrifugation and washing with phosphatebuffered saline,the lymphocytes were fixed with 2% paraformaldehyde.The cells were then examined using the BD FACSAria II flow cytometer (BD Biosciences,USA),and the data were analyzed using BD FACSDiva 8.0.3 (BD Biosciences,USA).

Genetic analysis

Genetic analysis was performed as previously described [7].Peripheral blood of patients and their parents was collected and sent to MyGenostics (Beijing,China) for whole exome sequencing after consent was obtained.The pathogenicity of the mutations was assessed following the American College of Medical Genetics and Genomics guidelines.The candidate mutations inADAwere confirmed by Sanger sequencing.

Minigene splicing assay

To investigate the effect of the synonymous mutation c.606G＞A (p.Q202=) on pre-mRNA splicing,theADAminigene model was constructed.The genomic fragment of 2612 nucleotides from exon 5 to exon 7 of wild-type (WT) and mutant-type (MT) was amplified using polymerase chain reaction (PCR),and then the fragment was cloned into the vector of pMini-CopGFP to construct a WT and MT minigene model,respectively.293 T cells were cultured in Dulbecco’s modified Eagle's medium supplemented with 10% fetal bovine serum (PNA,German) and transiently transfected with 4 μg of WT or MTADAminigene construct using Lipofectamine 2000 (Thermo,USA).The successfully constructed WT and MT minigene models were validated by Sanger sequencing.Cells were harvested in TRIzol reagent (Invitrogen,USA),and then total RNA was extracted and reverse-transcribed by reverse transcription-PCR (RT-PCR).PCR products were subsequently analyzed by agarose gel electrophoresis and Sanger sequencing.

Cytokine assays

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by density centrifugation.The cells were resuspended in RPMI 1640 supplemented with penicillin,streptomycin and 10% fetal calf serum.PBMCs were then stimulated for six hours with phorbol-12-myristate-13-acetate (PMA;100 ng/mL;Sigma-Aldrich,USA) and ionomycin (1 μg/mL;Millipore,USA) at 37 °C.Brefeldin A (5 μg/mL;Biolegend,USA) was present during the final five hours.Intracellular staining was performed after cell fixation and permeabilization,and anti-interferon (IFN)-γ (BD Pharmingen,USA) and anti-tumor necrosis factor (TNF)-α (Biolegend,USA) antibodies were used.

T and B lymphocyte proliferation assays

The patient’s PBMCs were isolated as described above.Carboxyfluorescein succinimidyl ester (CFSE) was added to the cells at 1 μmol/L and immediately incubated at 37 °C for 10 minutes.Cells were then incubated in ice-cold RPMI 1640 supplemented with 10% fetal calf serum for 10 minutes.After removing excess dye,CFSE-labeled PBMCs were seeded in a 96-well flat-bottom culture plate in RPMI 1640 complete medium and 100 U/mL penicillin,100 μg/mL streptomycin and stimulated with 12.5 μg/mL anti-CD3/anti-CD28 (Invitrogen,USA) or 2 μg/mL cytosine-phosphate-guanine (Hycult Biotech,The Netherlands),2 ng/mL interleukin (IL)-4 (Sigma,USA) and 2 μg/mL lipopolysaccharides (Sigma,USA).As baseline controls,unstimulated CFSE-labeled PBMCs were also cultured under the same conditions for each sample.Cells were cultured at 37 °C in the presence of 5% CO 2,harvested at day 3 and analyzed on a flow cytometer.

Search strategies

PubMed together with the Chinese databases “Wanfang” and “China Network Knowledge Infrastructure” were searched for literature written in English and Chinese.The key words included “adenosine deaminase deficiency”,“ADA deficiency”,“deficiency of adenosine deaminase”,and “deficiency of ADA”.We examined all the studies published up until December 2022;cases from China were included,and cases without clinical details were not included.

Results

Demographics

In this study,a total of nine Chinese patients with ADA deficiency were included and analyzed.Four of these patients were diagnosed and followed up in Beijing Children’s Hospital from July 2020 to December 2022.The other five cases were published previously from April 1989 to December 2022 in Chinese [8-11],two previously diagnosed by our group [9].The nine patients were from seven unrelated families [patient 2 (P2) and P3 were siblings,P7 and P8 were identical twins],including four males and five females.

