New markers and method for congenital adrenal hyperplasia diagnostic

Abstract

The present invention relates to a method of diagnosing Congenital Adrenal Hyperplasia (CAH) in newborn making use of the biomarkers 21-deoxycortisone (21DE) and / or 11-ketotestosterone (11KT).

Description

[0001]New markers and method for Congenital Adrenal Hyperplasia diagnostic The present invention relates to a method of diagnosing Congenital Adrenal Hyperplasia (CAH) in newborn or fetus making use of the biomarkers 21-deoxycortisone (21DE) and / or 11-ketotestosterone (11KT). Congenital Adrenal Hyperplasia (CAH) is a rare but significant genetic disorder affecting the adrenal glands' steroid hormone synthesis. It is an autosomal recessive condition caused by mutations in the genes that encode enzymes involved in cortisol and aldosterone production. The most common form of CAH is 21-hydroxylase deficiency (21OHD), accounting for approximately 95% of cases. The cortisol and aldosterone synthesis pathways involve a series of enzymatic reactions, and 21-hydroxylase deficiency leads to a block at a crucial step, preventing the production of these vital hormones (1). Neonatal screening for CAH has been in practice for several decades, primarily relying on one of 21-hydroxylase substrate measurement (17-hydroxyprogesterone, also designated 17OHP) using immunoassays. The Endocrine Society recommends this approach as a first-level screening strategy, with usual values defined for each gestational age (2). However, immunoassays still remain a source of false-positive results (3,4). In France, 80% of tests carried out each year are false positives using the 17OHP immunoassay technique (5). Positive results are confirmed by a second 17OHP assay (also by immunoassay) and therefore persist in generating false positives. The predictive positive value (PPV) is actually 16% with fluoro-immunoassay (FIA) technique for first and second- tier results (5). Finally, results are checked a third time by LC-MS / MS with a steroid profile on serum, where 17-hydroxyprogesterone measurement is always used as a first-line marker (5,6). Some international teams have progressively been using steroid profiling on dried blood spots (DBS) by mass spectrometry as a second-tier confirmation (7,8). In addition to the gain in specificity brought by LC-MS / MS method, the concept of profiling or multiplexing the circulating steroidome is another paradigm shift as it appears to be way more accurate than single 17OHP immunoassay quantitation to stratify patients (9,10). In recent years, thanks to advances in LC-MS / MS, 21-deoxycortisol (21DF), a steroid with a specific adrenal tropism, has been widely described as a performant discriminating marker (11–13). The association of 17OHP to ratios combining different steroids such as (21DF + 17OHP / F), (A4+17OHP / F), 17OHP / F, 17OHP / 11DF appears to improve the CAH diagnostic performances (7). However, the PPV, although improved after the introduction of a second- tier LC-MS / MS method in several countries, is still far from 100% (of 46% according to de Hora et al. (7), and of 56% according to Lind-Holst et al. (8)). To date, apart from 21-DF, the potential interest of 11-oxygen markers in the context of neonatal screening has been relatively unexplored. As mentioned, 21OHD deficiency leads to accumulation of androgens upstream. These are converted from androgens by a strictly adrenal enzyme, the 11-betahydroxylase into 11-oxygenated metabolites such as (11- ketotestosterone (11KT), 11-betahydroxytestosterone (11BOHT), 11- betahydroxyandrostenedione (11BOHA4)) (14). The Inventors identified in newborns other strictly adrenal steroids that accumulate in 21- hydroxylase deficiency in addition to 21-deoxycortisol, such as 21-deoxycortisone (21DE) and 11-ketotestosterone (11KT). In the specific context of neonatal screening, these biomarkers can significantly enrich the steroid signature and improve results in discriminating CAH newborns from the first blotting sample. The Inventors then demonstrated that, compared to 21DF, 17OHP, or ratios combining several steroids used as second tier in other countries, 21DE and 11KT have an unexpectedly better stratifying power as there is no overlap between 21-hydroxylase deficiency (21OHD) newborns and unaffected newborns. Accordingly, the Inventors have found new biomarkers