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The differential diagnosis of differences or disorders of sex development (DSD) belongs to the most complex fields in medicine. It requires a multidisciplinary team conducting a synoptic and complementary approach consisting of thorough clinical, hormonal and genetic workups. This position paper of EU COST (European Cooperation in Science and Technology) Action BM1303 'DSDnet' was written by leading experts in the field and focuses on current best practice in genetic diagnosis in DSD patients. Ascertainment of the karyotpye defines one of the three major diagnostic DSD subclasses and is therefore the mandatory initial step. Subsequently, further analyses comprise molecular studies of monogenic DSD causes or analysis of copy number variations (CNV) or both. Panels of candidate genes provide rapid and reliable results. Whole exome and genome sequencing (WES and WGS) represent valuable methodological developments that are currently in the transition from basic science to clinical routine service in the field of DSD. However, in addition to covering known DSD candidate genes, WES and WGS help to identify novel genetic causes for DSD. Diagnostic interpretation must be performed with utmost caution and needs careful scientific validation in each DSD case. [ABSTRACT FROM AUTHOR]

Background: GH release after stimuli classifies short children as severe idiopathic isolated GH deficiency (IIGHD), mild IIGHD, dissociated GH release (DGHR) and normal GH release (NGHR) and anthropometric birth data as adequate for gestational age (AGA) or small for gestational age (SGA). GH release after stimuli classifies AGA patients as IIGHD or as idiopathic short stature (ISS). Aim: To compare height gain induced by GH therapy (31.8 ± 3.5 µg/kg/day, 7.7 ± 1.6 years) started at prepubertal age and stopped at near adult-height age. Methods: A retrospective longitudinal multicenter study including184 short patients classified as severe IIGHD n = 25, mild IIGHD n = 75, DGHR n = 55 and NGHR n = 29; or as IIGHD n = 78, ISS n = 57 and SGA n = 49. Height gain was evaluated throughout GH therapy and at adult-height age. Results: Height-SDS gain at adult-height age was similar among severe IIGHD (1.8 ± 0.8 SDS), mild IIGHD (1.6 ± 0.6 SDS), DGHR (1.7 ± 0.7 SDS) and NGHR (1.6 ± 0.7 SDS), or among IIGHD (1.7 ± 0.7 SDS), ISS (1.7 ± 0.6 SDS) and SGA (1.6 ± 0.8 SD). Conclusion: GH-release stimuli are of little help for deciding on GH therapy in the clinical management of prepubertal children with IIGHD, ISS or SGA. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Presents several letters to the editor regarding issues on diabetes and diabetic medicine. Discussion on the influence of vitamin D receptor gene polymorphism at exon 2 start codon on type 1 diabetes mellitus susceptibility in Spanish populations; Study on the prevalence of misdiagnosis in diabetic peripheral neuropathy and spinal stenoses; Research on increased forefoot to rearfoot plantar pressure ratio in South Indian patients with diabetic foot ulceration; Consideration of pertussis infections, vaccines in type 1 diabetes.

Objectives: The development of female or male sex characteristics occurs during fetal life, when the genetic, gonadal, and internal and external genital sex is determined (female or male). Any discordance among sex determination and differentiation stages results in differences/disorders of sex development (DSD), which are classified based on the sex chromosomes found on the karyotype., Content: This chapter addresses the physiological mechanisms that determine the development of female or male sex characteristics during fetal life, provides a general classification of DSD, and offers guidance for clinical, biochemical, and genetic diagnosis, which must be established by a multidisciplinary team. Biochemical studies should include general biochemistry, steroid and peptide hormone testing either at baseline or by stimulation testing. The genetic study should start with the determination of the karyotype, followed by a molecular study of the 46,XX or 46,XY karyotypes for the identification of candidate genes., Summary: 46,XX DSD include an abnormal gonadal development (dysgenesis, ovotestes, or testes), an androgen excess (the most frequent) of fetal, fetoplacental, or maternal origin and an abnormal development of the internal genitalia. Biochemical and genetic markers are specific for each group., Outlook: Diagnosis of DSD requires the involvement of a multidisciplinary team coordinated by a clinician, including a service of biochemistry, clinical, and molecular genetic testing, radiology and imaging, and a service of pathological anatomy., Competing Interests: Competing interests: Authors state no conflict of interest., (© 2021 Maria Luisa Granada and Laura Audí, published by De Gruyter, Berlin/Boston.)