ADA gene defects in patients

Except for P1,who was diagnosed based on clinical manifestations and laboratory tests,ADAmutations were found in the other eight patients.Homozygous mutations were observed in two patients,and six patients were found to have compound heterozygous mutations.There were six missense mutations,three nonsense mutations and one synonymous mutation,of which two were novel: W272X and Q202=(Supplementary Fig.1).The synonymous mutation c.606G＞A (p.Q202=) alters the last nucleotide of exon 6,and the nucleotide substitution itself is silent with respect to the amino acid sequence.The variant was absent from the Exome Sequencing Project,1000 Genomes Project,or Exome Aggregation Consortium.To identify the pathological significance of the c.606G＞A mutation,a minigene splicing assay was performed.RT-PCR analysis results showed that the splicing products generated by the MT and WT minigenes were different.The WT lane demonstrated one fragment of 387 bp,whereas MT c.606G＞A generated a different fragment of 259 bp (Supplementary Fig.2a).Sanger sequencing of the products showed that the WT amplicons included transcripts of exon 5,exon 6,and exon 7,and the MT amplicons included transcripts of exon 5 and exon 7 (Supplementary Fig.2b,c).Taking these findings together,the splicing pattern of the mutant minigene indicated that synonymous mutation c.606G＞A (p.Q202=) resulted in skipping of exon 6,and this skipping caused a frameshift after amino acid 159,leading to premature termination at codon 207 within exon 8 (Supplementary Fig.2d).

Fig.1 Cranial angiography of patient 6.a A dissecting aneurysm was found in the left internal carotid artery C7,left anterior cerebral artery A1 and left middle cerebral artery M1 (arrow);b M1 moderate stenosis in the right middle cerebral artery (arrow)

Fig.2 Immunological characteristics of γδT cells in patient 9 (P9).a Phenotyping of P9 T cells shows increased proportions of γδT cells.Analysis by CD27 and CD45RA showed that the proportion of effector memory (EM,CD45RA -CD27 -) γδT cells was higher in P9.There were fewer center memory (CM,CD45RA -CD27 +) and naïve (CD45RA+CD27 +) γδT cells in P9 than in healthy controls,while terminal differentiated effector memory (TDEM,CD45RA+CD27 -) cells were comparable to control;b up to 90% of all γδT cells express the Vδ2 chain in P9;c upon stimulation with PMA/ionomycin,the cytokine (IFN-γ and TNF-α) production of γδT cells was increased in P9 compared with healthy control.TCR T-cell receptor,INF interferon,TNF tumor necrosis factor,PMA phorbol-12-myristate-13-acetate

Clinical manifestations of ADA-deficient patients

Five patients had disease onset in the neonatal period,and the latest time of onset was at 5 years of age.P6 and P9 were both diagnosed with the delayed-onset type at the age of 4 years.P5 was diagnosed with late-onset ADA deficiency at 37 years old.The median time to diagnosis was 5 months (range: 27 days to 32 years).All patients had lower respiratory tract infections and were diagnosed with pneumonia or bronchopneumonia.Klebsiella pneumoniaewas the most commonly reported organism (n=3).Pneumocystis jiroveciipneumonia was diagnosed in one patient.Autopsy of P1 revealed a large number of molds in the bronchus.Various bacterial (Streptococcus pneumoniae,Escherichia coli,Pseudomonas aeruginosa,Stenotrophomonas maltophilia,Haemophilus influenzae) and viral [respiratory syncytial viral,cytomegalovirus (CMV),rhinovirus] pathogens were also recorded.Urinary tract infections were identified in three patients,and the pathogens includedEscherichia coliand CMV.Three patients were diagnosed with infectious diarrhea.Salmonella typhimuriumwas detected in the stool culture of one patient,and rotavirus was detected in the other two patients.Septicaemia was diagnosed in two patients,and specific organisms were reported in one (Klebsiella pneumoniae).Omphalitis was diagnosed in one patient withKlebsiella pneumoniaedetected in umbilical secretion culture.More than half of the patients (5 of 9 cases) failed to thrive.The imaging findings showed reduced or absent thymic shadows in five patients.Of them,pathological biopsy was performed in P1 and indicated dysplasia of the thymus,spleen and mesenteric lymph nodes.Auditory neurologic deficits were found in three patients.Skeletal abnormalities were found in two patients,and these abnormalities manifested as morphological changes in the ribs.Autoimmune thyroiditis was detected in two delayed/late onset patients.In addition,P6 had language and fine motor regression starting in the third year after diagnosis.The vision of both eyes in P6 became gradually blurred,and there was only a sense of light at the latest follow-up visit.Notably,intracranial artery stenosis and multiple aneurysms were detected by cranial angiography in P6 (Fig.1 a,b),which were considered secondary to vasculitis.No other abnormalities were found in the systemic vascular ultrasonography.To the best of our knowledge,this has not been reported previously in ADA-deficient patients.The clinical manifestations of the nine patients with ADA deficiency are summarized in Tables 1,2.