specific of Congenital Adrenal Hyperplasia (CAH) in newborn. These biomarkers were validated in vivo on dried blood spot as shown in the Examples. The present invention thus relates to a method of diagnosing Congenital Adrenal Hyperplasia (CAH) in a subject, fetus or newborn, comprising the step of detecting the presence of 21-deoxycortisone (21DE) and / or 11-ketotestosterone (11KT) in a biological sample from said subject, wherein the presence of 21DE and / or 11KT in said sample identifies said subject as being affected or likely to develop Congenital Adrenal Hyperplasia, in particular Congenital Adrenal Hyperplasia due to 21-hydroxylase deficiency (21OHD). According to a preferred embodiment, the presence of both 21DE and 11KT is measured. Any analytical method may be used to measure the presence and the content of 21- deoxycortisone (21DE) and / or 11-ketotestosterone (11KT); preferably, the presence and the content of 21-deoxycortisone (21DE) and / or 11-ketotestosterone (11KT) are measured with chromatography-tandem mass spectrometry (LC-MS / MS). Such method is very advantageous as it is quick and allows a high throughput testing, it is also easy to use even with very small sample size. In addition, such method is more reliable than the immunoassay technology that lacks specificity because of cross-reactivity issues. Preferably, the biological sample can be a blood or saliva sample for diagnosing a newborn and amniotic liquid for diagnosing a fetus. Diagnosis of CAH is usually performed at birth, accordingly, the method of the invention is preferably applied on dried blood spot absorbed on a support such as blotting paper, thus avoiding venous sampling which can be very difficult for newborns, and causes considerable stress for parents. According to this embodiment, the dried blood spot is dissolved in a solvent and treated as described in the experimental part before performing the LC-MS / MS measure (see for example “sample preparation” part, corresponding to the protocol described in (5)). The accuracy of the method of the invention has been confirmed by the absence of detection of 21DE and 11KT in blood samples obtained from unaffected newborns. Knowing that the LC-MS / MS technique is a highly sensitive technique and can usually detect the presence of a compound from a level of approximately 0.1 ng / ml, according to a specific embodiment, the presence of at least 0.1 ng / ml of total blood of 21DE or 11KT indicates the tested subject, newborn or fetus, as being affected or likely to develop Congenital Adrenal Hyperplasia. Because it may happen that the content of 21DE and 11KT is measured with an apparatus having a sensitivity lower than the one of LC-MS / MS, according to a particular embodiment of the invention, when the measured content of 21DE is below 1 ng / ml of total blood, then the method of the invention comprises the measurement of the content of 11KT, if this measurement of the content of 11KT show a content of more than 1 ng / ml of the total blood, it confirms that the tested subject (newborn or fetus) is affected or likely to develop Congenital Adrenal Hyperplasia. In that same way, when the measured content of 11KT is below 1 ng / ml of total blood, then the method of the invention comprises the measurement of the content of 21DE, if this measurement of the content of 21DE show a content of more than 1 ng / ml of the total blood, it confirms that the tested subject is affected or likely to develop Congenital Adrenal Hyperplasia. According to this specific embodiment, when the measured content of 21DE is below 1 ng / ml, preferably, 0.5 ng / ml and more preferably 0.31 ng / ml of total blood, and the measured content of 11KT is below 1 ng / ml, preferably 0.6 ng / ml of total blood, then the method of the invention may further comprise additional steps, making use of a data processing system. The data processing system is designed for providing an assessment of a medical risk that a subject, newborn or fetus, is affected or likely to develop Congenital Adrenal Hyperplasia, by calculating a score from measurements of steroids from a subject, newborn or fetus, the score being representative of the risk. The steroids include at least 21DE, 11KT, 17-hydroxyprogesterone (17OHP), 11β- hydroxyandrostenedione (11βOHA4), androstenedione (A4), 16-hydroxyprogesterone (16OHP) and 21-deoxycortisol (21DF). The provided measurements may also include progesterone (P), 17-hydroxypregnenolone (17OHPreg), testosterone (T), 11β- hydroxytestosterone (11βOHT), Dehydroepiandrosterone (DHEA), cortisol (F), cortisone (E), and 11-deoxycortisol (11DF). Any analytical method may be used to measure the presence and the content of steroids, such as chromatography-tandem mass spectrometry (LC-MS / MS). The data processing system may comprise a man / machine interface comprising at least one information input device and at least one information presentation device, for example a visual presentation device such as a screen. For example, the man / machine interface comprises a touch screen playing the role of both the information input device and the information presentation device. The measurements may then for example be provided to the data processing system through the man / machine interface. The data processing system may further comprise a data reception module, designed for receiving the provided measurements. The data processing system may further comprise a calculation module designed for calculating the score from the received measurement according to a model previously trained by machine learning. For example, the model is a linear combination, so that the score is a linear combination of the received measurements. In this linear combination, the received measurements are respectively assigned to weights, the weighted measurements being summed to obtain the score. Thus, in this case, the score may be given by the following formula: where N is the number of measurements (for example 16 when all steroids are used), and ε is a constant representing a residue. This constant may be zero. The machine learning training comprises determining the weights, enabling the score to be representative of the risk of Congenital Adrenal Hyperplasia of the subject, newborn or fetus, to be evaluated. Alternatively, the model may comprise a neural network. Preferably, the model is trained so that the score is within a predefined range, for example between 0 and 1. The data processing system may further comprise a comparison module designed for comparing the score with a predefined threshold, e.g. 0.5. When the score is on a predefined side (above or below) of the threshold, it is considered that the subject, newborn or fetus, to be evaluated has a high risk of Congenital Adrenal Hyperplasia, and a low risk on the other side. Thus, the result of this comparison provides an assessment of the risk of Congenital Adrenal Hyperplasia. The additional steps are then the following. The method of the invention comprises a step of providing measurements of steroids from the subject, newborn or fetus, to the data processing system, which are received by the reception module. The method of the invention further comprises a step of the calculation module for calculating the score from the received measurements. The method of the invention may then further comprise a step of the comparison module comparing the score with the predefined threshold for determining the risk assessment. The method of the invention may then further comprise a step of the man / machine interface presenting the score and / or the risk assessment. The method of the invention, in particular, the sample collection, can be performed at any time after the birth, preferably after 48h to 72h after the birth. Newborn’s blood can be collected by any means known by the person skilled in the art, preferably, it is collected on blotting paper after doing a small puncture and squeezing out a few drops of blood. Advantageously, the method of the invention can be applied to premature newborns and remains accurate whatever is the preterm stage, in particular, as soon as 29 weeks of pregnancy as demonstrated in the experimental part. Alternatively, the method of the invention can be used for the diagnosis of fetus before birth; in such a case it is performed on amniotic fluid sample. The method of the present invention shows significant benefit as it can be performed from a very small blood sample such as a unique blood spot; it allows a very early and accurate diagnostic and with the testing of only one dried blood spot sample without any false positive (PPV of 100%). This method thus improves patient convenience, reduces health costs and care management as it avoids hospitalization for