Objectives: 46,XY differences/disorders of sex development (DSD) involve an abnormal gonadal and/or genital (external and/or internal) development caused by lack or incomplete intrauterine virilization, with or without the presence of Müllerian ducts remnants., Content: Useful biochemical markers for differential diagnosis of 46,XY DSD include hypothalamic-pituitary-gonadal hormones such as luteinizing and follicle-stimulating hormones (LH and FSH; in baseline or after LHRH stimulation conditions), the anti-Müllerian hormone (AMH), inhibin B, insulin-like 3 (INSL3), adrenal and gonadal steroid hormones (including cortisol, aldosterone, testosterone and their precursors, dihydrotestosterone and estradiol) and the pituitary ACTH hormone. Steroid hormones are measured at baseline or after stimulation with ACTH (adrenal hormones) and/or with HCG (gonadal hormones)., Summary: Different patterns of hormone profiles depend on the etiology and the severity of the underlying disorder and the age of the patient at diagnosis. Molecular diagnosis includes detection of gene dosage or copy number variations, analysis of candidate genes or high-throughput DNA sequencing of panels of candidate genes or the whole exome or genome., Outlook: Differential diagnosis of 46,XX or 46,XY DSD requires a multidisciplinary approach, including patient history and clinical, morphological, imaging, biochemical and genetic data. We propose a diagnostic algorithm suitable for a newborn with DSD that focuses mainly on biochemical and genetic data., Competing Interests: Competing interests: Authors state no conflict of interest., (© 2021 Maria Luisa Granada and Laura Audí, published by De Gruyter, Berlin/Boston.)

Objetivos: El desarrollo sexual anómalo o diferente (DSD) con cariotipo 46,XY incluye anomalías en el desarrollo gonadal y/o genital (externo y/o interno)., Contenido: Los marcadores bioquímicos útiles para el diagnóstico diferencial de los DSD con cariotipo 46,XY incluyen las hormonas del eje hipotálamo-hipófiso gonadal como son las gonadotropinas LH y FSH (en condiciones basales o tras la estimulación con LHRH), la hormona anti-Mülleriana, la inhibina B, el factor insulinoide tipo 3 y las hormonas esteroideas de origen suprarrenal (se incluirá la hormona hipofisaria ACTH) y testicular (cortisol, aldosterona y sus precursores, testosterona y sus precursores, dihidrotestosterona y estradiol). Las hormonas esteroideas se analizarán en condiciones basales o tras la estimulación con ACTH (hormonas adrenales) y/o con HCG (hormonas testiculares). Los patrones de variación de las distintas hormonas dependerán de la causa y la edad de cada paciente. El diagnóstico molecular debe incluir el análisis de un gen candidato, un panel de genes o el análisis de un exoma completo., Perspectivas: El diagnóstico diferencial de los DSD con cariotipos 46,XX ó 46,XY debe ser multidisciplinar, incluyendo los antecedentes clínicos, morfológicos, de imagen, bioquímicos y genéticos. Se han elaborado numerosos algoritmos diagnósticos., (© 2021 Maria Luisa Granada and Laura Audí, published by De Gruyter, Berlin/Boston.)

Disorders (or differences) of sex development (DSD) are congenital conditions characterized by atypical development of genetic, gonadal or phenotypic sex [...].