Table 2 Summary of the clinical manifestations of the nine patients with adenosine deaminase deficiency

Immunologic phenotype of ADA-deficient patients

The immunologic phenotype of ADA-deficient patients is summarized in Tables 3,4.Except for P5,the laboratory studies revealed both cellular and humoral immune deficiencies in all of the patients,as evidenced by the decreased absolute lymphocyte counts,including decreases in the T lymphocytes and B lymphocytes and low levels of serum immunoglobulin.The absolute number of T lymphocytes in P5 was normal with a decreased number of B lymphocytes as well as low levels of serum immunoglobulin.Notably,the relative and absolute number of γδT cells in P9 was significantly increased,which was normal or decreased in other patients.The phenotype of γδT cells was analyzed.The proportion of effector memory (EM;CD45RA -CD27 -) γδT cells was higher in P9.There were fewer center memory (CD45RA -CD27 +) and naïve (CD45RA+CD27 +) γδT cells in P9 than in healthy control,while terminally differentiated effector memory (CD45RA+CD27 -) cells were comparable to control (Fig.2 a).Further analysis revealed that up to 90% of the γδT cells expressed the Vδ2 chain (Fig.2 b).Upon stimulation with PMA/ionomycin,the cytokine (IFN-γ and TNF-α) production of γδT cells was increased in P9 compared with healthy control (Fig.2 c).T and B lymphocyte proliferation was tested and found to be decreased in P9 (Fig.3).The TCR/B-cell receptor (BCR) repertoire was examined in P6 and P9,which showed decreased TCR and BCR diversity and oligoclonal expansion of T cells in P6.The NK cell count was decreased in five of the nine patients.The isohemagglutinin titers (anti-A and/or anti-B) in the serum were evaluated in two patients and found to be reduced.Six of seven patients had neutropenia,and six patients presented with anemia and/or thrombocytopenia.ADA activity was assessed in six patients,and all of them demonstrated reduced ADA activity.

Fig.3 T and B lymphocyte proliferation assays of patient 9 (P9) by CFSE dilution assay.Representative proliferation of CD4+(left) and CD8+(middle) T cells and B cells (right) from healthy individual (top) and P9 (bottom) under different conditions.Stimulated (shown in gray): treated with anti-CD3/CD28 (for CD4+and CD8+T cells) or treated with the combination of CpG,LPS and IL-4 (for B cells).Unstimulated cells are shown as black dotted lines.CFSE carboxyfluorescein succinimidyl ester,CpG cytosine-phosphate-guanine,LPS lipopolysaccharides,IL-4 interleukin-4

Treatment and outcomes

Intravenous immunoglobulins were given in eight patients,and antibiotic prophylaxis was performed in seven patients.In total,five patients died.The median age at death was 4 months (range: 29 days to 10 months).P1 and P3 died of severe infection at 29 days and 3 months of age,respectively.P4 died after discontinuing treatment at 4 months because of economic reasons.Due to the lack of a matching donor and the difficulty of obtaining enzyme replacement therapy (ERT),P2 received a fresh irradiated red blood cell infusion to supplement ADA but finally died of infection at 6 months old.P7 and P8 suffered from recurrent infections with severe development retardation,and each received ERT only twice.P8 died of an infection at the age of 10 months.Hemopoietic stem cell transplantation (HSCT) was performed in P6 and P9 at the age of 5 years.P6 received cord blood stem cells from matched unrelated donors,and T-cell chimerism was 98.99% at the last follow-up (post-HSCT nine months).P9 received bone marrow stem cells from matched unrelated donors,and T-cell chimerism was 99.54% at the last followup (five months post-HSCT) (Table 5).

