RESUMO
Calcium (Ca2+) flux acts as a central signaling pathway in B cells, and its alterations are associated with autoimmune dysregulation and B-cell malignancies. We standardized a flow-cytometry-based method using various stimuli to investigate the Ca2+ flux characteristics of circulating human B lymphocytes from healthy individuals. We found that different activating agents trigger distinct Ca2+ flux responses and that B-cell subsets show specific developmental-stage dependent Ca2+ flux response patterns. Naive B cells responded with a more substantial Ca2+ flux to B cell receptor (BCR) stimulation than memory B cells. Non-switched memory cells responded to anti-IgD stimulation with a naive-like Ca2+ flux pattern, whereas their anti-IgM response was memory-like. Peripheral antibody-secreting cells retained their IgG responsivity but showed reduced Ca2+ responses upon activation, indicating their loss of dependence on Ca2+ signaling. Ca2+ flux is a relevant functional test for B cells, and its alterations could provide insight into pathological B-cell activation development.
Assuntos
Subpopulações de Linfócitos B , Linfócitos B , Humanos , Subpopulações de Linfócitos B/metabolismo , Células Produtoras de Anticorpos , Receptores de Antígenos de Linfócitos B/metabolismo , Diferenciação CelularRESUMO
ABCC6 promotes ATP efflux from hepatocytes to bloodstream. ATP is metabolized to pyrophosphate, an inhibitor of ectopic calcification. Pathogenic variants of ABCC6 cause pseudoxanthoma elasticum, a highly variable recessive ectopic calcification disorder. Incomplete penetrance may initiate disease heterogeneity, hence symptoms may not, or differently manifest in carriers. Here, we investigated whether incomplete penetrance is a source of heterogeneity in pseudoxanthoma elasticum. By integrating clinical and genetic data of 589 patients, we created the largest European cohort. Based on allele frequency alterations, we identified two incomplete penetrant pathogenic variants, c.2359G>A (p.Val787Ile) and c.1171A>G (p.Arg391Gly), with 6.5% and 2% penetrance, respectively. However, when penetrant, the c.1171A>G (p.Arg391Gly) manifested a clinically unaltered severity. After applying in silico and in vitro characterization, we suggest that incomplete penetrant variants are only deleterious if a yet unknown interacting partner of ABCC6 is mutated simultaneously. The low penetrance of these variants should be contemplated in genetic counseling.
Assuntos
Pseudoxantoma Elástico , Humanos , Mutação , Pseudoxantoma Elástico/genética , Pseudoxantoma Elástico/metabolismo , Pseudoxantoma Elástico/patologia , Penetrância , Trifosfato de Adenosina , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genéticaRESUMO
Anti-thyroid antibody (ATA) positivity affects 1 out of 9 women in childbearing age and presents a significant risk for infertility. Emerging evidence indicates that alterations in the B cell receptor induced calcium (Ca2+) signaling could be key in the development of autoimmunity. We aimed to investigate the Ca2+ flux response of B lymphocyte subsets to BCR stimulation in Hashimoto's thyroiditis and related infertility. We collected peripheral blood samples from ATA+, infertile, euthyroid patients (HIE), hypothyroid, ATA+ patients before (H1) and after levothyroxine treatment (H2), and age-matched healthy controls (HC). All B cell subsets of ATA+, infertile, euthyroid patients showed elevated basal Ca2+ level and hyper-responsivity to BCR ligation compared to the other groups, which could reflect altered systemic immune function. The Ca2+ flux of hypothyroid patients was similar to healthy controls. The levothyroxine-treated patients had decreased prevalence of CD25+ B cells and lower basal Ca2+ level compared to pre-treatment. Our results support the role of altered Ca2+ flux of B cells in the early phase of thyroid autoimmunity and infertility.
