Tratamiento con TRIAC en pacientes pediátricos con deficiencia de MCT8 / Treatment with TRIAC in pediatric patients with MCT8

Los transportadores de monocarboxilatos (MCT) permiten el ingreso celular de hormonas tiroideas, especialmente en el sistema nervioso central (SNC), donde son indispensables para el neurodesarrollo. La deficiencia de MCT8 produce la combinación de hipotiroidismo en SNC e hipertiroidismo periférico, caracterizada por T3 elevada. El único tratamiento actualmente disponible es el ácido 3,3',5-triyodotiroacético (TRIAC), un análogo de hormonas tiroideas que tiene como objetivo mejorar la tirotoxicosis periférica y prevenir la progresión del deterioro neurológico. En el presente artículo, se evalúan las características clínicas, imagenológicas, bioquímicas y genéticas de 4 pacientes con deficiencia de MCT8 tratados con TRIAC hasta la fecha, las dosis utilizadas y la respuesta al tratamiento.

Monocarboxylate transporters (MCTs) allow the cellular entry of thyroid hormones, especially into the central nervous system (CNS), where they are crucial for neurodevelopment. MCT8 deficiency results in the combination of hypothyroidism in the CNS and peripheral hyperthyroidism, characterized by elevated T3 levels. The only treatment currently available is 3,3',5-triiodothyroacetic acid (TRIAC), a thyroid hormone analogue aimed at improving peripheral thyrotoxicosis and preventing the progression of neurological impairment. Here we assess the clinical, imaging, biochemical, and genetic characteristics of 4 patients with MCT8 deficiency who have received TRIAC to date, the doses used, and the response to treatment.

Treatment with TRIAC in pediatric patients with MCT8. / Tratamiento con TRIAC en pacientes pediátricos con deficiencia de MCT8.

Monocarboxylate transporters (MCTs) allow the cellular entry of thyroid hormones, especially into the central nervous system (CNS), where they are crucial for neurodevelopment. MCT8 deficiency results in the combination of hypothyroidism in the CNS and peripheral hyperthyroidism, characterized by elevated T3 levels. The only treatment currently available is 3,3',5-triiodothyroacetic acid (TRIAC), a thyroid hormone analogue aimed at improving peripheral thyrotoxicosis and preventing the progression of neurological impairment. Here we assess the clinical, imaging, biochemical, and genetic characteristics of 4 patients with MCT8 deficiency who have received TRIAC to date, the doses used, and the response to treatment.

Los transportadores de monocarboxilatos (MCT) permiten el ingreso celular de hormonas tiroideas, especialmente en el sistema nervioso central (SNC), donde son indispensables para el neurodesarrollo. La deficiencia de MCT8 produce la combinación de hipotiroidismo en SNC e hipertiroidismo periférico, caracterizada por T3 elevada. El único tratamiento actualmente disponible es el ácido 3,3',5-triyodotiroacético (TRIAC), un análogo de hormonas tiroideas que tiene como objetivo mejorar la tirotoxicosis periférica y prevenir la progresión del deterioro neurológico. En el presente artículo, se evalúan las características clínicas, imagenológicas, bioquímicas y genéticas de 4 pacientes con deficiencia de MCT8 tratados con TRIAC hasta la fecha, las dosis utilizadas y la respuesta al tratamiento.

A Surgical and Clinical Approach to Persistent Müllerian Duct Syndrome: Laparoscopic, Histological, and Molecular Findings.

BACKGROUND: Persistent müllerian duct syndrome (PMDS) is characterized by the persistence of müllerian duct derivatives in otherwise normally virilized 46,XY males. Biallelic mutations of the anti-müllerian hormone (AMH) and AMH receptor type 2 (AMHR2) genes lead to PMDS type 1 and 2, respectively. AIM: The aims of the study were to report the clinical, hormonal, and genetic findings in a patient with PMDS and discuss surgical strategies to achieve successful orchidopexy. RESULTS: A 4-year-old boy was evaluated after the incidental finding of müllerian derivates during laparoscopy for nonpalpable gonads. Karyotype was 46,XY and laboratory tests revealed normal serum gonadotropin and androgen levels but undetectable serum AMH levels. PMDS was suspected. Molecular analysis revealed a novel variant c.902_929del in exon 5 and a previously reported mutation (c.367C>T) in exon 1 of the AMH gene. Successful orchidopexy was performed in two sequential surgeries in which the müllerian duct structure was preserved and divided to protect the vascular supply to the gonads. Histological evaluation of the testicular biopsy showed mild signs of dysgenesis. Doppler ultrasound showed blood flow in both testes positioned in the scrotum 1.5 years after surgery. CONCLUSION: PMDS is a rare entity that requires a high index of suspicion (from surgeons) when evaluating a patient with bilateral cryptorchidism. Surgical treatment is challenging and long-term follow-up is essential. Histological evaluation of the testis deserves further investigation.