Table 5 Treatment and outcome of the nine patients with adenosine deaminase deficiency

Discussion

ADA deficiency is an autosomal recessive genetic defect caused by mutations in theADAgene with a variable phenotypic spectrum.The most severe is early-onset SCID (known as ADA-SCID),which presents in infancy and usually results in early death.Ten to 15% of ADA-deficient patients have a less severe delayed onset combined immune deficiency (CID),usually diagnosed between the ages of one and ten years.A smaller percentage of patients have late-onset CID,diagnosed in the second to fourth decades.Benign partial ADA deficiency occurs in a subset of immunocompetent individuals with very low or absent ADA activity in erythrocytes but greater ADA activity in nucleated cells [5].Arredondo-Vega et al.[12] indicated that the clinical phenotype is affected significantly by the level of residual ADA catalytic activity and grouped alleles according to the potential of providing ADA activity (groups 1-4 with increasing ADA activity).The research demonstrated that deletions and nonsense mutations (group 0) and some missense variants,including R149W and R235Q (group 1),were associated with the SCID phenotype,which might explain the severe clinical phenotype of P2,P3,P4,P7 and P8 in our study.P5 with late-onset ADA deficiency was heterozygous for the missense mutations R235Q and P279Q,and the latter has been observed in partial ADA deficiency [13].The missense mutation V177M in P6 was found to be expressed at a low level of ADA activity (group 2) and has been reported in patients with a delayed onset of clinical manifestation [12,14].P9 with delayed-onset ADA deficiency was heteroallelic for R142X and a novel synonymous mutation of Q202=.R142X has been recorded in SCID patients homozygous for the variant,and ADA activity in T cells and red blood cells was ＜ 2% of normal [15].Synonymous mutations were historically thought to be of little significance,but over the past few decades,the importance of synonymous mutations has been pointed out [16-18],and a number of functional synonymous mutations have been reported in many human diseases [19-23].Here,we reported the first pathogenic synonymous mutation in theADAgene.This demonstrated that the synonymous mutation Q202=induced skipping of the entire exon 6 by affecting the splicing pattern inADA,resulting in a frameshift predicted to cause premature truncation of the protein.As discussed above,the less severe phenotype conflicted with the genotype of P9.It is possible that factors other than theADAgenotype are involved.

In this study,except for P5,all patients had a disease onset prior to one year of age,and the median time to diagnosis was five months.Similar to other reports of ADA deficiency [24],early-onset infection,thymic abnormalities and failure to thrive were the most common manifestations of our ADA-deficient patients,and infection was the main reason to suspect ADA deficiency.Notably,cerebral aneurysm was diagnosed in P6 at 4 years old by cranial angiography,which was considered to be secondary to vasculitis.To date,no ADA-deficient patient with cerebral aneurysms has been reported before.We hypothesized that this clinical condition might be associated with immune dysregulation accompanied by delayed onset.Adenosine is generally considered to have immune regulatory effects,which are mostly anti-inflammatory,through interaction with its four receptor subtypes,A1,A2a,A2b and A3.The tissue density of receptors,as well as the levels of endogenous adenosine,is closely related to the tissue-protective functions of adenosine.ADA is a key catabolic enzyme of endogenous adenosine,and blockade of ADA is an alternative strategy for the management of several pathological conditions,such as inflammation [25].However,A2bAR has a proinflammatory role in dendritic cells [26],and the activation of A2bAR is associated with pulmonary inflammation [27].A2bAR also plays an accelerative role in intestinal inflammation [28].In the central nervous system (CNS),the immunomodulatory effects of adenosine are mainly mediated by the A1 and A2aAR subtypes.Moreover,a study suggested that A2bAR had a detrimental effect on the CNS when it was inflamed [29].On the other hand,A2aAR antagonists showed a good ability to counteract neuroinflammation [30].Thus,the cerebral aneurysm secondary to vasculitis in P6 would be part of the clinical phenotype in ADA deficiency.The impaired purine metabolism and accumulation of substrates might lead to the development of neuroinflammation through interaction with A2aAR and/or A2bAR.It is worth mentioning that ADA contains two isoenzymes: ADA1 and ADA2.The two proteins are partially homologous but different in structure,cellular localization,and expression [31].We use “ADA” to refer to ADA1 in general.ADA2 is encoded by theADA2gene,and deficiency of ADA2 (DADA2) is a complex systemic autoinflammatory disorder [31].It is generally believed that ADA deficiency and DADA2 are two completely different diseases.In contrast to the role of ADA1 as the predominant metabolizer of adenosine in vivo,marked accumulation of deoxyadenosine nucleotides is not observed in DADA2 [32].Vasculopathy involving small-and medium-sized arteries is the major clinical feature of DADA2,and cerebral events,including cerebral vessel aneurysm,have been described [33].The functions of ADA2,including polarization of the monocyte phenotype,regulation of neutrophil extracellular trap formation and modulation of innate immunity,may play a role in vasculopathy formation,but the detailed mechanisms remain unclear [34].Kaljas et al.[35] demonstrated that adenosine deaminases ADA1 and ADA2 bind to distinct lymphocyte subsets in humans to regulate their activation and survival.Moreover,recent studies have shown that both B and T lymphocyte abnormalities are observed in DADA2 patients [36,37].Together,these overlap of the manifestations between ADA1 and ADA2 implies that there may be functional overlap or supplementation between the two enzymes to some degree.Further exploration of the mechanism underlying the effect of ADA deficiency on cerebral aneurysms is needed to confirm that cerebral aneurysm is a direct consequence of ADA deficiency.This is essential to provide accurate treatment for these patients.