more exploration. The present invention also relates to a kit for diagnosing CAH. Preferably, the kit is dedicated for a LC-MS / MS measurement of at least 21DE and 11KT and comprises: - isotope-labelled internal standard for 21DE and / or for 11KT; and - reagents for the preparation of a biological sample and for conducting the chromatography, such as wash buffer, elution buffer… Figures Figure 1: Scattergram of 17-hydroxyprogesterone, 21-deoxycortisol DBS concentrations and four steroids ratios measured by LC-MS / MS on the 1st DBS for 21OHD newborns (n=30) and on the 1st and 2nd DBS for false positive newborns (n=39) (i.e. positive on the first DBS then negative on the second both measured with FIA approach). All steroids are expressed in ng / mL (log scalling). NS: p-value>0,05; * p-value<0,05; ** p-value<0,01; *** p- value<0,001; **** p-value<0,0001 Figure 2: scattergram of new biomarkers 21-deoxycortisone and 11-ketotestosterone DBS concentration measured by LC-MS / MS on the 1st DBS for 21OHD newborns (n=30) and on the 1st and 2nd DBS for false positive newborns (n=39) (i.e. positive on the first DBS then negative on the second both measured with FIA approach). All steroids are expressed in ng / mL (log scalling). NS: p-value>0,05; * p-value<0,05; ** p-value<0,01; *** p-value<0,001; **** p-value<0,0001 Figure 3: score plot resulting from an OPLS-DA analysis on a modeling cohort comprising 21OHD newborns (n=30) and control newborn (n=75). Figure 4: variable importance projection (VIP) of the OPLS-DA analysis. Figure 5: score plot resulting from the OPLS-DA analysis, with observations from the modeling cohort and the first and second replication cohorts. Figure 6: model predictions results. Figure 7: similar to Fig. 5 with the observations sized according to their respective concentrations of 17OH-pregnenolone in DBS: the higher the 17OH-pregnenolone concentration, the larger the dot size. EXAMPLES Glossary : Pregnenolone: Preg; 17-hydroxypregnenolone: 17OHPreg; Progesterone: P; 17- hydroxyprogesterone: 17OHP; 16-hydroxyprogesterone: 16OHP; Androstenedione: A4; 11β-hydroxyandrostenedione: 11βOHA4; Testosterone: T; 11β-hydroxytestosterone: 11βOHT; Dehydroepiandrosterone: DHEA; Cortisol: F; Cortisone: E; 11-deoxycortisol: 11DF; 21-deoxycortisol: 21DF. I. Materials and methods - Patients The study was approved by the French national newborn screening program and a non- objection certificate has been obtained from all newborn’s relatives after full explanation of the purpose and nature of all procedures used. The study involved a retrospective analysis of dried blood spot (DBS) samples collected from a cohort of newborns (n=164) (original cohort). The DBS samples were obtained as part of routine neonatal screening (NBS) programs coordinated by the reference NBS center of Paris (CRDN-Ile de France, Necker hospital) and collected between April 2019 and March 2023. More precisely, one DBS sample was collected for each of 125 newborns of the original cohort and a second DBS sample was collected for each of the 39 FIA 17OHP- positive newborns of the original cohort. The original cohort included premature newborns, born between 29 and 38 weeks of pregnancy. All newborns treated with glucocorticoids were previously excluded, so that the original cohort included no newborn treated with glucocorticoids. All DBS samples were explored using a FIA approach for 17OHP, according to French newborn screening algorithm as described elsewhere (6). All DBS samples were further explored by LC-MS / MS dedicated for CAH screening to obtain a steroid profile composed of a panel of 16 steroids. The original cohort was then divided in 4 groups as follows, according to the results of the FIA and LC-MS / MS: - 21OHD confirmed newborns (1st and 2nd positive FIA screening and genotyping); - 21OHD false positives (1st FIA positive screening and 2nd FIA negative screening); -21OHD false positives at recalled (1st FIA and 2nd FIA positive screening and negative LC-MS / MS confirmation analysis in serum at recalled); - controls (1st FIA negative screening). The original cohort (n = 164) was then divided into a modeling cohort, a first replication cohort and a second replication cohort. The modeling cohort (n = 105) comprised 21OHD confirmed newborns (n=30, years 2019- 2021) and controls (n=75). The first replication cohort (n=20) comprised 