Gonadal tumor development in 46,XX disorders of gonadal development.

Background: Differences/disorders of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. Objective: The aim of this study is to report the histological characteristics and immunoexpression patterns of gonadal parenchyma in patients with 46,XX testicular and ovotesticular DSD, with a focus on the detection of germ cell malignancies. Design: Inclusion criteria were SRY-negative 46,XX testicular and ovotesticular DSD with available samples from gonadal biopsy or gonadectomy for the review of histological findings. Gonadal histology was assessed on hematoxylin and eosin-stained sections and immunohistochemical analysis. Histopathological criteria from the last World Health Organization classification of urogenital tumors were used to identify undifferentiated gonadal tissue, gonadoblastoma, and dysgerminoma. Results: Median age at first histological evaluation of gonadal samples was 1.46 years (range: 0.16-16 years). Totally 15 patients were classified as ovotesticular and only 1 as testicular DSD. Most individuals had bilateral ovotestes (12/15). No histological alterations were found in the ovarian parenchyma, while signs of dysgenesis were seen in all cases of testicular parenchyma. In 4/15 ovotesticular DSD, a prepubertal biopsy failed to identify ovarian parenchyma. We detected early prepubertal preinvasive and invasive malignancies in this cohort (five patients had undifferentiated gonadal tissue, five gonadoblastoma, and one dysgerminoma). Conclusion: 46,XX disorders of gonadal development are historically considered at a low risk for germ cell cancer, and the need for assessment of gonadal histology has been questioned. The finding of early germ cell malignancies in our cohort brings awareness and needs further research.

Congenital Adrenal Hyperplasia and Ehlers-Danlos Syndrome.

Congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase deficiency is an autosomal recessive disorder. The 21-hydroxylase enzyme P450c21 is encoded by the CYP21A2 gene located on chromosome 6p21.33 within the HLA major histocompatibility complex. This locus also contains the CYP21A1P, a non-functional pseudogene, that is highly homologous to the CYP21A2 gene. Other duplicated genes are C4A and C4B, that encode two isoforms of complement factor C4, the RP1 gene that encodes a serine/threonine protein kinase, and the TNXB gene that, encodes the extracellular matrix glycoprotein tenascin-X (TNX). TNX plays a role in collagen deposition by dermal fibroblasts and is expressed in the dermis of the skin and the connective tissue of the heart and skeletal muscle. During meiosis, misalignment may occur producing large gene deletions or gene conversion events resulting in chimeric genes. Chimeric recombination may occur between TNXB and TNXA. Three TNXA/TNXB chimeras have been described that differ in the junction site (CH1 to CH3) and result in a contiguous CYP21A2 and TNXB gene deletion, causing CAH-X syndrome. TNXB deficiency is associated with Ehlers Danlos syndrome (EDS). EDS comprises a clinically and genetically heterogeneous group of connective tissue disorders. As molecular analysis of the TNXB gene is challenging, the TNX-deficient type EDS is probably underdiagnosed. In this minireview, we will address the different strategies of molecular analysis of the TNXB-gene, as well as copy number variations and genetic status of TNXB in different cohorts. Furthermore, clinical features of EDS and clinical recommendations for long-term follow-up are discussed.

Growth hormone receptor gene polymorphism. Spontaneous catch up growth in small for gestational age patients.