As a key component of the purine metabolic pathway,the absence of ADA enzyme activity leads to the accumulation of poisonous purine degradation byproducts.These cytotoxic metabolites primarily affect lymphocyte development,viability,and function [1,38-41],resulting in impaired cellular and humoral immunity (T -B -).Herein,laboratory studies revealed cellular and humoral immune deficiencies in all of the patients except for P5.Our patients manifested with lymphopenia,hypogammaglobulinemia and dysplasia of the thymus,spleen and mesenteric lymph nodes in pathological biopsies.We further performed immune repertoire analysis in two patients and revealed decreased TCR and BCR diversity and oligoclonal T-cell expansion,indicating developmental abnormalities in T and B cells.Notably,γδT cells in P9 were significantly increased,and the frequency of effector γδT cells was higher than that in healthy control,which was associated with the enhanced ability to secrete TNF-α and IFN-γ in response to stimulation with PMA and ionomycin.γδT cells play a role in the defense against multiple viral and microbiological infections and tumoral antigens [42].Ou et al.[43] demonstrated that γδT cell functions correlate with their circulating levels in healthy individuals.We hypothesized that the increased number of γδT cells in peripheral blood might explain the milder clinical phenotype of P9.However,the potential mechanisms contributing to the dominance of γδT cells in P9 are not clear.Elevated proportions of γδT cells are a previously recognized phenomenon in some atypical SCID patients [44-46],and Tometten et al.[47] found that the expansion of γδT cells is a general feature of atypical SCID and not a response to αβT cell lymphopenia.In their cohort,Tometten et al.suggested that CMV infection may be a driver of γδT cell expansion,which may be a potential cause of autoimmune cytopenias.The lack of CMV infection in P9 suggests that other factors contributing to the elevated γδT cell counts remain to be elucidated.γδT cells and αβT cells arise from common T-cell progenitors in the thymus.The molecular events leading to the lineage decision of developing double-negative thymocytes to differentiate into γδT cells versus αβT cells or other unconventional T cells are not fully resolved [48].We hypothesize that the potential mechanisms contributing to the predominance of γδT cells in P9 might be due to abnormal molecular signals in lineage determination resulting from defective ADA activity.Further study should be carried out to resolve this issue.

In addition to the marked depletion of lymphocytes,neutropenia was observed in six patients (range from 0.1 to 0.78× 10 9/L).Kim et al.[49] demonstrated that the lack of ADA directly predisposes patients to myeloid abnormalities,which may be due to the toxic effect of purine metabolites,and neutropenia was identified in 65.0% of patients with ADA deficiency.Furthermore,due to the ubiquitous nature of ADA,nonimmunological symptoms were also observed in ADA deficiency.Radiologically detectable bone abnormalities were present in approximately 50% of ADA-SCID patients and may be associated with the imbalance of the receptor activator of nuclear factor-κB ligand and the osteoprotegerin axis [50].Rib abnormalities were observed in two of the seven patients in our study,and these lesions presented as trumpet-shaped changes of the ribs and rachitic rosary.Auditory neurologic deficits were found in three patients,which had a high prevalence (58%) in a previous study [51].In recent years,the effects of ADA deficiency on the nonimmune system have become more apparent because of the improved understanding of this disease and the development of treatment.In addition to the above observations in our patients,other nonimmunological manifestations,including behavioral abnormalities,non-infectious pulmonary disease,hyperbilirubinemia and noninfective hepatic failure,atypical hemolytic urinemic syndrome,myocardial hypertrophy and dermatofibrosarcoma protuberans,have also been identified [52-57].It is important to raise awareness about these nonimmunological manifestations among clinicians to optimize the management of this disease in a timely manner.

