Plasma pentraxin-3 levels and its role in childhood obesity-Is it anti-inflammatory? A matched group study.

OBJECTIVE: Obesity has been associated with chronic low-grade systemic inflammation. This study aimed to investigate the relationship of pentraxin-3 (PTX-3) with anthropometric measurements, dietary content and physical activity level in children. DESIGN: A matched group study. PATIENTS: This study was conducted with 91 children aged 6-17 years, divided into two groups: "non-obese group" (Body Mass Index Standard Deviation Score [BMI SDS] <95th percentile) and "obese group" (BMI SDS ≥95th percentile). MEASUREMENTS: Plasma PTX-3 levels. RESULTS: The mean age of 91 children included in the study was 12.34 ± 2.86 years. Plasma PTX-3 levels were significantly higher in obese children (p = .028). No significant correlation was found between BMI SDS and plasma PTX-3 values, but a weak positive correlation was found when physical activity level was controlled (r = .176, p = .049). In addition, it was found that fat mass was a partial mediator of plasma PTX-3 level, and an increase in the amount of subcutaneous adipose tissue negatively affected plasma PTX-3 level. Plasma PTX-3 level showed a weak positive correlation (r = .223, p = .017) with physical activity score and dietary polyunsaturated fatty acid intake, while a weak negative correlation with neutrophil-to-lymphocyte ratio. One unit increase in physical activity score or polyunsaturated fatty acid level caused 0.730 and 2.061 unit increases in plasma PTX-3 level, respectively; while one unit increase in dietary fat intake caused 0.413-unit decrease. CONCLUSION: There was an indirect relationship between the amount of subcutaneous adipose tissue and PTX-3 level. The results of our study suggested that plasma PTX-3 was associated with lower levels of inflammation in children.

Natural history of ENPP1 deficiency: Nationwide Turkish Cohort Study of autosomal-recessive hypophosphataemic rickets type 2.

OBJECTIVE: Autosomal-recessive hypophosphataemic rickets type 2 (ARHR2) is a rare disease that is reported in survivors of generalized arterial calcification of infancy (GACI). DESIGN, PATIENTS AND MEASUREMENT: The objective of this study was to characterize a multicenter paediatric cohort with ARHR2 due to ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) deficiency and with a diagnosis of GACI or GACI-related findings. The clinical, biochemical and genetic characteristics of the patients were retrospectively retrieved. RESULTS: We identified 18 patients from 13 families diagnosed with ARHR2. Fifteen of the patients had an ENPP1 variation confirmed with genetic analyses, and three were siblings of one of these patients, who had clinically diagnosed hypophosphataemic rickets (HRs) with the same presentation. From nine centres, 18 patients, of whom 12 (66.7%) were females, were included in the study. The mean age at diagnosis was 4.2 ± 2.2 (1.6-9) years. The most frequently reported clinical findings on admission were limb deformities (66.6%) and short stature (44.4%). At diagnosis, the mean height SD was -2.2 ± 1.3. Five of the patients were diagnosed with GACI in the neonatal period and treated with bisphosphonates. Other patients were initially diagnosed with ARHR2, but after the detection of a biallelic variant in the ENPP1 gene, it was understood that they previously had clinical findings associated with GACI. Three patients had hearing loss, and two had cervical fusion. After the treatment of HRs, one patient developed calcification, and one developed intimal proliferation. CONCLUSION: ARHR2 represents one manifestation of ENPP1 deficiency that usually manifests later in life than GACI. The history of calcifications or comorbidities that might be associated with GACI will facilitate the diagnosis in patients with ARHR2, and patients receiving calcitriol and phosphate medication should be carefully monitored for signs of calcification or intimal proliferation.

A novel homozygous nonsense NDNF variant in Kallmann syndrome.

Kallmann syndrome (KS) is a rare genetic disease characterized by pubertal failure and olfactory defects. Although many genes associated with KS have been reported, most are rare. Recently, heterozygous inactivating mutations in the neuron-derived neurotrophic factor gene (NDNF) were reported to cause KS. Here, we present a 14-year-old Kurdish boy with KS who has a novel homozygous nonsense c.1251C>A (p.Tyr417Ter) variant in NDNF. The variant was not observed in reference population databases and was predicted to be deleterious. Segregation analysis performed with Sanger sequencing indicated the autosomal recessive inheritance of the clinical phenotype. His heterozygous parents have experienced timely pubertal development and normal reproductive features. This study reported the first homozygous truncating NDNF variant, enabling the direct observation of the clinical consequences of predictively absent NDNF function. These results support the contention that the inactivating mutations in NDNF cause KS, and provide additional evidence for the complex inheritance of KS.

