Importance of TREC and KREC as molecular markers for immunological evaluation of down syndrome children

Recurrent and severe infections occurred in children with Down Syndrome (DS) due to immunological parameter defects have been reported. The aim of the study is to evaluate the importance of using T-cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) as molecular markers for immunological investigation of children with DS. The study included 40 non-disjunction trisomy 21 confirmed DS children, and 25 healthy controls. Peripheral blood (PB) was analyzed for lymphocyte subpopulations by flow cytometry, serum immunoglobulin levels, and TREC and KREC copy numbers using quantitative real-time PCR. DS patients showed significantly lower absolute counts of PB T lymphocytes, T helper lymphocytes, T cytotoxic lymphocytes, B lymphocytes, and Natural killer cells, and lower serum IgA, IgG, and IgM levels compared to healthy controls. Copy number of TREC and KREC showed no significant differences between DS patients and healthy controls. There is a significant positive correlation between TREC copy number with a percentage and absolute count of helper T lymphocytes in patients. Also, the KREC copy number was significantly negatively correlated with the age of patients. These findings suggest that copy numbers of TREC and KREC could be useful as molecular markers for immunological evaluation of patients with DS.


Association of Immunological profile with the clinical manifestations of DS patients.
Our association analysis showed that the percentage and absolute count of B lymphocytes and percentage of helper T cells have significant negative associations with the age of DS patients (Fig. 2).However, there is no association

TREC and KREC copy numbers and their correlation with the immunological parameters and clinical characteristics of DS patients.
Copy number of TREC and KREC showed no significant differences between patients and healthy controls.There is a significant positive correlation between TREC copy number with a percentage and absolute count of helper T cells in patients.Also, the KREC copy number was significantly negatively correlated with the age of patients (Fig. 3).Otherwise, no associations were found between TREC and KREC copy numbers and the associated clinical manifestations of patients, parental consanguinity, or patients' residence (rural or urban).The receiver operating characteristic (ROC) curve of TREC and KREC and their cut-off values.The ROC curve of TREC showed a good significant area under the curve (AUC) value of 0.837 with a sensitivity of 93% and specificity of 65% (P < 0.001); while KREC showed a ROC curve with a significant AUC value of 0.768, sensitivity 80% and specificity 70% (P ≤ 0.001) (Fig. 4, Table 2).

Discussion
The results presented in this study provide insights into the immunological profile of DS patients and their potential associations with clinical manifestations.The baseline characteristics of the study participants showed a predominantly male population with a median age of 1.8 years.The cumulative clinical manifestations of patients included thyroid, cardiac, and hernia disorders.The immunological profile analysis have revealed that the percentages of T lymphocytes, T helper lymphocytes, T cytotoxic lymphocytes, B lymphocytes, and natural killer cells in PB of DS patients are comparable to those of healthy controls, while their absolute counts are lower in patients than those of controls.Moreover, DS patients have lower immunoglobulin levels (IgA, IgG, and IgM) in serum compared to controls.Our findings are consistent with previous studies on the immune system in individuals with DS.Ram and Chinen 7 and Huggard et al. 23 have demonstrated that the lower absolute counts of immune cells and immunoglobulin levels observed in individuals with DS may contribute to an increased susceptibility to infections and a reduced ability to mount an effective immune response.Also, Dieudonné et al. 24 have reported immune dysfunction in DS patients.Our association analysis showed that the percentage www.nature.com/scientificreports/and absolute count of B lymphocytes and percentage of helper T cells have significant negative associations with the age of patients.This is in agreement with a study by Froňková et al. 18 who demonstrated a reduction of absolute B and T lymphocyte counts with aging.However, there are no significant associations between the immunological parameters of our DS patients and their associated hypothyroidism, congenital heart anomalies, or umbilical hernia.This study has found that there are no significant differences in TREC and KREC copy numbers between DS patients and healthy controls.Moreover, no associations have been found between TREC and KREC copy numbers and the clinical characteristics of DS patients.In contrast, other studies have found that the copy numbers of TREC and KREC were significantly lower in DS patients compared to healthy controls, suggesting a decrease in the output of newly produced T and B cells in DS patients which may contribute to immune defects and increased susceptibility to infections observed in individuals with DS [25][26][27] .
Interestingly, our study has found a significant positive correlation between TREC copy number and the percentage and absolute count of CD4 T lymphocytes in DS patients indicating a relationship between the molecular markers and the respective lymphocyte subsets.Similarly, a study by McCullough et al. 28 showed a positive association between TREC and the percentage of CD4 and CD8 T lymphocytes, recommending that TREC may be a useful biomarker for monitoring immunological insufficiency in DS patients.Additionally, we www.nature.com/scientificreports/observed a significant negative correlation between copy numbers of KREC and age in DS patients, which is consistent with previous studies which reported age-related declines in thymic and bone marrow outputs 18,29,30 , suggesting that KREC could be a helpful biomarker for monitoring age-related immune defects in DS patients.Furthermore, we have performed ROC curve analysis to assess the diagnostic accuracy of TREC and KREC copy numbers as molecular markers for immune insufficiency in DS patients.Our findings showed good significant AUC values, with TREC having a sensitivity of 93% and specificity of 65%, and KREC having a sensitivity of 80% and specificity of 70%.ROC curve analysis demonstrated that TREC and KREC have high sensitivity and specificity for detecting immune defects in DS patients, indicating that these biomarkers have potential diagnostic value for immune dysregulation in DS.This matched with other studies that found that TREC and KREC had high sensitivity and specificity for detecting immune deficiency, supporting their potential utility as molecular markers for immunological investigations in DS population 19,31 .
The main limitation of our study included the small sample size, which may limit the detection of significant associations between TREC/KREC and clinical data of DS patients.Therefore, further researches on a large sample size are needed in the future to investigate the clinical associations with TREC/KREC in DS patients.