The currently available therapeutic options for ADA deficiency include HSCT,ERT and ex vivo corrected autologous hematopoietic stem cell gene therapy (HSC-GT).All patients should have ERT initially before definitive treatment with HSCT or HSC-GT,which can alleviate the acute symptoms and enable partial immunological reconstitution [58].In this study,P7 and P8 received ERT only twice.P2 received an infusion of fresh irradiated red blood cells to supplement ADA;however,this patient’s condition did not improve.If matched sibling donors or matched family donors are available,HSCT should be the first choice for therapy [51].In this study,HSCT was performed in two patients from a matched unrelated donor,and the condition is stable thus far.A recent study showed that patients who received autologous ex vivo lentiviral gene therapy had high overall and event-free survival with sustained ADA expression,metabolic correction,and functional immune reconstitution,and the results of this therapy were comparatively favorable to the results from HSCT [59].To date,no individual has been treated with gene therapy in our study.

To date,only nine patients have been diagnosed with ADA deficiency in China.The incidence is far less than estimated in the world [5].On the basis of information from newborn screening (NBS) from different countries,the incidence of SCID differs among races and regions [60-64].Furthermore,the National Rare Diseases Registry System of China was established in 2006,which needs to be further improved to have meaningful inferences on disease prevalence [65].The lack of physician awareness and experience may be another main factor for the underestimated incidence,and some patients may not be diagnosed until death.However,early screening,diagnosis and treatment are essential for improving prognosis and decreasing morbidity.In North America,23.6% of ADA deficiency diagnoses were triggered by a positive SCID NBS result [66],which is not universally available in Chinese mainland.

This study has several limitations.First,the number of patients was small,as there were few ADA patients identified in China.Second,because it was a retrospective study,some patients’ information was not complete.Further prospective studies should be conducted on a large scale.In addition,five patients had the ADA level measured in the serum,which is less exact due to the lower ADA activity in the plasma than in the cells.

In conclusion,this study described the first case series of Chinese ADA-deficient patients to date.Early-onset infection,thymic abnormalities and failure to thrive were the most common manifestations of Chinese ADA-deficient patients.TheADAgenotype has a major effect on the clinical phenotype.A novel synonymous mutation (c.606G＞A,p.Q202=) was identified in a delayed-onset patient,which affected pre-mRNA splicing leading to a frameshift and premature truncation of the protein.Furthermore,the expansion of γδT lymphocytes with an increased effector phenotype was identified in the same patient.We hypothesized that γδT cell expansion is associated with delayed-onset ADA deficiency.In addition,we reported cerebral aneurysm and intracranial artery stenosis for the first time in delayed-onset ADA deficiency.Further study is warranted to investigate the underlying mechanisms.

Supplementary InformationThe online version contains supplementary material available at https:// doi.org/ 10.1007/ s12519-023-00729-3.

AcknowledgementsWe thank the patients and their families for their collaboration.We thank all the members of clinical team who provided care for patients.

Author contributionsZY and LW contributed equally to this work.ZY and LW contributed to original draft.SZ contributed to reviewing and editing.LY,SF and HTX contributed to data curation.LZG contributed to formal analysis.MHW and WTY guided the study and revised the manuscript critically.All authors read and approved the final manuscript.

FundingThis work was supported in part by the National Natural Science Foundation of China (81971547 and 81900136),Beijing Hospitals Authority’s Ascent Plan (DFL20221001),National Key Research and Development Program of China (2021YFC2702005),and Wu Jieping Medical Foundation (320.6750.2022-03-53).

Data availabilityAll data generated or analyzed during this study are included in this published article.

Declarations

Ethical approvalThis study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Beijing Children’s Hospital,Capital Medical University (2021-E-192-R).Informed consent to participate in the study has been obtained from the participants or their parent or legal guardian in the case of children under 16.

Conflict of interestNo financial or non-financial benefits have been received or will be received from any party related directly or indirectly to the subject of this article.The authors have no conflict of interest to declare.