Context: Mutations in cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We report a novel R550W mutation in POR identified in a 46,XX patient with signs of aromatase deficiency., Objective: Analysis of aromatase deficiency from the R550W mutation in POR., Design, Setting, and Patient: Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C > T/p.R550W in POR. Wild-type and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children's Hospital, Bern, Switzerland., Main Outcome Measure and Results: POR-R550W showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity, and CYP17A1 as well as CYP21A2 activities were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed., Conclusions: Pathological effects due to POR-R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sex development is a very complex biological event that requires the concerted collaboration of a large network of genes in a spatial and temporal correct fashion. In the past, much has been learned about human sex development from monogenic disorders/differences of sex development (DSD), but the broad spectrum of phenotypes in numerous DSD individuals remains a conundrum. Currently, the genetic cause of less than 50% of DSD individuals has been solved and oligogenic disease has been proposed. In recent years, multiple genetic hits have been found in individuals with DSD thanks to high throughput sequencing. Our group has been searching for additional genetic hits explaining the phenotypic variability over the past years in two cohorts of patients: 46,XY DSD patients carriers of NR5A1 variants and 46,XY DSD and 46,XX DSD with MAMLD1 variants. In both cohorts, our results suggest that the broad phenotypes may be explained by oligogenic origin, in which multiple hits may contribute to a DSD phenotype, unique to each individual. A search for an underlying network of the identified genes also revealed that a considerable number of these genes showed interactions, suggesting that genetic variations in these genes may affect sex development in concert.

Disorders/differences of sex development (DSD) are the result of a discordance between chromosomal, gonadal, and genital sex. DSD may be due to mutations in any of the genes involved in sex determination and development in general, as well as gonadal and/or genital development specifically. MAMLD1 is one of the recognized DSD genes. However, its role is controversial as some MAMLD1 variants are present in normal individuals, several MAMLD1 mutations have wild-type activity in functional studies, and the Mamld1 -knockout male mouse presents with normal genitalia and reproduction. We previously tested nine MAMLD1 variants detected in nine 46,XY DSD patients with broad phenotypes for their functional activity, but none of the mutants, except truncated L210X, had diminished transcriptional activity on known target promoters CYP17A1 and HES3 . In addition, protein expression of MAMLD1 variants was similar to wild-type, except for the truncated L210X. We hypothesized that MAMLD1 variants may not be sufficient to explain the phenotype in 46,XY DSD individuals, and that further genetic studies should be performed to search for additional hits explaining the broad phenotypes. We therefore performed whole exome sequencing (WES) in seven of these 46,XY patients with DSD and in one 46,XX patient with ovarian insufficiency, who all carried MAMLD1 variants. WES data were filtered by an algorithm including disease-tailored lists of MAMLD1 -related and DSD-related genes. Fifty-five potentially deleterious variants in 41 genes were identified; 16/55 variants were reported in genes in association with hypospadias, 8/55 with cryptorchidism, 5/55 with micropenis, and 13/55 were described in relation with female sex development. Patients carried 1-16 variants in 1-16 genes together with their MAMLD1 variation. Network analysis of the identified genes revealed that 23 genes presented gene/protein interactions with MAMLD1. Thus, our study shows that the broad phenotypes of individual DSD might involve multiple genetic variations contributing towards the complex network of sexual development.

Persistent müllerian duct syndrome (PMDS) is characterized by the presence of müllerian duct derivatives in otherwise phenotypically normal males. Homozygous or compound heterozygous alterations in AMH or AMHR2 have been identified in approximately 88% of PMDS cases. We report on a male patient with bilateral undescended gonads, müllerian derivatives, and normal serum AMH levels. A novel homozygous missense mutation, c.119G>C;p.Gly40Ala, in exon 2 of AMHR2 was detected that supported the clinical diagnosis of PMDS., (© 2019 S. Karger AG, Basel.)