The growth hormone receptor (GHR) mediates the effect of growth hormone (GH) on linear growth and metabolism. In humans, it exists as two isoforms differing by the retention or exclusion of exon 3; a full-length GHR isoform (GHRfl) and the exon 3-deleted isoform (GHRd3). The genotypic frequency of this polymorphism was analyzed in several studies and in different human populations. However scarce information in Argentinean population is available. Associations between GHRd3 and growth have been reported previously. Some studies have shown that the presence of GHRd3 polymorphism might be a potential variant that improves growth response to recombinant human GH (rhGH) therapy in patients born small for gestational age (SGA), among others. However, over the years the results have been controversial and inconclusive. Based on this, it would be proposed that variants at the genomic level are not completely reflected at the mRNA level. Our aim was to evaluate the genotypic frequencies (%) of the GHR gene polymorphism (GHRfl/GHRfl; GHRfl/GHRd3; GHRd3/GHRd3) in normal Argentinean population (n = 94) and SGA patients (n = 65), and the expression of these polymorphisms at mRNA level in the fetal side of placenta tissues was analyzed. In addition, their association with spontaneous postnatal catch-up growth in SGA patients was also evaluated. In this study, we show a significant increment of compensatory growth in small for gestational age children (SGA) associated to the presence of the GHRd3 allele polymorphism. In addition, the expression of GHR in healthy placentas revealed that no alternative splicing mechanism occurs.

El receptor de la hormona de crecimiento (GHR) media la acción de la hormona de crecimiento (GH) en el crecimiento lineal y el metabolismo. En los seres humanos, existen dos isoformas que difieren en la retención (GHRfl) o exclusión del exón 3 (GHRd3). La frecuencia genotípica de este polimorfismo fue analizada en varios estudios y en diferentes poblaciones. Sin embargo, la información disponible en la población argentina es escasa. Se ha reportado anteriormente asociación entre el polimorfismo GHRd3 y el crecimiento. Varios estudios ha n demostrado que la presencia del polimorfismo GHRd3 podría mejorar, en pacientes nacidos pequeños para la edad gestacional, entre otros, la respuesta a la terapia con GH humana recombinante (rhGH). Sin embargo, a lo largo de los años los resultados han sido controvertidos y no concluyentes. En base a esto, se propondría que las variantes a nivel genómico no se reflejan completamente a nivel del ARNm. Nuestro objetivo fue evaluar la frecuencia genotípica de los polimorfismos del gen del GHR (GHRfl/GHRfl; GHRfl/GHRd3; GHRd3/GHRd3) en la población argentina normal (n = 94) y en niños pequeños para la edad gestacional (n = 65), y se analizó la expresión de estos polimorfismos a nivel de ARNm en la porción fetal de placentas sanas. Además, se evaluó la asociación de este polimorfismo con el crecimiento postnatal espontáneo en pacientes pequeños para la edad gestacional. En este estudio, mostramos un incremento significativo del crecimiento compensatorio en niños pequeños para la edad gestacional asociado a la presencia del polimorfismo del alelo GHRd3. Además, los ensayos de expresión de GHR en placentas sanas revelaron que no se produciría ningún mecanismo de splicing alternativo.

Growth hormone receptor gene polymorphism. Spontaneous catch up growth in small for gestational age patients / Polimorfismo del gen del receptor de la hormona de crecimiento. Crecimiento postnatal espontáneo en niños pequeños para la edad gestacional

Abstract The growth hormone receptor (GHR) mediates the effect of growth hormone (GH) on linear growth and metabolism. In humans, it exists as two isoforms differing by the retention or exclusion of exon 3; a full-length GHR isoform (GHRfl) and the exon 3-deleted isoform (GHRd3). The genotypic frequency of this polymorphism was analyzed in several studies and in different human populations. However scarce information in Argentinean population is available. Associations between GHRd3 and growth have been reported previously. Some studies have shown that the presence of GHRd3 polymorphism might be a potential variant that improves growth response to recombinant human GH (rhGH) therapy in patients born small for gestational age (SGA), among others. However, over the years the results have been controversial and inconclusive. Based on this, it would be proposed that variants at the genomic level are not completely reflected at the mRNA level. Our aim was to evaluate the genotypic frequencies (%) of the GHR gene polymorphism (GHRfl/GHRfl; GHRfl/GHRd3; GHRd3/GHRd3) in normal Argentinean population (n = 94) and SGA patients (n = 65), and the expression of these polymorphisms at mRNA level in the fetal side of placenta tissues was analyzed. In addition, their asso ciation with spontaneous postnatal catch-up growth in SGA patients was also evaluated. In this study, we show a significant increment of compensatory growth in small for gestational age children (SGA) associated to the presence of the GHRd3 allele polymorphism. In addition, the expression of GHR in healthy placentas revealed that no alternative splicing mechanism occurs.

