X-linked congenital adrenal hypoplasia: Report of long clinical follow-up and description of a new complex variant in the NR0B1 gene.

Adrenal hypoplasia congenita, attributed to NR0B1 pathogenic variants, accounts for more than 50% of the incidence of primary adrenal insufficiency in children. Although more than 250 different deleterious variations have been described, no genotype-phenotype correlation has been defined to date. We report a case of an adopted boy who reported the onset of an adrenal crisis at 2 weeks of age, requiring replacement therapy with mineralocorticoids and glucocorticoids for 4 months. For 3 years, he did well without treatment. At almost 4 years of age, the disorder was restarted. A long follow-up showed the evolution of hypogonadotropic hypogonadism. Molecular studies on NR0B1 revealed a novel and deleterious deletion-insertion-inversion-deletion complex rearrangement sorted in the 5'-3' direction, which is described as follows: (1) deletion of the intergenic region (between TASL and NR0B1 genes) and 5' region, (2) insertion of a sequence containing 37 bp at the junction of the intergenic region of the TASL gene and a part of exon 1 of the NR0B1 gene, (3) inversion of a part of exon 1, (4) deletion of the final portion of exon 1 and exon 2 and beginning of the 3'UTR region, (5) maintenance of part of the intergenic sequence (between genes MAGEB1 and NR0B1, telomeric sense), (6) large posterior deletion, in the same sense. The path to molecular diagnosis was challenging and involved several molecular biology techniques. Evaluating the breakpoints in our patient, we assumed that it was a nonrecurrent rearrangement that had not yet been described. It may involve a repair mechanism known as nonhomologous end-joining (NHEJ), which joins two ends of DNA in an imprecise manner, generating an "information scar," represented herein by the 37 bp insertion. In addition, the local Xp21 chromosome architecture with sequences capable of modifying the DNA structure could impact the formation of complex rearrangements.

DHX37 and the implications in Disorders of Sex Development: an update review.

BACKGROUND: DHX37 is an autosomal gene responsible for encoding a helicase from the DExD/H-box family that plays an essential role in ribosome biogenesis. Variants in this gene were previously reported in two different phenotypes: neurodevelopmental disorders and disorders/differences of sex development (DSD). Particularly for the DSD group, variants were mainly reported associated with gonadal dysgenesis and testicular regression syndrome. SUMMARY: Focusing specific in the DSD group, we revised the 21 DHX37 variants described across a total of 55 cases published in the literature so far. We summarized the most important clinical and molecular features of all cases, trying to have a better comprehensiveness about this gene in the sexual development. KEY MESSAGES: The trick question regarding DHX37 is how a helicase involved in basic cell function could have a specific role in testis development. Little is known about the impact of DHX37 variants in DSD individuals. Nevertheless, current research strongly suggests that DHX37 is involved in the male sex development pathway, particularly in testis determination and maintenance. This is evidenced by the predominant assignment of affected individuals as males and the presence of Wolffian structures in most of the cases. Advancements in molecular techniques, such as the generation of induced pluripotent stem cells, and the digenic inheritance for DHX37 cases, are also addressed in this paper. This represents the first comprehensive review of all DHX37 variants published in the literature to date.

DHX37 and NR5A1 Variants Identified in Patients with 46,XY Partial Gonadal Dysgenesis.

The group of disorders known as 46,XY gonadal dysgenesis (GD) is characterized by anomalies in testis determination, including complete and partial GD (PGD) and testicular regression syndrome (TRS). Several genes are known to be involved in sex development pathways, however approximately 50% of all cases remain elusive. Recent studies have identified variants in DHX37, a gene encoding a putative RNA helicase essential in ribosome biogenesis and previously associated with neurodevelopmental disorders, as a cause of PGD and TRS. To investigate the potential role of DHX37 in disorders of sexual development (DSD), 25 individuals with 46,XY DSD were analyzed and putative pathogenic variants were found in four of them. WES analyses were performed on these patients. In DHX37, the variant p.(Arg308Gln), recurrent associated with DSD, was identified in one patient; the p.(Leu467Val), predicted to be deleterious, was found together with an NR5A1 loss-of-function variant in patient 2; and, the p.(Val999Met) was identified in two unrelated patients, one of whom (patient 3) also carried a pathogenic NR5A1 variant. For both patients carrying DHX37 and NR5A1 pathogenic variants, a digenic inheritance is suggested. Our findings support the importance of DHX37 variants as a cause of disorders of sex development, implying a role in testis development.