Clinical features of generalized lipodystrophy in Turkey: A cohort analysis.

AIM: To describe the Turkish generalized lipodystrophy (GL) cohort with the frequency of each complication and the death rate during the period of the follow-up. METHODS: This study reports on 72 patients with GL (47 families) registered at different centres in Turkey that cover all regions of the country. The mean ± SD follow-up was 86 ± 78 months. RESULTS: The Kaplan-Meier estimate of the median time to diagnosis of diabetes and/or prediabetes was 16 years. Hyperglycaemia was not controlled in 37 of 45 patients (82.2%) with diabetes. Hypertriglyceridaemia developed in 65 patients (90.3%). The Kaplan-Meier estimate of the median time to diagnosis of hypertriglyceridaemia was 14 years. Hypertriglyceridaemia was severe (≥ 500 mg/dl) in 38 patients (52.8%). Seven (9.7%) patients suffered from pancreatitis. The Kaplan-Meier estimate of the median time to diagnosis of hepatic steatosis was 15 years. Liver disease progressed to cirrhosis in nine patients (12.5%). Liver disease was more severe in congenital lipodystrophy type 2 (CGL2). Proteinuric chronic kidney disease (CKD) developed in 32 patients (44.4%) and cardiac disease in 23 patients (31.9%). Kaplan-Meier estimates of the median time to diagnosis of CKD and cardiac disease were 25 and 45 years, respectively. Females appeared to have a more severe metabolic disease, with an earlier onset of metabolic abnormalities. Ten patients died during the follow-up period. Causes of death were end-stage renal disease, sepsis (because of recurrent intestinal perforations, coronavirus disease, diabetic foot infection and following coronary artery bypass graft surgery), myocardial infarction, heart failure because of dilated cardiomyopathy, stroke, liver complications and angiosarcoma. CONCLUSIONS: Standard treatment approaches have only a limited impact and do not prevent the development of severe metabolic abnormalities and early onset of organ complications in GL.

Inactivating NHLH2 variants cause idiopathic hypogonadotropic hypogonadism and obesity in humans.

Metabolism has a role in determining the time of pubertal development and fertility. Nonetheless, molecular/cellular pathways linking metabolism/body weight to puberty/reproduction are unknown. The KNDy (Kisspeptin/Neurokinin B/Dynorphin) neurons in the arcuate nucleus of the hypothalamus constitute the GnRH (gonadotropin-releasing hormone) pulse generator. We previously created a mouse model with a whole-body targeted deletion of nescient helix-loop-helix 2 (Nhlh2; N2KO), a class II member of the basic helix-loop-helix family of transcription factors. As this mouse model features pubertal failure and late-onset obesity, we wanted to study whether NHLH2 represents a candidate molecule to link metabolism and puberty in the hypothalamus. Exome sequencing of a large Idiopathic Hypogonadotropic Hypogonadism cohort revealed obese patients with rare sequence variants in NHLH2, which were characterized by in-silico protein analysis, chromatin immunoprecipitation, and luciferase reporter assays. In vitro heterologous expression studies demonstrated that the variant p.R79C impairs Nhlh2 binding to the Mc4r promoter. Furthermore, p.R79C and other variants show impaired transactivation of the human KISS1 promoter. These are the first inactivating human variants that support NHLH2's critical role in human puberty and body weight control. Failure to carry out this function results in the absence of pubertal development and late-onset obesity in humans.

Loss-of-function variants in SEMA3F and PLXNA3 encoding semaphorin-3F and its receptor plexin-A3 respectively cause idiopathic hypogonadotropic hypogonadism.