Resumen El receptor de la hormona de creci miento (GHR) media la acción de la hormona de crecimiento (GH) en el crecimiento lineal y el metabolismo. En los seres humanos, existen dos isoformas que difieren en la retención (GHRfl) o exclusión del exón 3 (GHRd3). La frecuencia genotípica de este polimorfismo fue analizada en varios estudios y en diferentes poblaciones. Sin embargo, la información disponible en la población argentina es escasa. Se ha reportado anteriormente asociación entre el polimorfismo GHRd3 y el crecimiento. Varios estudios ha n demostrado que la presencia del polimorfismo GHRd3 podría mejorar, en pacientes nacidos pequeños para la edad gestacional, entre otros, la respuesta a la terapia con GH humana recombinante (rhGH). Sin embargo, a lo largo de los años los resultados han sido con trovertidos y no concluyentes. En base a esto, se propondría que las variantes a nivel genómico no se reflejan completamente a nivel del ARNm. Nuestro objetivo fue evaluar la frecuencia genotípica de los polimorfismos del gen del GHR (GHRfl/GHRfl; GHRfl/GHRd3; GHRd3/GHRd3) en la población argentina normal (n = 94) y en niños pequeños para la edad gestacional (n = 65), y se analizó la expresión de estos polimorfismos a nivel de ARNm en la porción fetal de placentas sanas. Además, se evaluó la asociación de este polimorfismo con el cre cimiento postnatal espontáneo en pacientes pequeños para la edad gestacional. En este estudio, mostramos un incremento significativo del crecimiento compensatorio en niños pequeños para la edad gestacional asociado a la presencia del polimorfismo del alelo GHRd3. Además, los ensayos de expresión de GHR en placentas sanas revelaron que no se produciría ningún mecanismo de splicing alternativo.

Hereditary hypophosphatemic rickets and craniosynostosis.

BACKGROUND: Craniosynostosis is an underdiagnosed complication associated with hypophosphatemic rickets. The study aims to describe the clinical and auxological characteristic of children with hypophosphatemic rickets and craniosynostosis, describe the usual treatment, and compare the characteristics with those of children without craniosynostosis. METHODS AND PATIENTS: An observational and retrospective cohort study was conducted. Clinical notes and cranial images were reviewed. Out of 96 children, only the 50 patients who had skull images were included. RESULTS: Out of 50 patients, 26 (15 males) had craniosynostosis (52%). No differences were observed in birth size, age, height, body proportions, alkaline phosphatase, serum phosphate, or percent tubular reabsorption of phosphate at first appointment among children with or without craniosynostosis. Among patients with craniosynostosis, dolichocephaly was prevalent. The sagittal suture was affected in all patients with craniosynostosis, with 19 of 26 children (73%) affected with isolated scaphocephaly. Pan-sutural craniosynostosis was present in 7 children (27%). None of the children had microcephaly, 7 of them presented macrocephaly and, in the remaining subjects, head circumference was normal. Five patients had undergone at least 1 cranial remodeling surgery. One patient with craniosynostosis was diagnosed with a Chiari I malformation. Molecular characterization of PHEX gene was performed in 14 cases. CONCLUSIONS: Craniosynostosis is an underdiagnosed complication of hypophosphatemic rickets. Many patients with normal head size and growth may go undiagnosed, thus it is important to consider this association for early diagnosis and possible surgical treatment. A multidisciplinary approach is necessary for a correct long-term follow-up.

Comprehensive Identification of Pathogenic Gene Variants in Patients With Neuroendocrine Disorders.

PURPOSE: Congenital hypopituitarism (CH) can present in isolation or with other birth defects. Mutations in multiple genes can cause CH, and the use of a genetic screening panel could establish the prevalence of mutations in known and candidate genes for this disorder. It could also increase the proportion of patients that receive a genetic diagnosis. METHODS: We conducted target panel genetic screening using single-molecule molecular inversion probes sequencing to assess the frequency of mutations in known hypopituitarism genes and new candidates in Argentina. We captured genomic deoxyribonucleic acid from 170 pediatric patients with CH, either alone or with other abnormalities. We performed promoter activation assays to test the functional effects of patient variants in LHX3 and LHX4. RESULTS: We found variants classified as pathogenic, likely pathogenic, or with uncertain significance in 15.3% of cases. These variants were identified in known CH causative genes (LHX3, LHX4, GLI2, OTX2, HESX1), in less frequently reported genes (FOXA2, BMP4, FGFR1, PROKR2, PNPLA6) and in new candidate genes (BMP2, HMGA2, HNF1A, NKX2-1). CONCLUSION: In this work, we report the prevalence of mutations in known CH genes in Argentina and provide evidence for new candidate genes. We show that CH is a genetically heterogeneous disease with high phenotypic variation and incomplete penetrance, and our results support the need for further gene discovery for CH. Identifying population-specific pathogenic variants will improve the capacity of genetic data to predict eventual clinical outcomes.