SOX3 duplication in a boy with 46,XX ovotesticular disorder of sex development and his 46,XX sister with atypical genitalia: Probable germline mosaicism.

Ovotesticular disorders of sex development (OT-DSD) are characterized by ovarian follicles and seminiferous tubules in the same individual, with a wide range of atypical genitalia. We report on two sibs with atypical genitalia and SRY-negative 46,XX DSD, OT-DSD was confirmed only in the boy, while the girl had bilateral ovaries. Chromosome microarray analysis (CMA) showed a 737-kb duplication at Xq27.1 including the entire SOX3 gene in both sibs, which was confirmed by quantitative real time PCR. Also, X chromosome inactivation assay showed random inactivation in both sibs. Whole exome sequencing revealed no pathogenic or likely pathogenic variant. CMA of the parents showed normal results for both, suggesting that germline mosaicism could be the reason of recurrence of this duplication in the siblings. Our results support a pathogenic role of SOX3 overexpression in 46,XX subjects leading to variable DSD phenotypes.

So, and if it is not congenital adrenal hyperplasia? Addressing an undiagnosed case of genital ambiguity.

BACKGROUND: The Congenital Adrenal Hyperplasia due to 21 hydroxylase deficiency is the most common cause of genital ambiguity in persons with XX sexual chromosomes. Genital ambiguity among persons with XY sexual chromosomes comprises diverse and rare etiologies. The deficiency of 17-beta-hydroxysteroid dehydrogenase type 3 enzyme (HSD17B3) is a rare autosomal recessive disorder due to functionally altered variants of the HSD17B3 gene. In this disorder/difference of sex development, the conversion of androstenedione into testosterone is impaired. The appearance of external genitalia of 46,XY individuals varies from typically male to almost female. CASE PRESENTATION: We report on a child presenting severe ambiguous genitalia. Due to access constraints, specialized care did not start until the child was 10 months old. Parents are consanguineous and were born in an area of high isonymy that is a cluster for rare recessive diseases. A new homozygous missense variant c.785G > T was found in exon 10 of the HSD17B3 gene. CONCLUSIONS: Researchers-clinicians and researchers-researchers collaborative efforts to elucidate the genetic basis of this disease were critical since this etiologic investigation is not available through the public health system. This case exemplifies the families' pilgrimage in cases of genital ambiguity due to a rare genetic condition. Recognizing the etiology was the baseline to provide information on prognosis and treatment options, and to shelter family and child doubts and hopes in order to better support their decisions.

APOL1 in an ethnically diverse pediatric population with nephrotic syndrome: implications in focal segmental glomerulosclerosis and other diagnoses.

BACKGROUND: APOL1 high-risk genotypes (HRG) are associated with increased risk of kidney disease in individuals of African ancestry. We analyzed the effects of APOL1 risk variants on an ethnically diverse Brazilian pediatric nephrotic syndrome (NS) cohort. METHODS: Multicenter study including 318 NS patients, categorized as progressors to advanced CKD [estimated glomerular filtration rate (eGFR)] < 30 mL/min/1.73 m2] and slow/non-progressors (eGFR > 30 mL/min/1.73 m2 through the study). We employed Cox regression with progression time as the outcome and APOL1 genotype as the independent variable. We tested this association in the entire cohort and three subgroups; (1) focal segmental glomerulosclerosis (FSGS), (2) steroid-resistant NS (SRNS), and (3) those who underwent kidney biopsy. RESULTS: Nineteen patients (6%) had an HRG. Of these, 47% were self-reported White. Patients with HRG manifested NS at older ages and presented higher frequencies of FSGS and SRNS. HRG patients progressed to advanced CKD more often than low-risk-genotype (LRG) children in the whole NS cohort (p = 0.001) and the three subgroups. In SRNS and biopsied patients, a single risk variant was associated with trends of higher CKD progression risk. CONCLUSIONS: Novel discoveries include a substantial prevalence of HRG among patients self-reported White, worse kidney outcomes in HRG versus LRG children in the FSGS subgroup, and a trend of higher CKD progression risk associated with a single risk variant in the SRNS cohort. These findings suggest APOL1-associated NS extends beyond patients self-reported non-White, the HRG effect is independent of FSGS, and a single risk variant may have a detrimental impact in children with NS.

Androgens by immunoassay and mass spectrometry in children with 46,XY disorder of sex development.