SF-1/NR5A1 is a transcriptional regulator of adrenal and gonadal development. NR5A1 disease-causing variants cause disorders of sex development (DSD) and adrenal failure, but most affected individuals show a broad DSD/reproductive phenotype only. Most NR5A1 variants show in vitro pathogenic effects, but not when tested in heterozygote state together with wild-type NR5A1 as usually seen in patients. Thus, the genotype-phenotype correlation for NR5A1 variants remains an unsolved question. We analyzed heterozygous 46,XY SF-1/NR5A1 patients by whole exome sequencing and used an algorithm for data analysis based on selected project-specific DSD- and SF-1-related genes. The variants detected were evaluated for their significance in literature, databases and checked in silico using webtools. We identified 19 potentially deleterious variants (one to seven per patient) in 18 genes in four 46,XY DSD subjects carrying heterozygous NR5A1 disease-causing variants. We constructed a scheme of all these hits within the landscape of currently known genes involved in male sex determination and differentiation. Our results suggest that the broad phenotype in these heterozygous NR5A1 46,XY DSD subjects may well be explained by an oligogenic mode of inheritance, in which multiple hits, individually non-deleterious, may contribute to a DSD phenotype unique to each heterozygous SF-1/NR5A1 individual.

The CYP17A1 gene regulates sex steroid biosynthesis in humans through 17α-hydroxylase/17,20 lyase activities and is a target of anti-prostate cancer drug abiraterone. In a 46, XY patient with female external genitalia, together with a loss of function mutation S441P, we identified a novel missense mutation V366M at the catalytic center of CYP17A1 which preferentially impaired 17,20 lyase activity. Kinetic experiments with bacterially expressed proteins revealed that V366M mutant enzyme can bind and metabolize pregnenolone to 17OH-pregnenolone, but 17OH-pregnenolone binding and conversion to dehydroepiandrosterone (DHEA) was impaired, explaining the patient’s steroid profile. Abiraterone could not bind and inhibit the 17α-hydroxylase activity of the CYP17A1-V366M mutant. Molecular dynamics (MD) simulations showed that V366M creates a “one-way valve” and suggests a mechanism for dual activities of human CYP17A1 where, after the conversion of pregnenolone to 17OH-pregnenolone, the product exits the active site and re-enters for conversion to dehydroepiandrosterone. The V366M mutant also explained the effectiveness of the anti-prostate cancer drug abiraterone as a potent inhibitor of CYP17A1 by binding tightly at the active site in the WT enzyme. The V366M is the first human mutation to be described at the active site of CYP17A1 that causes isolated 17,20 lyase deficiency. Knowledge about the specificity of CYP17A1 activities is of importance for the development of treatments for polycystic ovary syndrome and inhibitors for prostate cancer therapy.

Scientific knowledge to understand the biological basis of sex development was prompted by the observation of variants different from the 2 most frequent body types, and this became one of the fields first studied by modern pediatric endocrinology. The clinical observation was supported by professionals working in different areas of laboratory sciences which led to the description of adrenal and gonadal steroidogenesis, the enzymes involved, and the different deficiencies. Steroid hormone measurements evolved from colorimetry to radioimmunoassay (RIA) and automated immunoassays, although gas and liquid chromatography coupled to mass spectrometry are now the gold standard techniques for steroid measurements. Peptide hormones and growth factors were purified, and their measurement evolved from RIA to automated immunoassays. Hormone action mechanisms were described, and their specific receptors were characterized and assayed in experimental materials and in patient tissues and cell cultures. The discovery of the genetic basis for variant sex developments began with the description of the sex chromosomes. Molecular technology allowed cloning of genes coding for the different proteins involved in sex determination and development. Experimental animal models aided in verifying the roles of proteins and also suggested new genes to be investigated. New candidate genes continue to be described based on experimental models and on next-generation sequencing of patient DNAs., (© 2017 S. Karger AG, Basel.)