Testis formation in XX individuals resulting from novel pathogenic variants in Wilms' tumor 1 (WT1) gene.

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10-6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10-4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor ß-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

Molecular analysis of the CYP21A2 gene in dried blood spot samples

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder due to a deficiency of enzymes involved in cortisol biosynthesis. In more than 90% of cases, CAH is secondary to deleterious mutations in the CYP21A2 gene leading to 21-hydroxilase deficiency (21OHD). The CYP21A2 gene is located on the short arm of chromosome 6 (6p21·3) and encodes the cytochrome P450C21 enzyme. Neonatal screening programs detect the classic forms of CAH-21OHD quantifying 17OH-progesterone in dried blood spots (DBS). This test is very sensitive, but it has a low specificity, requiring a second sample to confirm the result. In these cases, a second-tier test in the same sample may be useful. Our aim was to evaluate a DNA extraction method from DBS and assess the performance of such DNA in the molecular analysis of the CYP21A2 gene mutations. Twelve individuals, who presumably had CAH based on the initial neonatal screening results, were analyzed using DNA extracted from freshly collected blood on EDTA and DBS. The CYP21A2 gene was analyzed by automated sequencing of all exons and intron boundaries and MLPA analysis in DBS. Molecular analysis results from both extraction methods were compared. In this study, we show that DNA extracted from neonatal screening DBS is a useful tool to define CYP21A2 gene mutations in 21-OHD diagnostic confirmation for the newborn screening program and that its results are comparable to traditional genotyping.

La hiperplasia suprarrenal congénita (HSC) es un desorden autosómico recesivo producido por la deficiencia de alguna de las enzimas involucradas en la biosíntesis de cortisol. Más del 90% se debe a mutaciones en el gen CYP21A2 que genera deficiencia de 21 hidroxilasa (21OHD). Este gen se encuentra en el brazo corto del cromosoma 6 (6p21·3) y codifica para la enzima citocromo P450C21. Los programas de pesquisa neonatal detectan la forma clásica de la HSC-21OHD cuantificando 17OH-progesterona en gota de sangre en papel de filtro (GSPF). Este test es muy sensible, pero tiene baja especificidad , por lo que se utiliza una segunda muestra para confirmar el resultado. En estos casos, una segunda determinación en la misma muestra podría ser de utilidad. Nuestro objetivo fue evaluar el método de extracción de ADN y posterior análisis molecular del gen CYP21A2 en muestras de GSPF. Analizamos doce individuos presumiblemente afectados por HSC en la pesquisa neonatal usando ADN extraído de sangre fresca recolectada sobre EDTA y de GSPF. Realizamos el análisis del gen CYP21A2 mediante secuenciación automática de todos los exones y regiones intrónicas flanqueantes y MLPA en GSPF, y comparamos los resultados con ambos métodos de extracción. En este estudio demostramos que el ADN extraído de GSPF es una herramienta muy útil para analizar las mutaciones del gen CYP21A2 en la confirmación diagnóstica de 21-OHD para los programas de pesquisa neonatal y que los resultados son comparables con la genotipificación tradicional.

Molecular analysis of the CYP21A2 gene in dried blood spot samples.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder due to a deficiency of enzymes involved in cortisol biosynthesis. In more than 90% of cases, CAH is secondary to deleterious mutations in the CYP21A2 gene leading to 21-hydroxilase deficiency (21OHD). The CYP21A2 gene is located on the short arm of chromosome 6 (6p21·3) and encodes the cytochrome P450C21 enzyme. Neonatal screening programs detect the classic forms of CAH-21OHD quantifying 17OH-progesterone in dried blood spots (DBS). This test is very sensitive, but it has a low specificity, requiring a second sample to confirm the result. In these cases, a second-tier test in the same sample may be useful. Our aim was to evaluate a DNA extraction method from DBS and assess the performance of such DNA in the molecular analysis of the CYP21A2 gene mutations. Twelve individuals, who presumably had CAH based on the initial neonatal screening results, were analyzed using DNA extracted from freshly collected blood on EDTA and DBS. The CYP21A2 gene was analyzed by automated sequencing of all exons and intron boundaries and MLPA analysis in DBS. Molecular analysis results from both extraction methods were compared. In this study, we show that DNA extracted from neonatal screening DBS is a useful tool to define CYP21A2 gene mutations in 21-OHD diagnostic confirmation for the newborn screening program and that its results are comparable to traditional genotyping.