PURPOSE: Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by absent puberty and subsequent infertility due to gonadotropin-releasing hormone (GnRH) deficiency. IHH can be accompanied by normal or compromised olfaction (Kallmann syndrome). Several semaphorins are known potent modulators of GnRH, olfactory, and vomeronasal system development. In this study, we investigated the role of Semaphorin-3F signaling in the etiology of IHH. METHODS: We screened 216 IHH patients by exome sequencing. We transiently transfected HEK293T cells with plasmids encoding wild type (WT) or corresponding variants to investigate the functional consequences. We performed fluorescent IHC to assess SEMA3F and PLXNA3 expression both in the nasal region and at the nasal/forebrain junction during the early human fetal development. RESULTS: We identified ten rare missense variants in SEMA3F and PLXNA3 in 15 patients from 11 independent families. Most of these variants were predicted to be deleterious by functional assays. SEMA3F and PLXNA3 are both expressed along the olfactory nerve and intracranial projection of the vomeronasal nerve/terminal nerve. PLXNA1-A3 are expressed in the early migratory GnRH neurons. CONCLUSION: SEMA3F signaling through PLXNA1-A3 is involved in the guidance of GnRH neurons and of olfactory and vomeronasal nerve fibers in humans. Overall, our findings suggest that Semaphorin-3F signaling insufficiency contributes to the pathogenesis of IHH.

Prevalence and associated phenotypes of PLXNA1 variants in normosmic and anosmic idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) can be divided into two major forms, normosmic IHH and Kallmann syndrome (KS). Genetic mutations are responsible for the majority of IHH. PLXNA1 has recently been implicated in the GnRH neuron migration and the etiology of KS. We aimed to investigate the prevalence and associated phenotypes of PLXNA1 variants in a large cohort of IHH patients. We screened the whole exome data of 215 IHH patients in a single center for causative PLXNA1 variants. Our studies showed eight novel (p.Arg836His, p.Lys1451Arg, p.Val287Met, p.Val536Ile, p.Ser1850Arg, p.Ile1701Val, p.Arg319Trp, and p.Pro485Leu) and two previously described (p.Arg528Trp and p.Gly720Glu) heterozygous PLXNA1 variants in nine affected individuals from seven unrelated families. Only three of nine patients were anosmic (KS) while the remaining patients showed normal olfactory function (nIHH). Seven of nine patients (77.7%) harbored additional one or two variants in other nIHH/KS-associated genes, including PROKR2, IGSF10, HS6ST1, SEMA3E, CCDC141, FGFR1, NRP1, POLR3A, and SRA1. Our findings indicate that PLXNA1 variants cause not only anosmic but also normosmic IHH with a relatively high prevalence (3.9%). Heterozygous missense PLXNA1 variants appear to be involved together with other IHH gene variants in bringing about the IHH disease phenotype.

Molecular genetic studies in a case series of isolated hypoaldosteronism due to biosynthesis defects or aldosterone resistance.

BACKGROUND AND AIM: Hypoaldosteronism is associated with either insufficient aldosterone production or aldosterone resistance (pseudohypoaldosteronism). Patients with aldosterone defects typically present with similar symptoms and findings, which include failure to thrive, vomiting, hyponatremia, hyperkalemia and metabolic acidosis. Accurate diagnosis of these clinical conditions therefore can be challenging. Molecular genetic analyses can help to greatly clarify this complexity. The aim of this study was to obtain an overview of the clinical and genetic characteristics of patients with aldosterone defects due to biosynthesis defects or aldosterone resistance. DESIGN AND PATIENTS: We investigated the clinical and molecular genetic features of 8 consecutive patients with a clinical picture of aldosterone defects seen in our clinics during the period of May 2015 through October 2017. We screened CYP11B2 for aldosterone synthesis defects and NR3C2 and the three EnaC subunits (SCNN1A, SCNN1B and SCNN1G) for aldosterone resistance. RESULTS: We found 4 novel and 2 previously reported mutations in the genes CYP11B2, NR3C2, SCNN1A and SCNN1G in 9 affected individuals from 7 unrelated families. CONCLUSION: Molecular genetic investigations can help confidently diagnose these conditions and clarify the pathogenicity of aldosterone defects. This study may expand the clinical and genetic correlations of defects in aldosterone synthesis or resistance.

17α Hydroxylase/17,20 lyase deficiency: clinical features and genetic insights from a large Turkey cohort.