OBJECTIVE: Steroid measurement is a challenge in pediatric endocrinology. Currently, liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered a gold standard for this purpose. The aim of this study was to compare both LC-MS/MS and immunoassay (IA) for androgens before and after human recombinant chorionic gonadotropin (rhCG) stimulus in children with 46,XY disorders of sex development (DSD). METHODS: Nineteen patients with 46,XY DSD were evaluated; all of them were prepubertal and non-gonadectomized. Testosterone, dihydrotestosterone (DHT), DHEA and androstenedione were measured by IA and LC-MS/MS before and 7 days after rhCG injection. The correlation between IA and LC-MS/MS was analyzed by the intraclass correlation coefficient (ICC) and Spearman's rank correlation coefficient (SCC). For concordance analysis the Passing and Bablok (PB) regression and the Bland and Altman (BA) method were used. RESULTS: Testosterone showed excellent correlation (ICC = 0.960 and SCC = 0.964); DHT showed insignificant and moderate correlations as indicated by ICC (0.222) and SCC (0.631), respectively; DHEA showed moderate correlation (ICC = 0.585 and SCC = 0.716); and androstenedione had poor and moderate correlations in ICC (0.363) and SCC (0.735), respectively. Using the PB method, all hormones showed a linear correlation, but proportional and systematic concordance errors were detected for androstenedione, systematic errors for testosterone and no errors for DHEA and DHT. By the BA method, there was a trend of IA to overestimate testosterone and androstenedione and underestimate DHEA and DHT when compared to LC-MS/MS. CONCLUSION: Traditional IA should be replaced by LC-MS/MS for the androgens measurement in prepubertal children whenever is possible.

Mutation update for the NR5A1 gene involved in DSD and infertility.

Nuclear receptor subfamily 5 group A member 1 (NR5A1), also named steroidogenic factor 1, is an essential transcription factor that regulates a number of target genes crucial for normal reproductive physiology and endocrine function. It is encoded by NR5A1 gene and is expressed in high doses mainly in steroidogenic tissues, where it controls several steps of adrenal and gonadal development. NR5A1 mutations are associated with a wide phenotypic spectrum of disorders/differences of sex development (DSD), a group of conditions in which development of chromosomal, gonadal, or anatomic sex is atypical. Here, we reviewed 188 NR5A1 mutations from 238 cases reported in literature so far. Additionally, we report the variations p.Ser4*, p.(Cys55Ser), p.(Met78Leu), and p.Met98Glyfs*45, which have not been annotated for NR5A1 before and were identified in some of the 205 46,XY patients of our own cohort. This is the first NR5A1 mutation review which includes both 46,XX and 46,XY karyotype, with the purpose of discussing the complexity of genotype-phenotype correlations among DSD and infertile male patients and also females with primary ovarian failure.

Promises and pitfalls of whole-exome sequencing exemplified by a nephrotic syndrome family.

High-throughput techniques such as whole-exome sequencing (WES) show promise for the identification of candidate genes that underlie Mendelian diseases such as nephrotic syndrome (NS). These techniques have enabled the identification of a proportion of the approximately 54 genes associated with NS. However, the main pitfall of using WES in clinical and research practice is the identification of multiple variants, which hampers interpretation during downstream analysis. One useful strategy is to evaluate the co-inheritance of rare variants in affected family members. Here, we performed WES of a patient with steroid-resistant NS (SRNS) and intermittent microhematuria. Currently, 15 years after kidney transplantation, this patient presents normal kidney function. The patient was found to be homozygous for a rare MYO1E stop-gain variant, and was heterozygous for rare variants in NS-associated genes, COL4A4, KANK1, LAMB2, ANLN, E2F3, and APOL1. We evaluated the presence or absence of these variants in both parents and 11 siblings, three of whom exhibited a milder phenotype of the kidney disease. Analysis of variant segregation in the family, indicated the MYO1E stop-gain variant as the putative causal variant underlying the kidney disease in the patient and two of her affected sisters. Two secondary variants in COL4A4-identified in some other affected family members-require further functional studies to determine whether they play a role in the development of microhematuria in affected family members. Our data illustrate the difficulties in distinguishing the causal pathogenic variants from incidental findings after WES-based variant analysis, especially in heterogenous genetic conditions, such as NS.

Prevalence of Testicular Adrenal Rest Tumor and Factors Associated with Its Development in Congenital Adrenal Hyperplasia.