Background: The prevalence of vitamin D (vitD) deficiency presenting as rickets is increasing worldwide. Insufficient sun exposure, vitD administration, and/or calcium intake are the main causes. However, vitD system-related genes may also have a role., Methods: Prospective study: 109 rachitic children completed a 6-mo study period or until rachitic manifestations disappeared. Thirty children were selected as controls. Clinical and biochemical data were evaluated at baseline in patients and controls and biochemistry re-evaluated at radiological healing. Therapy was stratified in three different protocols. Fifty-four single-nucleotide polymorphisms (SNPs) of five vitD system genes (VDR, CP2R1, CYP27B1, CYP24A1, and GC) were genotyped and their association with clinical and biochemcial data was analyzed., Results: Therapy response was similar in terms of radiological healing although it was not so in terms of biochemical normalization. Only VDR gene (promoter, start-codon, and intronic genotypes) was rickets-associated in terms of serum 25-OH-D, calcium, radiological severity and time needed to heal. Eight patients with sufficient calcium intake and 25-OH-D levels carried a VDR genotype lacking minor allele homozygous genotypes at SNPs spread along the gene., Conclusion: Although patients presented epidemiologic factors strongly contributing to rickets, genetic modulation affecting predisposition, severity, and clinical course is exerted, at least in part, by VDR gene polymorphic variation.

17α-hydroxylase/17,20-lyase deficiency is a rare form of congenital adrenal hyperplasia caused by mutations in CYP17A1. Two phenotypic female sisters, aged 17 and 15 years and with 46,XY and 46,XX karyotypes, respectively, presented with primary amenorrhea and absent secondary sexual characteristics. The elder sib also presented with high blood pressure. Both patients had elevated levels of ACTH, gonadotropins, progesterone, corticosterone, and deoxycorticosterone, and reduced levels of estradiol, testosterone, androstenedione, 17-OH-P, DHEA-S, cortisol, aldosterone, and renin activity. The CYP17A1 gene was sequenced, and polymorphic haplotypes were further analyzed in the Spanish family and in Brazilian patients. The 2 sisters were compound heterozygous for p.Arg362Cys and p.Trp406Arg mutations, previously described as the most prevalent mutations in Brazilian families of Spanish (p.Trp406Arg) or Portuguese (p.Arg362Cys) origin. Analysis of polymorphisms in CYP17A1 suggested that the paternal allele with p.Arg362Cys may share a common origin with the Brazilian carriers, while the maternal allele with p.Trp406Arg did not. Hydrocortisone and sex hormone replacement therapy was initiated in both patients. In conclusion, one CYP17A1 mutation (p.Arg362Cys) may share a common ancestry in Brazilian and our present Spanish patients, while p.Trp406Arg may have arisen separately. The elder patient (46,XY) developed a more severe phenotype and a poorer response to estradiol replacement therapy., (© 2017 S. Karger AG, Basel.)

MAMLD1 is thought to cause disordered sex development in 46,XY patients. But its role is controversial because some MAMLD1 variants are also detected in normal individuals, several MAMLD1 mutations have wild-type activity in functional tests, and the male Mamld1-knockout mouse has normal genitalia and reproduction. Our aim was to search for MAMLD1 variations in 108 46,XY patients with disordered sex development, and to test them functionally. We detected MAMDL1 variations and compared SNP frequencies in controls and patients. We tested MAMLD1 transcriptional activity on promoters involved in sex development and assessed the effect of MAMLD1 on androgen production. MAMLD1 expression in normal steroid-producing tissues and mutant MAMLD1 protein expression were also assessed. Nine MAMLD1 mutations (7 novel) were characterized. In vitro, most MAMLD1 variants acted similarly to wild type. Only the L210X mutation showed loss of function in all tests. We detected no effect of wild-type or MAMLD1 variants on CYP17A1 enzyme activity in our cell experiments, and Western blots revealed no significant differences for MAMLD1 protein expression. MAMLD1 was expressed in human adult testes and adrenals. In conclusion, our data support the notion that MAMLD1 sequence variations may not suffice to explain the phenotype in carriers and that MAMLD1 may also have a role in adult life.