PURPOSE: 17α Hydroxylase/17,20 lyase deficiency (17OHD) is a rare form of congenital adrenal hyperplasia, typically diagnosed in late adolescence with symptoms of pubertal delay and hypertension. This study aimed to determine the clinical and laboratory characteristics of 17OHD cases and gather data on disease management. METHODS: Data from 97 nationwide cases were analyzed using the CEDD-NET web system. Diagnostic, follow-up findings, and final heights of patients were evaluated. RESULTS: Mean age at admission was 13.54 ± 4.71 years, with delayed puberty as the most common complaint. Hypertension was detected in 65% at presentation; hypokalemia was present in 34%. Genetic analysis revealed Exon 1-6 homozygous deletion as the most frequent mutation, identified in 42 cases. Hydrocortisone replacement was universal; pubertal replacement was administered to 66 cases. Antihypertensive treatment was required in 57 (90%) patients. Thirty-seven cases reached final height, with an average SD of 0.015 in 46,XX and -1.43 in 46,XY. Thelarche and pubarche did not develop properly in some cases despite estradiol treatment. CONCLUSION: This study represents the largest cohort of pediatric cases of 17-hydroxylase deficiency (17OHD) documented in the literature. Hypertension and hypokalemia can serve as guiding indicators for early diagnosis.The final height is typically considered to be normal. The relationship between genotype and phenotype remains elusive. The initial genetic test for exon 1-6 deletions may be MLPA in our region.

POU6F2 mutation in humans with pubertal failure alters GnRH transcript expression.

Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by the absence of pubertal development and subsequent impaired fertility often due to gonadotropin-releasing hormone (GnRH) deficits. Exome sequencing of two independent cohorts of IHH patients identified 12 rare missense variants in POU6F2 in 15 patients. POU6F2 encodes two distinct isoforms. In the adult mouse, expression of both isoform1 and isoform2 was detected in the brain, pituitary, and gonads. However, only isoform1 was detected in mouse primary GnRH cells and three immortalized GnRH cell lines, two mouse and one human. To date, the function of isoform2 has been verified as a transcription factor, while the function of isoform1 has been unknown. In the present report, bioinformatics and cell assays on a human-derived GnRH cell line reveal a novel function for isoform1, demonstrating it can act as a transcriptional regulator, decreasing GNRH1 expression. In addition, the impact of the two most prevalent POU6F2 variants, identified in five IHH patients, that were located at/or close to the DNA-binding domain was examined. Notably, one of these mutations prevented the repression of GnRH transcripts by isoform1. Normally, GnRH transcription increases as GnRH cells mature as they near migrate into the brain. Augmentation earlier during development can disrupt normal GnRH cell migration, consistent with some POU6F2 variants contributing to the IHH pathogenesis.

The utility of annual growth velocity standard deviation scores and measurements of biochemical parameters in long-term treatment monitoring of children with 21-hydroxylase deficiency.

PURPOSE: This study aimed to investigate the utility of annual growth velocity (GV) standard deviation scores (SDSs) and compatibility and effectiveness of biochemical parameters in long-term treatment monitoring and management of 21-hydroxylase deficiency (21-OHD) in children. METHODS: Fifty children with 21-OHD were included in this study, and the biochemical parameters obtained during 402 visits were retrospectively evaluated. The follow-up period was divided between two GV SDS groups (GV SDS < 2 and GV SDS ≥ 2) and compared with auxological, biochemical, and clinical findings. RESULTS: Elevation of 17-hydroxyprogesterone (17-OHP) values was observed at 193/402 visits, and both adrenocorticotropic hormone (ACTH) and total testosterone (tT) were observed at 53 of 193 (27.5%) visits. The calculated cut-off value for 17-OHP was > 4.3 ng/ml, with a sensitivity of 85.48% and specificity of 37.59% in the GV SDS ≥ 2 group. In the GV SDS ≥ 2 group, the corrected final height SDS (cFH SDS) was lower, and the delta height was higher than in the GV SDS < 2 group (p = 0.005 and p = 0.008, respectively). Linear regression analysis of the GV SDSs revealed that 17-OHP values and the hydrocortisone dose (mg/m2) were affected (ß = 0.037, p = 0.035, and ß = - 0.147, p = 0.001, respectively). CONCLUSIONS: Annual GV was critical in the final height (FH) of children with 21-OHD. However, we observed inconsistency between the biochemical parameters in the follow-ups, and there were difficulties in evaluating these markers. Therefore, annual GV SDSs and biochemical findings should be used together in patients with 21-OHD at follow-ups.