Conclusion
Our study provides evidence of immune defects in DS patients, as well as the potential utility of TREC and KREC as molecular markers for immunological evaluation of those patients.The findings also highlight the importance of considering age-related changes in the immunological profile of those patients when interpreting immunological data.However, there is a need for large-scale studies to confirm these findings.

Methods
Ethics approval and consent to participate.This study was approved by the ethics committee of the National Research Centre, Egypt with an approval number 19267 and has therefore been performed under the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.All samples were obtained with the written informed consent of the parent/guardian of all children involved in this study before their enrollment.Determination of serum immunoglobulin levels.Serum immunoglobulin levels (IgA, IgG, and IgM) were determined in patients and healthy controls using Human ELISA kits (SunRed Biotechnology Company, Shanghai, China) according to the manufacturer's instructions.

Study participants.
DNA isolation.DNA was isolated from 200 µl of PB using QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) by following the manufacturer's instructions.
TREC and KREC quantification.Copy numbers of TREC and KREC were determined using Quantitative Real-Time PCR assay performed on the 7500 Fast Real-Time PCR (Applied Biosystems).Primers and probes of TREC, KREC, and the reference gene TRAC were designed according to Sottini et al. (2010).TaqMan Universal master mix (Applied Biosystems) was used in the reaction.The protocol of reaction was as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s and 60 °C for 1 min.TREC, KREC, and TRAC copy numbers were obtained by deducing the respective sample quantities from the standard curve obtained by serial dilutions (10 6 , 10 5 , 10 4 , 10 3 , 10 2 , and 10) of the linearized triple-insert plasmid DNA (obtained from Alessandra Sottini and Luisa Imberti, Italy) that amplified in each PCR plate 33 .
Statistical analysis.Data were statistically analyzed using SPSS version 27.0 software (SPSS Inc., Chicago, Illinois, USA).The Non-parametric Mann-Whitney U test was used for comparing the immunological parameters and immunoglobulin levels between groups of the study.Associations among the immunological parameters, immunoglobulin levels, TRECs and KRECs, age, and the clinical manifestations of patients were analyzed using Spearman's rank correlation.Data were presented as mean ± SD or median (IQR).A P-value of less than 0.05 was considered statistically significant.ROC curve was constructed for TREC and KREC to evaluate the efficiency of those markers for immunological investigation in DS patients.AUC values, specificity, sensitivity, and 95% confidence intervals were calculated 34 .

Figure 1 .
Figure 1.Immunological parameters of Down syndrome patients compared to healthy controls.Data were expressed as a median.*Statistically significant at P < 0.05 (by Mann-Whitney U test).

Figure 2 .
Figure 2. Association of Immunological profile with the age of Down syndrome patients.*Statistically significant at P < 0.05 (by Spearman correlation analysis).

Figure 3 .
Figure 3. Correlation of TREC and KREC copy numbers with the immunological parameters and age of Down syndrome patients.*Statistically significant at P < 0.05 (by Spearman correlation analysis).
32is study included 40 DS patients and 25 healthy subjects matched for age and sex (age range: 6 months to 7 years).They were recruited from the Developmental Assessment and Genetic Disorders Clinic, National Research Centre, Egypt.Enrolled children were subjected to history taking, pedigree analysis, and meticulous clinical examinations.G-banding chromosomal analysis was performed on all patients to confirm the diagnosis of trisomy 21.A thyroid profile, electrocardiogram (ECG), and abdominal sonar were done for all DS patients to exclude or confirm any associated anomalies.Demographic and cumulative clinical manifestations were recorded.Subjects with missing values were excluded during collecting data before beginning of the study.Follow-up observation time was one year.Flow cytometry analysis.PB samples were collected from patients and healthy controls in tubes containing EDTA.50 μL of EDTA-treated PB were incubated for 30 min at 4 °C in the dark with fluorochrome-labeled monoclonal antibodies (mAbs): Fluorescein Isothiocyanate (FITC)-conjugated CD3, Allophycocyanin (APC)-conjugated CD19, Phycoerythrin (PE)-conjugated CD4, FITC-conjugated CD8, and PE-conjugated CD16 (Becton Dickinson, USA).Red blood cells were lysed using BD FACS Lysing Solution (Becton Dickinson, USA).The stained cells were then washed and resuspended in phosphate-buffered saline.Approximately 30,000 stained cells in each sample were analyzed with a BD Accuri™ C6 Flow Cytometer (BD Biosciences).The lymphocytes were gated by setting the appropriate forward scatter/side scatter axes.Data were acquired and data analysis was performed by the BD Accuri™ C6 software program32.