BACKGROUND: Testicular adrenal rest tumors (TART) can cause infertility in congenital adrenal hyperplasia (CAH) males. AIMS: To determine TART prevalence in patients with CAH due to 21-hydroxylase deficiency (21-OHD) and evaluate possible factors associated with its development. METHODS: This is a descriptive and analytical cross-sectional study evaluating males with the classical form of 21-OHD through testicular ultrasonography and serum inhibin B dosages. Data on prescribed glucocorticoid dose and serum levels of 17- hydroxyprogesterone (17-OHP), androstenedione (Andro), ACTH, renin, and LH were obtained from medical records. RESULTS: Thirty-eight males were evaluated. The mean age on ultrasonography was 15.2 ± 6.7 (3-27) years. Nine patients (23.7%) had TART, 4 of them were prepubertal and the youngest was 5 years old. No association was found between TART and 21-OHD phenotype, glucocorticoid dose, or 17-OHP, ACTH, LH, renin, and inhibin B levels measured in the 6 preceding years. However, 50% of the patients who presented increased Andro 2 years prior to the evaluation had TART (p = 0.018, OR = 8.00 [95% CI: 1.42-44.92]), whereas in the normal Andro group only 16.7% had tumors. CONCLUSION: This study showed that TART can occur in prepubertal patients and that disease control could be a factor associated with its development. Therefore, we suggest investigating TART development early in childhood, mainly in poorly controlled 21-OHD patients.

Three new Brazilian cases of 17α-hydroxylase deficiency: clinical, molecular, hormonal, and treatment features.

BACKGROUND: Deficiency of 17α-hydroxylase (17OHD) is a rare form of adrenal hyperplasia. Diagnosis is generally delayed, impairing appropriate treatment. CASE PRESENTATION: Here, we report the clinical, molecular, hormonal, and treatment data of three unrelated 17OHD patients, aged 14-16 years with hypergonadotrophic hypogonadism; uncontrolled hypertension; primary adrenal insufficiency; and high progesterone, low to normal potassium, and low dehydroepiandrosterone, androstenedione, and testosterone levels. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) at baseline and after an adrenocorticotropic hormone test showed low cortisol and cortisone and high deoxycorticosterone (DOC) and corticosterone levels; both DOC/21-deoxycortisol and costicosterone/cortisol ratios were very high. Patient 2 had 46,XX karyotype and patients 1 and 3, had 46,XY. A molecular analysis showed that two of the patients were homozygous for p.W406R mutation and the other patient was compound heterozygous for p.W406R and p.P428L. Hypertension was controlled only after the administration of both prednisone and mineralocorticoid antagonist. CONCLUSIONS: Hypertension in young women must lead to diagnostic suspicion, even in the pre-pubertal period. The basal level of progesterone is an indicator of 17OHD. Mineral and glucocorticoid ratios obtained from LC-MS/MS can reinforce the diagnosis. Hypertension can be controlled using glucocorticoid replacement therapy and mineralocorticoid antagonist.

Dopamine D2 receptor gene polymorphisms and externalizing behaviors in children and adolescents.

BACKGROUND: Dopamine is involved in several cerebral physiological processes, and single nucleotide polymorphisms (SNP) in the dopamine D2 receptor gene (DRD2) have been associated with numerous neurological and mental disorders, including those involving alterations in cognitive and emotional processes. METHODS: The aim of this study was to evaluate the association between the SNPs c.957C > T (rs6277) and c.-585A > G (rs1799978) in the DRD2 gene and behavioral characteristics of children and adolescents based on an inventory of the Child Behavior Checklist (CBCL). Children and adolescents between 8 and 20 years old who were clinically followed-up were genotyped for the SNPs c.957C > T and c.-585A > G, and related to data of the CBCL/6-18 scale assessment performed with the help of caregivers. The chi-squared test was used to assess the differences in the frequencies of the C and T alleles in the polymorphism c.957C > T and of the A and G alleles in the polymorphism c.-585A > G with respect to the grouped CBCL scores at a significance level of 5%. Multiple logistic regression models were performed, to control whether sex and/or ethnicity could influence the results. RESULTS: Eighty-five patients were assessed overall, and the presence of the T allele (C/T and T/T) of DRD2 c.957C > T polymorphism was found to be significantly associated with the occurrence of defiant and oppositional problems and with attention and hyperactivity problems. There were no associations detected with polymorphism DRD2 c.-585A > G polymorphism. Both SNPs were in Hardy-Weinberg-equilibrium. CONCLUSIONS: Although the findings of this study are preliminary, due to its small number of participants, the presence of T allele (C/T, T/T) in c.957C > T SNP was associated with difficulty in impulse control, self-control of emotions, and conduct adjustment, which can contribute to improving the identification of mental and behavioral phenotypes associated with gene expression.