PLXNB1 mutations in the etiology of idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) comprises a group of rare genetic disorders characterized by pubertal failure caused by gonadotropin-releasing hormone (GnRH) deficiency. Genetic factors involved in semaphorin/plexin signaling have been identified in patients with IHH. PlexinB1, a member of the plexin family receptors, serves as the receptor for semaphorin 4D (Sema4D). In mice, perturbations in Sema4D/PlexinB1 signaling leads to improper GnRH development, highlighting the importance of investigating PlexinB1 mutations in IHH families. In total, 336 IHH patients (normosmic IHH, n = 293 and Kallmann syndrome, n = 43) from 290 independent families were included in the present study. Six PLXNB1 rare sequence variants (p.N361S, p.V608A, p.R636C, p.V672A, p.R1031H, and p.C1318R) are described in eight normosmic IHH patients from seven independent families. These variants were examined using bioinformatic modeling and compared to mutants reported in PLXNA1. Based on these analyses, the variant p.R1031H was assayed for alterations in cell morphology, PlexinB1 expression, and migration using a GnRH cell line and Boyden chambers. Experiments showed reduced membrane expression and impaired migration in cells expressing this variant compared to the wild-type. Our results provide clinical, genetic, molecular/cellular, and modeling evidence to implicate variants in PLXNB1 in the etiology of IHH.

Co-existence of Congenital Adrenal Hyperplasia and Familial Hypokalemic Periodic Paralysis due to CYP21A2 and SCN4A Pathogenic Variants

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia (CAH), usually due to biallelic variants in CYP21A2. Classical 21-hydroxylase deficiency is characterised by virilisation of the external genitalia in females and hypocortisolism. Hyponatremia and hyperkalemia are among the common biochemical findings. Familial hypokalemic periodic paralysis (FHPP) is a rare disorder in which affected individuals may experience paralytic episodes associated with hypokalemia, caused by pathogenic variants in SCN4A or CACNA1S. A 14-year-old female, who had been diagnosed with classical 21-hydroxylase deficiency and treated with hydrocortisone and fludrocortisone since early infancy, presented with acute onset weakness. The laboratory results revealed a remarkably low serum potassium level. The family history revealed that both her father and uncle had the same hypokalemic symptoms, which suggested an FHPP diagnosis. We found two previously reported homozygous variants in the CYP21A2 (p.Ile173Asn) and SCN4A (p.Arg672His) genes in the patient. Therefore, diagnoses of simple virilising 21-hydroxylase deficiency and FHPP were genetically confirmed. Here, FPHH and chronic overtreatment with fludrocortisone may explain the presentation of our patient with severe hypokalemia. The family's medical history, which is always a valuable clue, should be investigated in detail since rare inherited conditions may co-occur in geographies where consanguineous marriages are common and the genetic pool is diverse. In patients with CAH, care should be taken to avoid overtreatment with fludrocortisone. Androgens may have triggered the hypokalemic attack in FHPP, as supported in a previous study.

Changes in the presentation of newly diagnosed type 1 diabetes in children during the COVID-19 pandemic in a tertiary center in Southern Turkey.

OBJECTIVES: The COVID-19 pandemic is a global health problem with high morbidity and mortality. This study aimed to investigate patients who were diagnosed with type 1 diabetes during the pandemic and evaluate the effect of the pandemic on the clinical findings of these patients by comparing them with findings from a year prior. METHODS: Patients diagnosed with type 1 diabetes mellitus between 2019 and 2021 were separated into two groups: Patients diagnosed prepandemic and those diagnosed during the pandemic. RESULTS: The number of newly diagnosed diabetes cases increased from 46 in the prepandemic period to 74 in the pandemic period. The number of cases diagnosed with diabetic ketoacidosis (DKA) in the clinic increased from 58.7 to 91.9%. We found that moderate and severe DKA rates from 18.5 and 14.8% to 23.5 and 22.1%, respectively. Besides, the average HbA1c was higher, while the average bicarbonate was lower in cases diagnosed during the pandemic period compared to the prepandemic period (p=0.048 and p<0.001, respectively). We found that celiac autoantibody positivity antibodies to glutamic acid decarboxylase (anti GAD) positivity, and islet cell antibodies (ICA), ICA and anti GAD positivity coexistence were higher (p=0.045, p=0.008, and p=0.007, respectively) among the patients diagnosed during the pandemic. CONCLUSIONS: We observed an increase in the number of patients newly diagnosed with type 1 diabetes mellitus, an increase in autoantibody positivity, and higher rates and severity of DKA during the COVID-19 pandemic period compared to the prepandemic period.

DLG2 Mutations in the Etiology of Pubertal Delay and Idiopathic Hypogonadotropic Hypogonadism.