La hiperplasia suprarrenal congénita (HSC) es un desorden autosómico recesivo producido por la deficiencia de alguna de las enzimas involucradas en la biosíntesis de cortisol. Más del 90% se debe a mutaciones en el gen CYP21A2 que genera deficiencia de 21 hidroxilasa (21OHD). Este gen se encuentra en el brazo corto del cromosoma 6 (6p21·3) y codifica para la enzima citocromo P450C21. Los programas de pesquisa neonatal detectan la forma clásica de la HSC-21OHD cuantificando 17OH-progesterona en gota de sangre en papel de filtro (GSPF). Este test es muy sensible, pero tiene baja especificidad , por lo que se utiliza una segunda muestra para confirmar el resultado. En estos casos, una segunda determinación en la misma muestra podría ser de utilidad. Nuestro objetivo fue evaluar el método de extracción de ADN y posterior análisis molecular del gen CYP21A2 en muestras de GSPF. Analizamos doce individuos presumiblemente afectados por HSC en la pesquisa neonatal usando ADN extraído de sangre fresca recolectada sobre EDTA y de GSPF. Realizamos el análisis del gen CYP21A2 mediante secuenciación automática de todos los exones y regiones intrónicas flanqueantes y MLPA en GSPF, y comparamos los resultados con ambos métodos de extracción. En este estudio demostramos que el ADN extraído de GSPF es una herramienta muy útil para analizar las mutaciones del gen CYP21A2 en la confirmación diagnóstica de 21-OHD para los programas de pesquisa neonatal y que los resultados son comparables con la genotipificación tradicional.

Accelerated Pubertal Tempo in a 46,XY Aromatase-Deficient Patient.

BACKGROUND: Aromatase deficiency is a rare autosomal recessive disorder. 46,XY-affected patients often remain undiagnosed until late puberty. Only 2 pediatric cases have been reported. Data on pubertal development in affected males are scarce. AIM: To report the clinical phenotype and hormonal studies of an aromatase-deficient boy during the prepubertal and early pubertal period. RESULTS: The patient was the older brother of a 46,XX girl with aromatase deficiency. Molecular analysis revealed a previously reported homozygous mutation (Arg192Cys) in the CYP19A1 gene. Pubertal onset was at 9.8 years. At 11.3 years of age, signs of rapidly progressive puberty were seen. Laboratory tests revealed normal pubertal basal and GnRH-stimulated gonadotropin levels, normal Sertoli cell markers, and increased testosterone. The prepubertal lumbar spine bone mineral density (BMD) was normal but pubertal bone mineral accrual was incomplete, leading to osteopenia. CONCLUSION: Estrogen restraint on gonadotropin secretion has been demonstrated in animal and human models. Interestingly, our patient presented with accelerated puberty and apparently normal pituitary gonadal function. These findings suggest that aromatase activity may be required to define pubertal progression in boys. Estrogen deficiency due to aromatase deficiency is responsible for insufficient bone mineral accrual during puberty.

DNA methylation is not involved in specific down-regulation of HSD3B2, NR4A1 and RARB genes in androgen-secreting cells of human adrenal cortex.

We hypothesized that DNA methylation is involved in human adrenal functional zonation. mRNAs expression and methylation pattern of RARB, NR4A1 and HSD3B2 genes in human adrenal tissues (HAT) and in pediatric virilizing adrenocortical tumors (VAT) were analyzed. For analysis of the results samples were divided into 3 age groups according to FeZ involution, pre and post-adrenarche ages. In all HAT, similar RARB mRNA was found including microdissected zona reticularis (ZR) and zona fasciculata, but HSD3B2 and NR4A1 mRNAs were lower in ZR (p < 0.05). NR4A1 and RARB promoters remained unmethylated in HAT and VAT. No adrenal zone-specific differences in NR4A1 methylation were observed. In summary, RARB was not associated with ZR-specific downregulation of HSD3B2 in postnatal human adrenocotical zonation. DNA methylation would not be involved in NR4A1 adrenocortical cell-type specific downregulation. Lack of CpG islands in HSD3B2 suggested that HSD3B2 ZR-specific downregulation would not be directly mediated by DNA methylation.