21OHD confirmed newborns (n=9, year 2022) and false positive (n=11, year 2022). The second replication cohort (n=39, year 2023) included false positive newborns for which steroids were measured with LC-MS / MS on both 1st and 2nd DBS. These had the same inclusion and exclusion criteria as patients in the modeling cohort. - LC-MS / MS Steroid profile measurement A panel of 16 steroids was measured by LC-MS / MS on the DBS of 166 newborns used for CAH screening. The aim was to test the relevance of two new markers 21DE and 11KT to improve second-tier. In comparison with serum steroid profiles used in routine clinical practice, it was not possible to quantify certain steroids, particularly those of the mineralocorticoid pathway. In addition to 21-deoxycortisone and 11-ketotestosterone, 14 other commonly assayed steroids were included in the steroid profile: pregnenolone, 17-hydroxyprogesterone, progesterone, 17-hydroxypregnenolone, 16-hydroxyprogesterone, androstenedione, 11β- hydroxyandrostenedione, testosterone, 11β-hydroxytestosterone, DHEA, cortisol, cortisone, 11-dezoxycortisol and 21-deoxycortisol. - Sample preparation The extraction protocol was adapted from a previously published study (5). Briefly, 1 spot (6 mm) of DBS is dissolved in 300 μL of NaCl. The solution was mixed and left standing for 20 min. Then 30 μL of a mixture of the deuterium-labeled internal standard was added. Sample were extract with 2 ml of a 90 / 10 hexane ethyl acetate solution after incubated 30 min at room temperature and centrifuged for 10 min at 4°C at 5000 tours / min. The supernatant (150 μL) was dried at 37°C in a gentle flow of air for 20 min. The residue was dissolved in 100 μL of a methanol / water (50 / 50, v / v) solution. As proposed by De hora et all (15). - LC-MS / MS Instruments and Settings Steroids were chromatographically separated by high-performance liquid chromatography using a Shimadzu Nexera XR system (Shimazu France, Marne la Vallee, France) and a Coreshell C18 column (Kinetex, 2.6 μm 100 Å 100 × 2.1 mm; Phenomenex, Le Pecq, France). Detection was performed using a triple quadripole mass spectrometer (Triple Quad 6500, ABSciex, FosterCity, CA). Upon collection, the LC-MS / MS data were analyzed using MultiQuant software (ABSciex, Foster City, CA version 3.0) with built-in queries or quality control rules allowing us to set compound-specific criteria for flagging outlier results. Flagging criteria included accuracies for standards and quality controls, quantifier ion / qualifier ion ratios, and lower / upper calculated concentration limits (16). For each calibration curve, the regression line used for quantitation was calculated using least- squares weighting (1 / x). - Statistical analysis Univariate analysis was performed using Kruskal-Wallis (XLSTAT, lumivero, France) testing to evaluate the significance of the differences in steroid levels between 21OHD newborns, controls, and false positive newborns (statistical significance threshold p < 0.05). Principal component analysis (PCA) was used as a first unsupervized exploration of the system variability with SIMCA 18 software (version 18, Umetrics, Vasterbotten, Sweden). Then orthogonal partial least square discriminant analysis (OPLS-DA) was performed on the modeling cohort for the construction of the model to assess the combined effect of multiple steroids in differentiating between 21OHD affected and non-affected newborns. The 2 or 3 first components were considered to capture the variability through a linear combination of the input of 16 steroids features. The number of components was determined based on cumulative explained variance with a minimum of 60% explained variability threshold. The model’s performance was evaluated using confusion matrix and accuracy score (the fraction of correct predictions), using the first and second replication cohorts. II. Results - FIA-LC-MS / MS algorithm The discriminating power of these two steroids were compared with respect to the biomarkers classically used in LC-MS / MS, as well as ratios combining different steroids such as (21DF + 17OHP / F), (A4+17OHP / F), 17OHP / F, 17OHP / 11DF recently described in literature in second tier. For this purpose, 21OHD confirmed newborns (n=30) were compared with newborns initially false-positive on the first DBS (FP 1st DBS) and then negative with the control DSB (FP 2nd