Assuntos
Doença de Hashimoto , Hipotireoidismo , Infertilidade Feminina , Humanos , Feminino , Tiroxina , AutoanticorposRESUMO
Acquired hemophilia A (AHA) is a rare severe autoimmune bleeding disorder with significant morbidity and mortality. Although critical for disease control, there is no consensus for the best immunosuppressive regimen. Most authors use steroids first line, followed by other agents for steroid failures. Upfront combined regimens offer the advantage of reduced steroid exposure and toxicity as well as increased efficacy. We retrospectively analyzed data from 32 patients with AHA treated on an identical such institutional protocol: cyclophosphamide 1000 mg on days 1 and 22, dexamethasone 40 mg on days 1, 8, 15, and 22, and rituximab 100 mg on days 1, 8, 15, and 22 (the regimen was termed CyDRi). All patients received at least 1 cycle of CyDRi. If necessary, CyDRi was repeated until remission, no sooner than day 43 of the previous cycle. Bleeding control was rapidly achieved. The median time for bleeding control was 15.5 days (range, 0-429 days; interquartile range, 2.5-29.5 days). Thirty-one (96.8%) of 32 patients achieved durable complete remission (CR); 29 (90.6%) of 32 patients were alive at last follow-up, all of them in CR. The median time to reach first CR was 77 days (range, 19-939 days; interquartile range, 31-115 days). Toxicity and side effects were acceptable and milder than those of commonly used, prolonged steroid therapies. In conclusion, the CyDRi regimen produced markedly higher CR rates and overall survival than currently used sequential regimens. Taken together, CyDRi proved to be an attractive option for the immunosuppression of elderly patients with AHA.
Assuntos
Hemofilia A , Humanos , Idoso , Estudos Retrospectivos , Ciclofosfamida/efeitos adversos , Hemorragia/tratamento farmacológico , Hemorragia/etiologia , Terapia de Imunossupressão , Esteroides/uso terapêuticoRESUMO
We aimed to identify incompletely penetrant (IP) variants and interallelic interactions in autosomal recessive disorders by a population-genetic approach. Genotype and clinical data were collected from 9038 patients of European origin with ASL, ATP7B, CAPN3, CFTR, CTNS, DHCR7, GAA, GALNS, GALT, IDUA, MUT, NPHS1, NPHS2, PAH, PKHD1, PMM2, or SLC26A4-related disorders. We calculated the relative allele frequency of each pathogenic variant (n = 1936) to the loss-of-function (LOF) variants of the corresponding gene in the patient ( ACptV/ACptLOF ) and the general population ( ACgnomADV/ACgnomADLOF ) and estimated the penetrance of each variant by calculating their ratio: (ACptV/ACptLOF)(ACgnomADV/ACgnomADLOF) (V/LOF ratio). We classified all variants as null or hypomorphic based on the associated clinical phenotype. We found 25 variants, 29% of the frequent 85 variants, to be underrepresented in the patient population (V/LOF ratio <30% with p < 7.22 × 10-5 ), including 22 novel ones in the ASL, CAPN3, CFTR, GAA, GALNS, PAH, and PKHD1 genes. In contrast to the completely penetrant variants (CP), the majority of the IP variants were hypomorphic (IP: 16/18, 88%; CP: 177/933, 19.0%; p = 5.12 × 10-10 ). Among them, only the NPHS2 R229Q variant was subject to interallelic interactions. The proposed algorithm identifies frequent IP variants and estimates their penetrance and interallelic interactions in large patient cohorts.
Assuntos
Alelos , Genes Recessivos , Doenças Genéticas Inatas/genética , Genética Populacional , Estudos de Coortes , Feminino , Genes Letais , Humanos , MasculinoRESUMO
INTRODUCTION: Osteoarthritis (OA) as the most common joint disease is a major public health concern. AIM: To investigate the effect of multimorbidity on functional and quality of life outcomes in women with generalized osteoathritis (hand and knee arthritis, GOA). METHOD: In this cross-sectional study, patients according to the American College of Rheumatology (ACR) criteria for OA were invited. The control group consisted of subjects without any musculoskeletal symptoms, osteoarthritis or inflammatory rheumatic disease. Comorbidity count was calculated from the investigated comorbidities. In the GOA group, the function was assessed by Western Ontario and McMaster Universities Arthritis Index (WOMAC), Cochin Hand Scale, Knee Injury and Osteoarthritis Outcome Score (KOOS), Health Assessment Questionnaire (HAQ), while quality of life was measured in both groups with the EuroQol-5D Scale. Interaction between summarized comorbidity count, age, body mass index (BMI) and scores were analysed. Descriptive statistics, two-sample t-test and Pearson's correlation test were used for data analysis. RESULTS: The study groups included 200-200 participants with a similar age spread. Significant correlation was demonstrated in both study groups between higher comorbidity count and older age (0.37, p<0.001, and 0.24, p<0.001 in the GOA and the control group, respectively) and higher BMI (0.18, p: 0.01, and 0.45, p<0.001 in the GOA and the control group, respectively). In GOA, the increasing comorbidity number had a negative effect on the measured outcomes. CONCLUSIONS: Age and BMI showed strong correlation with multimorbidity in both groups. The lower correlation between BMI and comorbidity count in the GOA group requires further investigation and may suggest different interactions. Orv Hetil. 2020; 161(32): 1332-1340.