Comparison between two inhibin B ELISA assays in 46,XY testicular disorders of sex development (DSD) with normal testosterone secretion.

BACKGROUND: Inhibin B is a hormone produced by the Sertoli cells that can provide important information for the investigation of disorders of sex development (DSD) with 46,XY karyotype. The aim of this study is to compare two enzyme-linked immunosorbent assay (ELISA) assays for dosage of serum inhibin B in patients with 46,XY DSD with normal testosterone secretion. METHODS: Twenty-nine patients with 46,XY DSD and normal testosterone secretion (partial androgen insensitivity syndrome [PAIS] [n=8]; 5α-reductase deficiency [n=7] and idiopathic 46,XY DSD [n=14]) were included. Molecular analysis of the AR and SRD5A2 genes were performed in all patients and the NR5A1 gene analysis in the idiopathic group. Measurements of inhibin B were performed by two second-generation ELISA assays (Beckman-Coulter and AnshLabs). Assays were compared using the interclass correlation coefficient (ICC) and the Bland-Altman method. RESULTS: ICC was 0.915 [95% confidence interval (CI): 0.828-0.959], however, a discrepancy was observed between trials, which is more evident among higher values when analyzed by the Bland-Altman method. CONCLUSIONS: It is recommended to perform the inhibin B measurement always using the same ELISA kit when several evaluations are required for a specific patient.

Functional characterization of five NR5A1 gene mutations found in patients with 46,XY disorders of sex development.

Steroidogenic factor-1 (SF1), encoded by the NR5A1 gene, is a key regulator of steroidogenesis and reproductive development. NR5A1 mutations described in 46,XY patients with disorders of sex development (DSD) can be associated with a range of conditions of phenotypes; however, the genotype-phenotype correlation remains elusive in many cases. In the present study, we describe the impact of five NR5A1 variants (three novel: p.Arg39Cys, p.Ser32Asn, and p.Lys396Argfs*34; and two previously described: p.Cys65Tyr and p.Cys247*) on protein function, identified in seven patients with 46,XY DSD. In vitro functional analyses demonstrate that NR5A1 mutations impair protein functions and result in the DSD phenotype observed in our patients. Missense mutations in the DNA binding domain and the frameshift mutation p.Lys396Argfs*34 lead to both, markedly affected transactivation assays, and loss of DNA binding, whereas the mutation p.Cys247* retained partial transactivation capacity and the ability to bind a consensus SF1 responsive element. SF1 acts in a dose-dependent manner and regulates a cascade of genes involved in the sex determination and steroidogenesis, but in most cases reported so far, still lead to a sufficient adrenal steroidogenesis and function, just like in our cases, in which heterozygous mutations are associated to 46,XY DSD with intact adrenal steroid biosynthesis.

A study of splicing mutations in disorders of sex development.

The presence of splicing sequence variants in genes responsible for sex development in humans may compromise correct biosynthesis of proteins involved in the normal development of gonads and external genitalia. In a cohort of Brazilian patients, we identified mutations in HSD17B3 and SRD5A2 which are both required for human sexual differentiation. A number of these mutations occurred within regions potentially critical for splicing regulation. Minigenes were used to validate the functional effect of mutations in both genes. We evaluated the c.277 + 2 T > G mutation in HSD17B3, and the c.544 G > A, c.548-44 T > G and c.278delG mutations in SRD5A2. We demonstrated that these mutations altered the splicing pattern of these genes. In a genomic era these results illustrate, and remind us, that sequence variants within exon-intron boundaries, which are primarily identified for diagnostic purposes and have unknown pathogenicity, need to be assessed with regards to their impact not only on protein expression, but also on mRNA splicing.

Development of CYP21A2 Genotyping Assay for the Diagnosis of Congenital Adrenal Hyperplasia.