DBS) using the FIA method (n=39). Gold Standard markers, 17OHP and 21DF showed significant differences between the 21OHD and false positive groups with the 1st DBS. Moreover, the 2nd DBS improved screening accuracy with 17OH-progesterone. Nevertheless, theses markers show an overlap between 21OHD and false-positive newborns with the initial and control DBS. Surprisingly, 5 newborns are falsely negative with 21DF marker (fig.1). There are four ratios commonly used to confirm results of 17OHP and 21DF in second tier. False-positive FIA newborns are significantly different from confirmed 21OHD newborns, but with a very high degree of overlap. The ratios (21DF + 17OHP / F), (A4+17OHP / F), 17OHP / F show even greater overlap with the second sample, partly due to the fact that cortisol also decreases with age in newborns.17OHP / 11DF is the ratio that shows the least overlap, and decreases significantly with the second DBS. Univariate analysis was performed on all steroids of the panel to evaluate the significance of the differences in steroids levels between 21OHD newborns, controls, and FIA false positive newborns. As expected from the literature, androgens in the control group were significantly different than those in the 21OHD group, as are all the other steroids in the panel with the exception of pregnenolone. Similar results were found in the FIA false positive group. Additional biomarkers: 11-ketotestosterone and 21-deoxycortisone In a second step, the study has been focused on 2 others biomarkers. For this purpose, we specifically compared the diagnostic performance of 11-ketotestosterone and 21- deoxycortisone with the others steroids previously described according the same methodology (fig.2). While 17OHP, 21DF and steroid ratios showed significant differences between the 21OHD and non-21OHD groups, this 2 markers demonstrated no overlap between the two groups. As observed for 21DF, 21DE and 11KT, DBS concentrations were bellow LLOQ (0.1 ng / mL) in the two false positive groups, and in controls (not shown). However, unlike 21DF (or other steroid ratios), the absence of 21DE and 11KT overlapping concentrations between the 21OHD and the two false positive groups showed that they have superior discriminative power. Two isolated 21OHD newborns presented low 21DE concentrations (0.31ng / mL and 0.45 ng / mL) but rather high concentrations of 11KT (2.24 ng / mL and 2.01ng / mL). Similarly, one 21OHD newborn exhibited a lower concentration of 11KT (0.58 ng / mL) but a higher concentration of 21DE (3.91 ng / mL). Statistical analysis - PCA The PCA analysis showed a good separation between the 21OHD group and Controls (R2X = 0.721). - OPLS-DA Fig. 3 shows the projection of the observations onto the maximum variability plan (R2X = 0.732 and Q2X = 0.941) on two components, which have been selected. The capability to predict the model was good according to internal cross validation Q2X = 0.941. 21OHD newborns were segregated on the right side of the score plot (purple dots) while controls were segregated on the right quadrants (black dots). There was a good separation of the groups without any overlap. Fig.4 shows the contribution of each variable in the model. The higher the absolute value of the coefficient of a variable, the more discriminatory and important the variable was for prediction and group separation. The coefficients were calculated using the algorithm established by the OPLS-DA regression. The variables 21-deoxycortisone, 11- ketotestosterone, 17OH-progesterone, 11βOH-Androstenedione, and 21-déoxycortisol were the top 5 highest contribution coefficients for 21OHD group discrimination. Fig. 5 and Fig. 6 shows the results of the evaluation of the model by the first and second replication cohorts. The first and second replication cohort were not used to refit the model; they were only used to test its accuracy. On Fig.5, newborns from the replication cohort were labeled according their retrospective final diagnosis: 21OHD (grey triangles) and false positive (black triangles).All 21OHD newborns from the first replication cohort were segregated in the same area of the score plot as 21OHD newborns from the modeling cohort (grey dots). All false positive were correctly segregated, on the left side as controls (black dots). Fig. 6 summarized the number of observations in each group, the FIA screening result in