Assuntos
Atividades Cotidianas , Osteoartrite do Joelho/psicologia , Qualidade de Vida , Fatores Etários , Idoso , Índice de Massa Corporal , Estudos de Casos e Controles , Comorbidade , Estudos Transversais , Feminino , Humanos , Multimorbidade , Dor/epidemiologiaRESUMO
NPHS2, encoding podocin, is the major gene implicated in steroid-resistant nephrotic syndrome. Its c.686G>A, p.R229Q variant is the first human variant with a mutation-dependent pathogenicity; it is only pathogenic when trans-associated to specific mutations. Secondary to its high allele frequency in the European, South Asian, African, and Latino populations, its benign trans-associations can be accidentally identified in affected patients. Distinguishing pathogenic and benign p.R229Q associations can be challenging. In this paper, we present the currently known pathogenic and benign associations, and show that a rare p.R229Q association can be considered pathogenic if the variant in trans meets the following criteria; it affects the 270-351 residues and alters but does not disrupt the oligomerization, its p.R229Q association is found in a family with slowly progressing focal segmental glomerulosclerosis, but is expected to be rare in the general population (<1:106 ). We show that >15% of the p.R229Q associations identified so far in patients are benign.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Síndrome Nefrótica/genética , Polimorfismo de Nucleotídeo Único , Motivos de Aminoácidos , Frequência do Gene , Predisposição Genética para Doença , Glomerulosclerose Segmentar e Focal/genética , Humanos , Conformação Proteica , Multimerização Proteica , Medição de RiscoRESUMO
The transient increase of the cytoplasmic free calcium level in T lymphocytes plays a key role in initiating and maintaining the autoimmune reaction in rheumatoid arthritis (RA). Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. We aimed to compare peripheral T lymphocyte calcium influx kinetics upon activation in patients with recently diagnosed and established RA, and to demonstrate the differences in analysis of kinetic flow cytometry data when using two different algorithms. We took peripheral blood samples from nine patients with recently diagnosed and six patients with established RA. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2, and CD8 cells applying an approach based on smoothing of median fluorescence values (FlowJo) and an algorithm based on function fitting (FacsKin). We assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. Th2 cells of patients with established RA react slower to activating stimuli, whereas CD8 cells show a faster reaction than in patients with recently diagnosed RA. While initially Th1 cells are less sensitive to the inhibition of Kv1.3 and IKCa1 channels in RA, their sensitivity increases along with the duration of the disease. With the algorithm of function fitting instead of smoothing, more statistically significant differences of potassium channel inhibition between the two RA groups could be demonstrated. The function fitting algorithm applied by FacsKin is suitable to provide a common basis for evaluating and comparing flow cytometry kinetic data.
Assuntos
Artrite Reumatoide/imunologia , Transporte Biológico/fisiologia , Cálcio/metabolismo , Canais de Potássio/metabolismo , Adulto , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Sinalização do Cálcio/imunologia , Feminino , Citometria de Fluxo , Humanos , Cinética , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Bloqueadores dos Canais de Potássio/farmacologia , Células Th1/citologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/metabolismoRESUMO
OBJECTIVE: The transient increase of the cytoplasmic free calcium level plays a key role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. DESIGN: Case-control study. SUBJECTS AND METHODS: We took peripheral blood samples from 10 healthy individuals and 9 recently diagnosed rheumatoid arthritis (RA) patients receiving no anti-rheumatic treatment. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2 and CD8 cells applying a novel flow cytometry approach. We also assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. RESULTS: The peak of calcium influx in lymphocytes isolated from RA patients is reached more rapidly, indicating that they respond more quickly to stimulation compared to controls. In healthy individuals, the inhibition of the IKCa1 channel decreased calcium influx in Th2 and CD4 cells to a lower extent than in Th1 and CD8 cells. On the contrary, the inhibition of Kv1.3 channels resulted in a larger decrease of calcium entry in Th2 and CD4 than in Th1 and CD8 cells. No difference was detected between Th1 and Th2 or CD4 and CD8 cells in the sensitivity to IKCa1 channel inhibition among lymphocytes of RA patients. However, specific inhibition of the Kv1.3 channel acts differentially on calcium influx kinetics in RA lymphocyte subsets. Th2 and particularly CD8 cells are inhibited more dominantly than Th1 and CD4 cells. CONCLUSION: The inhibition of Kv1.3 channels does not seem to be specific enough in peripheral RA lymphocytes, since anti-inflammatory Th2 cells are also affected to a noteworthy extent.