Androgen Insensitivity Syndrome at Prepuberty: Marked Loss of Spermatogonial Cells at Early Childhood and Presence of Gonocytes up to Puberty.

Androgen insensitivity syndrome (AIS) is a hereditary condition in patients with a 46,XY karyotype in which loss-of-function mutations of the androgen receptor (AR) gene are responsible for defects in virilization. The aim of this study was to investigate the consequences of the lack of AR activity on germ cell survival and the degree of testicular development reached by these patients by analyzing gonadal tissue from patients with AIS prior to Sertoli cell maturation at puberty. Twenty-three gonads from 13 patients with AIS were assessed and compared to 18 testes from 17 subjects without endocrine disorders. The study of the gonadal structure using conventional microscopy and the ultrastructural characteristics of remnant germ cells using electron microscopy, combined with the immunohistochemical analysis of specific germ cell markers (MAGE-A4 for premeiotic germ cells and of OCT3/4 for gonocytes), enabled us to carry out a thorough investigation of germ cell life in an androgen-insensitive microenvironment throughout prepuberty until young adulthood. Here, we show that germ cell degeneration starts very early, with a marked decrease in number after only 2 years of life, and we demonstrate the permanence of gonocytes in AIS testis samples until puberty, describing 2 different populations. Additionally, our results provide further evidence for the importance of AR signaling in peritubular myoid cells during prepuberty to maintain Sertoli and spermatogonial cell health and survival.

An Intron 9 CYP19 Gene Variant (IVS9+5G>A), Present in an Aromatase-Deficient Girl, Affects Normal Splicing and Is Also Present in Normal Human Steroidogenic Tissues.

BACKGROUND/AIMS: Splicing CYP19 gene variants causing aromatase deficiency in 46,XX disorder of sexual development (DSD) patients have been reported in a few cases. A misbalance between normal and aberrant splicing variants was proposed to explain spontaneous pubertal breast development but an incomplete sex maturation progress. The aim of this study was to functionally characterize a novel CYP19A1 intronic homozygote mutation (IVS9+5G>A) in a 46,XX DSD girl presenting spontaneous breast development and primary amenorrhea, and to evaluate similar splicing variant expression in normal steroidogenic tissues. METHODS: Genomic DNA analysis, splicing prediction programs, splicing assays, and in vitro protein expression and enzyme activity analyses were carried out. CYP19A1 mRNA expression in human steroidogenic tissues was also studied. RESULTS: A novel IVS9+5G>A homozygote mutation was found. In silico analysis predicts the disappearance of the splicing donor site in intron 9, confirmed by patient peripheral leukocyte cP450arom and in vitro studies. Protein analysis showed a shorter and inactive protein. The intron 9 transcript variant was also found in human steroidogenic tissues. CONCLUSIONS: The mutation IVS9+5G>A generates a splicing variant that includes intron 9 which is also present in normal human steroidogenic tissues, suggesting that a misbalance between normal and aberrant splicing variants might occur in target tissues, explaining the clinical phenotype in the affected patient.

Five new cases of 46,XX aromatase deficiency: clinical follow-up from birth to puberty, a novel mutation, and a founder effect.

CONTEXT: Aromatase is the key enzyme for estrogen biosynthesis and is encoded by the CYP19A1 gene. Since 1991, several molecular CYP19A1 gene alterations associated with aromatase deficiency have been described in both sexes. OBJECTIVE: The objective of the study was to detect CYP19A1 mutations in five aromatase-deficient 46,XX patients, to describe the clinical follow-up from birth to puberty and to perform haplotype analysis associated with the high-frequency c.628G>A splice mutation in Argentinean patients. DESIGN: The design of the study was the sequencing of the coding and flanking intronic regions of the CYP19A1 gene in all patients and parents. Haplotype analysis of patients carrying the c.628G>A mutation was also performed. PATIENTS: Clinical and biochemical findings in five new cases and one previously reported female aromatase-deficient patient (46,XX) are described. All patients presented with ambiguous genitalia at birth. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency as well as other steroidogenic defects were ruled out. RESULTS: Phenotypic variability among the affected patients was found during follow-up. Direct sequencing of the CYP19A1 gene from genomic DNA revealed one novel mutation (c.574C>T) in two patients. In silico analysis predicted the c.574C>T mutation to be probably damaging. Four of six nonrelated patients presented with the c.628G>A splice mutation. Haplotype analysis showed that the c.628G>A splice mutation is associated with the same haplotype in our population. CONCLUSIONS: Increased knowledge on phenotypical variability found in female aromatase-deficient patients is useful to improve the detection rate in this disorder. In our population, a genetic founder defect has probably contributed to an increase in the incidence of the c.628G>A splice mutation.