INTRODUCTION: Idiopathic hypogonadotropic hypogonadism (IHH) is caused by dysfunction of the hypothalamic-pituitary-gonadal axis. DLG2 was recently implicated as a gene associated with delayed puberty and which may also contribute to IHH. The confirmation of the candidate puberty genes in independent IHH cohorts has become crucial due to the lack of proper genotype-phenotype segregations in reported pedigrees. Therefore, we aimed to screen DLG2 in patient variants in a large cohort of IHH patients. METHODS: The present study included a total of 336 IHH patients from 290 independent families. The coding and flanking regions of DLG2 were screened for potentially important variants in the WES data. Candidate variants were evaluated in the -gnomAD and GME databases according to their allele frequencies, and only those with a frequency <0.0001 were considered rare. Detected variants were classified according to the ACMG/AMP criteria. RESULTS: We found 1 homozygous and 2 heterozygous missense variants in 3 independent pedigrees. Identified variants were found extremely rare or not reported in gnomAD. Two variants were categorized as "uncertain significance," and the other one was "likely pathogenic" according to the ACMG criteria. All patients were normosmic, and in 2 of the 3 families, there were no causal variants in other IHH-related genes. CONCLUSION: We detected 3 rare sequencing variants in DLG2 in 5 patients with IHH or delayed puberty in a large IHH cohort. Our results support the contention that the DLG2 mutations are associated with IHH in human puberty.

Experience with the targeted next-generation sequencing in the diagnosis of hereditary hypophosphatemic rickets.

OBJECTIVES: Hereditary Hypophosphatemic Rickets (HHR) is a heterogeneous group of disorders characterized by hypophosphatemia. Although the X-linked dominant HHR is the most common form, the genetic etiology of HHR is variable. Recently, developed next-generation sequencing techniques may provide opportunities for making HHR diagnosis in a timely and efficient way. METHODS: We investigated clinical and genetic features for 18 consecutive probands and their 17 affected family members with HHR. All patient's clinical and biochemical data were collected. We first analyzed a single gene with Next-generation sequencing if the patients have a strong clue for an individual gene. For the remaining cases, a Hypophosphatemic Rickets gene panel, including all known HHR genes by Next-generation sequencing, was employed. RESULTS: We were able to diagnosis all of the consecutive 35 patients in our tertiary care center. We detected nine novel and 10 previously described variants in PHEX (9; 50%), SLC34A3 (3; 17%), ENPP1 (3; 17%), SLC34A1 (1; 5%), CLCN5 (1; 5%), and DMP1 (1; 5%). CONCLUSIONS: To delineate the etiology of HHR cases in a cost and time-efficient manner, we propose single gene analysis by next-generation sequencing if findings of patients indicate a strong clue for an individual gene. If that analysis is negative or for all other cases, a Next-generation Sequence gene panel, which includes all known HHR genes, should be employed.

21-Hydroxylase deficiency: Mutational spectrum and Genotype-Phenotype relations analyses by next-generation sequencing and multiplex ligation-dependent probe amplification.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is autosomal recessive disorder of cortisol biosynthesis. Genetic defects in CYP21A2 cause 21OHD. The aim of this study was to determine spectrum of mutations in CYP21A2 in a large cohort and analyze the genotype-phenotype correlation to assess predictive characteristics of genotype. We investigated a total of 113 patients with 21OHD. Next-generation sequencing and Multiplex ligation-dependent probe amplification of the CYP21A2 gene were performed in patients and their parents. The genotypes were categorized into Groups 0, A, B, and C according to the residual 21-hydroxylase activities. In this study, the group A was divided into two subgroups as A1 and A2. Three novel variants were found. The genotype-phenotype correlation of the mutation classification was 91.5%. Positive predictivity of subgroups A1 was higher than groups A and subgroups A2. Our study reports genotype-phenotype correlations in the largest 21OHD cohort in Turkey. This correlation sustained when we analyzed our data in combination with metadata from other published studies. This study confirms that CYP21A2 genotyping with next-generation sequencing and MLPA can accurately and reliably confirm the diagnosis of 21OHD. We propose a new classification by dividing group A into two new subgroups to better predict the phenotype. In light of this very high genotype-phenotype correlation, with their ever-increasing availability, declining cost, and turnaround time, we propose that molecular genetic studies can be more economical and practical alternative to the current initial diagnostic laboratory studies based on assays of intermediary steroid metabolites.