Assuntos
Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canal de Potássio Kv1.3/antagonistas & inibidores , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Sinalização do Cálcio/efeitos dos fármacos , Estudos de Casos e Controles , Feminino , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canal de Potássio Kv1.3/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Bloqueadores dos Canais de Potássio/farmacologia , Venenos de Escorpião/farmacologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Preliminary data suggest different intracellular calcium handling of Th1 and Th2 lymphocytes that may contribute to distinct cytokine production patterns. In this study we explored the contribution of the main mechanisms in charge of the elevation and decrease of cytoplasmic free calcium levels, i.e., the endoplasmic calcium release, the calcium release activated calcium (CRAC) channel, the mitochondrial calcium uniporter (MCU), the sarco/endoplasmic reticulum calcium ATPase (SERCA), and the plasma membrane calcium ATPase (PMCA) during the first 10 min of activation in human Th1 and Th2 lymphocytes applying a kinetic flow cytometry approach. We isolated peripheral blood mononuclear cells from 10 healthy individuals. Cells were stained with CD4, CXCR3 and CCR4 cell surface markers to identify Th1 and Th2 cells, respectively and loaded with Fluo-3/AM calcium sensitive dye. Cells were activated with phytohemagglutinine and alterations of cytoplasmic free calcium levels were monitored for 10 min after specific inhibition of the above mechanisms. Our results revealed delicate differences in calcium flux kinetics of Th1 and Th2 lymphocytes. The lower activity of MCU, and therefore of CRAC channels, along with the higher activity of the SERCA pump account for the notion that Th2 cells go through a lower level of lymphocyte activation compared with Th1 cells upon identical activating stimuli. The observed differences in calcium flux of Th1 and Th2 cells may contribute to different calcium handling kinetics and, hence, to distinct cytokine production patterns by these subsets.
Assuntos
Sinalização do Cálcio/imunologia , Cálcio/metabolismo , Citocinas/imunologia , Citosol/metabolismo , Imunidade Celular , Células Th1/metabolismo , Células Th2/metabolismo , Adulto , Compostos de Anilina/análise , Biomarcadores/metabolismo , Canais de Cálcio/imunologia , Canais de Cálcio/metabolismo , Citocinas/biossíntese , Citosol/imunologia , Feminino , Citometria de Fluxo , Humanos , Cinética , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Proteína ORAI1 , Fito-Hemaglutininas/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/imunologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/imunologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th2/citologia , Células Th2/imunologia , Xantenos/análiseRESUMO
The aim of our work was to establish flow cytometry methods for the characterization of mitochondrial Ca(2+) levels, plasma membrane potential, and superoxide generation and to relate kinetics to that of cytoplasmic Ca(2+) levels during short-term activation of T-lymphocytes. We monitored the change of fluorescence absorbance of sequentially measured Jurkat cells for 12 min. The cells were stained with the fluorescent dyes Fluo3-AM, Rhod2/AM, di-BA-C4-(5), or dihydroethidium and then were stimulated with increasing doses of phytohemagglutinin (PHA) or were treated with rotenone. Double-logistic function was fitted to cytoplasmic Ca(2+) signal and mitochondrial Ca(2+) levels, whereas logistic function was fitted to plasma membrane potential and superoxide levels. The calculated function parameters were area under the curve (AUC), maximum (Max), time to reach maximum (t(max)), slope at the first 50% value of Max (Slope), and ending (End) values, respectively. We found significant dose-response relationship between PHA dose and cytoplasmic Ca(2+) signals (AUC, Max, Slope: P<0.05), mitochondrial Ca(2+) levels (AUC and Max: P<0.05), and plasma membrane potential (AUC and End values: P<0.05). In rotenone-treated cells, superoxide generation increased in a dose-dependent manner (P<0.05 for AUC and End values, respectively). The present methodology provides an opportunity for monitoring and characterizing mitochondrial Ca(2+) levels, plasma membrane potential, and superoxide generation in PHA-activated or rotenone-treated Jurkat cells with flow cytometry.