BACKGROUND: Steroid 21-hydroxylase deficiency due to CYP21A2 gene mutations represents more than 90% of all congenital adrenal hyperplasia cases. This deficiency is screened by measuring levels of 17-hydroxyprogesterone, which may vary, causing false positive or false negative results. In order to assist the diagnosis, molecular methodologies have been employed. This work aimed to perform genotyping assays to detect mutations in the CYP21A2 gene and compare the findings with other population studies. METHODS: The SNaPshot assay was developed to simultaneously detect 12 frequent point mutations in the CYP21A2 gene (p.Arg409Cys, p.Gln319Ter, p.Arg357Trp, p.Leu308PhefsTer6, p.Val237Glu, IVS2-13A/C > G, p.Ile173Asn, p.Pro31Leu, p.Pro454Ser, p.Val282Leu, p.Gly111ValfsTer21 and p.His63Leu). The direct sequencing and multiplex ligation-dependent probe amplification assays were used to confirm point mutations present in the developed method. The latter was also used to search large deletions and gene conversion, complementing the investigation. A total of 166 cases were studied. RESULTS: The SNaPshot assay was successfully developed to detect the 12 mutations. The results of mutation analysis indicated 84 pathogenic alleles in 48 cases, with p.Val282Leu (27.1%) and IVS2-13A/C > G (20.8%) being the most frequently found mutations. Between the findings of this study and those of other South American studies, there were significant differences in frequency for p.Pro31Leu and p.Val282Leu (p < 0.001). A new variant T in IVS2-13A/C > G was identified in two patients via the SNaPshot assay. CONCLUSION: The molecular strategy developed for CYP21A2 gene mutation screening allowed us to detect the principle mutations described around the world. Furthermore, the first Southern Brazilian mutation frequencies concerning the CYP21A2 gene were obtained.

Corrigendum to "Pharmacogenetics of Risperidone and Cardiovascular Risk in Children and Adolescents".

[This corrects the article DOI: 10.1155/2016/5872423.].

408 Cases of Genital Ambiguity Followed by Single Multidisciplinary Team during 23 Years: Etiologic Diagnosis and Sex of Rearing.

Objective. To evaluate diagnosis, age of referral, karyotype, and sex of rearing of cases with disorders of sex development (DSD) with ambiguous genitalia. Methods. Retrospective study during 23 years at outpatient clinic of a referral center. Results. There were 408 cases; 250 (61.3%) were 46,XY and 124 (30.4%) 46,XX and 34 (8.3%) had sex chromosomes abnormalities. 189 (46.3%) had 46,XY testicular DSD, 105 (25.7%) 46,XX ovarian DSD, 95 (23.3%) disorders of gonadal development (DGD), and 19 (4.7%) complex malformations. The main etiology of 46,XX ovarian DSD was salt-wasting 21-hydroxylase deficiency. In 46,XX and 46,XY groups, other malformations were observed. In the DGD group, 46,XY partial gonadal dysgenesis, mixed gonadal dysgenesis, and ovotesticular DSD were more frequent. Low birth weight was observed in 42 cases of idiopathic 46,XY testicular DSD. The average age at diagnosis was 31.7 months. The final sex of rearing was male in 238 cases and female in 170. Only 6.6% (27 cases) needed sex reassignment. Conclusions. In this large DSD sample with ambiguous genitalia, the 46,XY karyotype was the most frequent; in turn, congenital adrenal hyperplasia was the most frequent etiology. Malformations associated with DSD were common in all groups and low birth weight was associated with idiopathic 46,XY testicular DSD.

Functional and Structural Consequences of Nine CYP21A2 Mutations Ranging from Very Mild to Severe Effects.

We present the functional and structural effects of seven novel (p.Leu12Met, p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, p.Gln389_Ala391del, and p.Thr450Met) and two previously reported but not studied (p.Ser113Phe and p.Thr450Pro) CYP21A2 mutations. Functional analyses were complemented with in silico prediction of mutation pathogenicity based on the recently crystallized human CYP21A2 structure. Mutated proteins were transiently expressed in COS-1 cells and enzyme activities towards 17-hydroxyprogesterone and progesterone were determined. Residual enzyme activities between 43% and 97% were obtained for p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, and p.Thr450Met, similar to the activities of the well-known nonclassic mutations p.Pro453Ser and p.Pro482Ser, whereas the p.Leu12Met variant showed an activity of 100%. Conversely, the novel p.Ser113Phe, p.Gln389_Ala391del, and p.Thr450Pro mutations drastically reduced the enzyme function below 4%. The Km values for all novel variants were in the same order of magnitude as for the wild-type protein except for p.The450Met. The maximum velocity was decreased for all mutants except for p.Leu12Met. We conclude that p.Leu12Met is a normal variant; the mutations p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, and p.Thr450Met could be associated with very mild nonclassic CAH, and the mutations p.Ser113Phe, p.Gln389_Ala391del, and p.Thr450Pro are associated with classic CAH. The obtained residual activities indicated a good genotype-phenotype correlation.