the 1st and 2nd DBS, and the correct classification rate after measurement of steroids profile with LC-MS / MS and a modeling approached. All newborns with concentration of 17OH-progesterone closed to cut-off values with FIA-FIA algorithm were correctly classified based on the LC-MS / MS machine learning screening test on the first DBS. Interestingly, even on the same side of the quadrant as controls, false positive newborns from the 1st DBS segregated below. Then, with the 2nd DBS they segregated in the same area as controls. Fig.7 shows a very marked vertical gradient of the label size running from top (low 17OH- Preg concentrations) to bottom (high 17OH-Preg concentrations) in the left quadrants (False positives / ctrls). As anticipated, FIA false-positive newborns showed high 17OH- pregnenolone concentrations. Bibliography 1. Charmandari E, Brook C, Hindmarsh P. Classic congenital adrenal hyperplasia and puberty. Eur J Endocrinol.1 nov 2004;151(Supplement_3):U77-82. 2. Speiser PW, Arlt W, Auchus RJ, Baskin LS, Conway GS, Merke DP, et al. Congenital Adrenal Hyperplasia Due to Steroid 21-Hydroxylase Deficiency: An Endocrine Society* Clinical Practice Guideline. J Clin Endocrinol Metab.1 nov 2018;103(11):4043-88. 3. Taylor AE, Keevil B, Huhtaniemi IT. Mass spectrometry and immunoassay: how to measure steroid hormones today and tomorrow. Eur J Endocrinol. août 2015;173(2):D1-12. 4. Wudy SA, Schuler G, Sánchez-Guijo A, Hartmann MF. The art of measuring steroids: Principles and practice of current hormonal steroid analysis. J Steroid Biochem Mol Biol. mai 2018;179:88-103. 5. Houang M, Nguyen-Khoa T, Eguether T, Ribault B, Brabant S, Polak M, et al. Analysis of a pitfall in congenital adrenal hyperplasia newborn screening: evidence of maternal use of corticoids detected on dried blood spot. Endocr Connect. 15 juin 2022;11(6):e220101. 6. Kariyawasam D, Nguyen-Khoa T, Briceño LG, Polak M. Le dépistage néonatal de l’hyperplasie congénitale des glandes surrénales. médecine / sciences. 1 mai 2021;37(5):500-6. 7. de Hora M, Heather N, Webster D, Albert B, Hofman P. The use of liquid chromatography-tandem mass spectrometry in newborn screening for congenital adrenal hyperplasia: improvements and future perspectives. Front Endocrinol [Internet]. 2023 [cité 26 déc 2023];14. Disponible sur: https: / / www.frontiersin.org / articles / 10.3389 / fendo.2023.1226284 8. Lind-Holst M, Bækvad-Hansen M, Berglund A, Cohen AS, Melgaard L, Skogstrand K, et al. Neonatal Screening for Congenital Adrenal Hyperplasia in Denmark: 10 Years of Experience. Horm Res Paediatr.2022;95(1):35-42. 9. Lacey JM, Minutti CZ, Magera MJ, Tauscher AL, Casetta B, McCann M, et al. Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry. Clin Chem. mars 2004;50(3):621-5. 10. Seo JY, Park HD, Kim JW, Oh HJ, Yang JS, Chang YS, et al. Steroid profiling for congenital adrenal hyperplasia by tandem mass spectrometry as a second-tier test reduces follow- up burdens in a tertiary care hospital: a retrospective and prospective evaluation. J Perinat Med. janv 2014;42(1):121-7. 11. Held PK, Bialk ER, Lasarev MR, Allen DB.21-Deoxycortisol is a Key Screening Marker for 21-Hydroxylase Deficiency. J Pediatr. mars 2022;242:213-219.e1. 12. Miller WL. Congenital Adrenal Hyperplasia: Time to Replace 17OHP with 21- Deoxycortisol. Horm Res Paediatr.2019;91(6):416-20. 13. Watanabe K, Tsuji-Hosokawa A, Hashimoto A, Konishi K, Ishige N, Yajima H, et al. The High Relevance of 21-Deoxycortisol, (Androstenedione + 17α- Hydroxyprogesterone) / Cortisol, and 11-Deoxycortisol / 17α-Hydroxyprogesterone for Newborn Screening of 21-Hydroxylase Deficiency. J Clin Endocrinol Metab. 25 nov 2022;107(12):3341-52. Turcu AF, Rege J, Auchus RJ, Rainey WE. 11-Oxygenated androgens in health and disease. Nat Rev Endocrinol. mai 2020;16(5):284-96. de Hora MR, Heather NL, Webster DR, Albert BB, Hofman PL. Evaluation of a New Laboratory Protocol for Newborn Screening for Congenital Adrenal Hyperplasia in New Zealand. Int J Neonatal Screen.21 oct 2022;8(4):56. J F, Y LB, J G, N H, Ma M, D F, et al. A Liquid Chromatography / Tandem Mass Spectometry Profile of 16 Serum Steroids, Including 21-Deoxycortisol and 21-Deoxycorticosterone, for Management of Congenital Adrenal Hyperplasia. J Endocr Soc [Internet].2 oct 2017 [cité 8 janv 2024];1(3). Disponible sur: https: / / pubmed.ncbi.nlm.nih.gov / 29264476 /