A novel missense mutation in the HSD3B2 gene, underlying nonsalt-wasting congenital adrenal hyperplasia. new insight into the structure-function relationships of 3ß-hydroxysteroid dehidrogenase type II.

CONTEXT: 3ßHSD2 is a bifunctional microsomal NAD+-dependent enzyme crucial for adrenal and gonad steroid biosynthesis, converting Δ5-steroids to Δ4-steroids. 3ßHSD2 deficiency is a rare cause of congenital adrenal hyperplasia caused by recessive loss-of-function HSD3B2 mutations. OBJECTIVE: The aim was to define the pathogenic consequences of a novel missense mutation in the HSD3B2 gene. PATIENT: We report a 7-month-old 46,XX girl referred because of precocious pubarche and postnatal clitoromegaly. Hormonal profile showed inadequate glucocorticoid levels, increased 17OHP and renin levels, and very high DHEAS levels, suggestive of compensated nonsalt-losing 3ßHSD2 deficiency. DESIGN AND RESULTS: Direct sequencing revealed a novel, homozygous, pG250V HSD3B2 mutation. In vitro analysis in intact COS-7 cells showed impaired enzymatic activity for the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione (20% and 27% of WT at 6 h, respectively). G250V-3ßHSD2 decreased the Vmax for progesterone synthesis without affecting the Km for pregnenolone. Western blot and immunofluorescence suggested that p.G250V mutation has no effect on the expression and intracellular localization of the mutant protein. Molecular homology modeling predicted that mutant V250 affected an L239-Q251 loop next to a ß-sheet structure in the NAD+-binding domain. CONCLUSIONS: We identified a novel p.G250V mutation of HSD3B2 which causes an incomplete loss of enzymatic activity, explaining the compensated nonsalt loss phenotype. In vitro and in silico experiments provided insight into the structure-function relationship of the 3ßHSD2 protein suggesting the importance of the L239-Q251 loop for the catalytic activity of the otherwise stable 3ßHSD2 enzyme.

Steroid 21-hydroxylase gene mutational spectrum in 454 Argentinean patients: genotype-phenotype correlation in a large cohort of patients with congenital adrenal hyperplasia.

OBJECTIVE: To report genotype-phenotype correlation in a large cohort of patients. CONTEXT: Study of the CYP21A2 gene in 866 unrelated chromosomes of 21-hydroxylase deficiency in Argentinean patients with classic and nonclassic (NC) forms of congenital adrenal hyperplasia (CAH). METHODS: Eleven most common mutations were analysed by allele-specific polymerase chain reaction, restriction fragment length polymorphism (RFLP) or southern blot analysis. Gene sequencing was performed when no mutation was detected in one allele or the genotype-phenotype correlation was lacking. RESULTS: The 11-most-common-mutation screening allowed for the detection of 88·1% of affected alleles (80·3% in the NC and 95·2% in the classic forms). p.V281L, IVS2-13A/C>G (In2) and gene deletions and large gene conversions were the most prevalent mutations. In2 (35·2%) in salt wasting (SW), p.I172N (37·3%) in simple virilizing and p.V281L (54·1%) in NC CAH were the most prevalent mutations within the clinical forms. In 7/15 p.P30L mutation alleles, a chimeric CYP21A1P/CYP21A2 gene [PromCYP21A1P; p.P30L] was detected, while 6/15 represented a single-nucleotide substitution, and in 2/15 linkage with mutations, p.[P30L; V281L] and [p.P30L; IVS2-13A/C > G; p.Q318X] was found. In two SW patients, a novel nonsense mutation, p.Q41X, was observed. In three p.V281L mutation patients, the phenotype was more severe than predicted by genotype. Sequence analysis revealed an intronic alteration in the allele carrying the p.V281L mutation [IVS2 + 5G > A; p.V281L]. An aberrant splicing in this p.V281L mutated allele explains the clinical phenotype. CONCLUSIONS: A high percentage of CYP21A2 affected alleles is detected by the 11-mutation screening study. Genotype-phenotype correlation was high, but when the phenotype is more severe than predicted by genotype, presence of two alterations in one allele should be ruled out.