Assuntos
Membrana Celular/metabolismo , Citometria de Fluxo/métodos , Líquido Intracelular/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/química , Humanos , Líquido Intracelular/química , Células Jurkat , Potenciais da Membrana/fisiologia , Superóxidos/metabolismoRESUMO
Kv1.3 and IKCa1 potassium channels play an important role in the maintenance of calcium-influx during lymphocyte activation and present a possible target for selective immunomodulation. We investigated the calcium-influx characteristics of Th1, Th2, CD4, CD8 T-lymphocytes isolated from multiple sclerosis patients without or with interferon-beta therapy, and its modulation by Kv1.3 and IKCa1 channel inhibitors using flow cytometry. Specific immunomodulation of the CD8 subset can be reached through inhibition of Kv1.3 channels in multiple sclerosis patients without interferon-beta. However, this effect is not specific enough concerning all lymphocyte subsets influencing the autoimmune response, since it also affects anti-inflammatory Th2 cells.
Assuntos
Sinalização do Cálcio/imunologia , Cálcio/metabolismo , Interferon beta/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/terapia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/patologia , Projetos Piloto , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Adulto JovemRESUMO
Kv1.3 and IKCa1 potassium channels participate in the maintenance of calcium-influx during lymphocyte activation. Kv1.3 channels have a prominent role in specific T cell subsets, presenting a possible target for selective immunomodulation. We investigated the impact of Kv1.3 and IKCa1 channel inhibitors on calcium-influx characteristics in human T cells in type 1 diabetes mellitus. We isolated lymphocytes from 9 healthy and 9 type 1 diabetic individuals and measured the alteration of calcium-influx with flow cytometry in the Th1, Th2, CD4 and CD8 subsets after treatment of samples with specific channel inhibitors. Our results indicate an increased reactivity of type 1 diabetes lymphocytes, which is correlated to their increased sensitivity to Kv1.3 channel inhibition. However, the contribution of Kv1.3 channels to calcium flux is not exclusive for a specific lymphocyte subset as previous reports suggest, but is characteristic for each subset investigated. Therefore, the proposed inhibition of Kv1.3 channels as a novel therapeutic approach for the treatment of type 1 diabetes mellitus may have a major effect on overall lymphocyte function in this disease.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Canal de Potássio Kv1.3/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Adulto , Antígenos CD4/biossíntese , Antígenos CD8/biossíntese , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/fisiopatologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/imunologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/imunologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Pirazóis/farmacologia , Venenos de Escorpião/farmacologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/patologia , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/patologiaRESUMO
Studies have been performed to describe the significance of genetic polymorphisms in complications associated with disturbed perinatal adaptation. Due to the large number of interacting factors, the results of classic statistical methods are often inconsistent. The random forest technique (RFT) is a robust nonparametric statistical approach that overcomes this problem through the calculation of the importance of each factor. We used RFT to reanalyze the importance of 24 genetic polymorphisms in the classification of preterm infants (birth weight, 680-1460 g, n = 100) to affected and unaffected groups according to the presence of acute perinatal complications. The accuracy of classification was between 0.5 and 0.8 for each complication when only birth data were considered. However, when genetic polymorphisms with the highest importance scores (ISs) were included in the analysis, the accuracy of classification according overall morbidity, necrotizing enterocolitis (NEC), acute renal failure (ARF), infant respiratory distress syndrome (IRDS), cardiac failure (CF), and patent ductus arteriosus (PDA) improved from 0.69, 0.60, 0.70, 0.72, 0.68, and 0.57 to 0.77, 0.70, 0.76, 0.77, 0.76, and 0.64, respectively. Our findings suggest that genetic polymorphisms identified by RFT as predictors may improve the risk assessment of preterm infants. RFT is a suitable tool to develop risk factor patterns in this population.