Claims

CLAIMS 1. Method of diagnosing Congenital Adrenal Hyperplasia (CAH) in a subject comprising the step of detecting the presence of 21-deoxycortisone (21DE) and / or 11-ketotestosterone (11KT) in a biological sample from said subject, wherein the presence of 21DE and / or 11KT in said sample identifies said subject as being affected or likely to develop Congenital Adrenal Hyperplasia.

2. The method of claim 1, wherein the presence of 21-deoxycortisone (21DE) and / or 11- ketotestosterone (11KT) is measured with chromatography-tandem mass spectrometry (LC-MS / MS).

3. The method of claim 1 or claim 2, wherein the subject is a newborn and the biological sample is a blood sample or saliva.

4. The method of claim 1 or claim 2, wherein the subject is a fetus and the biological sample is a amniotic liquid.

5. The method of any of the preceding claims, further comprising, when the measured content of 21DE is below 1 ng / ml, preferably, 0,5 ng / ml and more preferably 0.31 ng / ml of total blood, and the measured content of 11KT is below 1 ng / ml, preferably 0.6 ng / ml of total blood: providing measurements of steroids from the subject, the steroids including at least 21DE, 11KT, 17-hydroxyprogesterone (17OHP), 11β-hydroxyandrostenedione (11βOHA4), androstenedione (A4), 16-hydroxyprogesterone (16OHP) and 21-deoxycortisol (21DF), to a data processing system, designed for providing an assessment of a medical risk that the subject is affected or likely to develop Congenital Adrenal Hyperplasia, by calculating a score from the measurements of steroids, the score being representative of the risk.

6. The method of claim 5, wherein the steroids further include: progesterone (P), 17- hydroxypregnenolone (17OHPreg), testosterone (T), 11β-hydroxytestosterone (11βOHT), Dehydroepiandrosterone (DHEA), cortisol (F), cortisone (E), and 11-deoxycortisol (11DF).

7. The method of claim 5 or 6, wherein the score is calculated according to a model previously trained by machine learning.

8. The method of claim 7, wherein the model is a linear combination or a neural network.

9. The method of any of claims 5 to 8, further comprising the data processing system comparing the score with a predefined threshold.

10. The method of anyone of the preceding claims, wherein it is performed between 24h to 48h after the birth of said subject.

11. Kit for diagnosing Congenital Adrenal Hyperplasia comprising: - isotope-labelled internal standard for 21DE and / or for 11KT; and - reagents for the preparation of a biological sample and for conducting a LC-MS / MS.

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