[Show abstract][Hide abstract] ABSTRACT: Down Syndrome is the most common chromosomal disease and is also known for its decreased incidence of solid tumors and its progeroid phenotype. Cellular and systemic oxidative stress has been considered as one of the Down Syndrome phenotype causes. We correlated, in a preliminary study, the fibroblast proliferation rate and different cell proliferation key regulators, like Rcan1 and the telomere length from Down syndrome fetuses, with their oxidative stress profile and the RNA and protein expression of the main antioxidant enzymes together with their activity. Increased GSSG/GSH ratio and high peroxide production were found in our cell model. These results correlated with a distorted antioxidant shield. The mRNA (SOD1) and protein levels of Copper/Zinc superoxide dismutase were increased together with a decreased mRNA expression and protein levels of glutathione peroxidase (GPx). As a consequence the [Cu/ZnSOD/(catalase+GPx)] activity ratio increases which explains the oxidative stress generated in the cell model. In addition, the expression of thioredoxin 1 and glutaredoxin 1 is decreased. The results obtained show a decreased antioxidant phenotype that correlates with an increased levels of Rcan1 and attrition of telomeres, both related to oxidative stress and cell cycle impairment. Our preliminary results may explain the proneness to a progeroid phenotype.
Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The steroidogenic acute regulatory protein (StAR) transports cholesterol to the mitochondria for steroidogenesis. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH) which is characterized by impaired synthesis of adrenal and gonadal steroids causing adrenal insufficiency, 46,XY disorder of sex development (DSD) and failure of pubertal development. Partial loss of StAR activity may cause adrenal insufficiency only.
A newborn girl was admitted for mild dehydration, hyponatremia, hyperkalemia and hypoglycemia and had normal external female genitalia without hyperpigmentation. Plasma cortisol, 17OH-progesterone, DHEA-S, androstendione and aldosterone were low, while ACTH and plasma renin activity were elevated, consistent with the diagnosis of primary adrenal insufficiency. Imaging showed normal adrenals and cytogenetics revealed a 46,XX karyotype. She was treated with fluids, hydrocortisone and fludrocortisone.
Genetic studies revealed a novel homozygous STAR mutation in the 3' acceptor splice site of intron 4, c.466-1G>A (IVS4-1G>A). To test whether this mutation would affect splicing, we performed a minigene experiment with a plasmid construct containing wild-type or mutant StAR gDNA of exons-introns 4 to 6 in COS-1 cells. The splicing was assessed on total RNA using RT-PCR for STAR cDNAs. The mutant STAR minigene skipped exon 5 completely and changed the reading frame. Thus, it is predicted to produce an aberrant and shorter protein (p.V156GfsX19). Computational analysis revealed that this mutant protein lacks wild-type exons 5 to 7 which are essential for StAR-cholesterol interaction.
STAR c.466-1A skips exon 5 and causes a dramatic change in the C-terminal sequence of the protein, which is essential for StAR-cholesterol interaction. This splicing mutation is a loss-of-function mutation explaining the severe phenotype of our patient. Thus far, all reported splicing mutations of STAR cause a severe impairment of protein function and phenotype. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Steroidogenic factor-1 (SF-1/NR5A1) is a nuclear receptor that regulates adrenal and reproductive development and function. NR5A1 mutations have been detected in 46,XY individuals with disorders of sexual development (DSD) but apparently normal adrenal function and in 46,XX women with normal sexual development yet primary ovarian insufficiency (POI).
A group of 100 46,XY DSD and two POI was studied for NR5A1 mutations and their impact.
Clinical, biochemical, histological, genetic, and functional characteristics of the patients with NR5A1 mutations are reported.
Patients were referred from different centers in Spain, Switzerland, and Turkey. Histological and genetic studies were performed in Barcelona, Spain. In vitro studies were performed in Bern, Switzerland.
A total of 65 Spanish and 35 Turkish patients with 46,XY DSD and two Swiss 46,XX patients with POI were investigated.
Ten novel heterozygote NR5A1 mutations were detected and characterized (five missense, one nonsense, three frameshift mutations, and one duplication).
The novel NR5A1 mutations were tested in vitro by promoter transactivation assays showing grossly reduced activity for mutations in the DNA binding domain and variably reduced activity for other mutations. Dominant negative effect of the mutations was excluded. We found high variability and thus no apparent genotype-structure-function-phenotype correlation. Histological studies of testes revealed vacuolization of Leydig cells due to fat accumulation.
SF-1/NR5A1 mutations are frequently found in 46,XY DSD individuals (9%) and manifest with a broad phenotype. Testes histology is characteristic for fat accumulation and degeneration over time, similar to findings observed in patients with lipoid congenital adrenal hyperplasia (due to StAR mutations). Genotype-structure-function-phenotype correlation remains elusive.
The Journal of Clinical Endocrinology and Metabolism 05/2012; 97(7):E1294-306. · 6.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular causes of isolated severe growth hormone deficiency (ISGHD) in several genes have been established. The aim of this study was to analyse the contribution of growth hormone-releasing hormone receptor (GHRHR) gene sequence variation to GH deficiency in a series of prepubertal ISGHD patients and to normal adult height.
A systematic GHRHR gene sequence analysis was performed in 69 ISGHD patients and 60 normal adult height controls (NAHC). Four GHRHR single-nucleotide polymorphisms (SNPs) were genotyped in 248 additional NAHC. An analysis was performed on individual SNPs and combined genotype associations with diagnosis in ISGHD patients and with height-SDS in NAHC.
Twenty-one SNPs were found. P3, P13, P15 and P20 had not been previously described. Patients and controls shared 12 SNPs (P1, P2, P4-P11, P16 and P21). Significantly different frequencies of the heterozygous genotype and alternate allele were detected in P9 (exon 4, rs4988498) and P12 (intron 6, rs35609199); P9 heterozygous genotype frequencies were similar in patients and the shortest control group (heights between -2 and -1 SDS) and significantly different in controls (heights between -1 and +2 SDS). GHRHR P9 together with 4 GH1 SNP genotypes contributed to 6·2% of height-SDS variation in the entire 308 NAHC.
This study established the GHRHR gene sequence variation map in ISGHD patients and NAHC. No evidence of GHRHR mutation contribution to ISGHD was found in this population, although P9 and P12 SNP frequencies were significantly different between ISGHD and NAHC. Thus, the gene sequence may contribute to normal adult height, as demonstrated in NAHC.
[Show abstract][Hide abstract] ABSTRACT: One hundred and forty-six index patients with 46,XY DSD in whom gonads were confirmed as testes were consecutively studied for a molecular diagnosis during the period 2002-2010. AR gene was analysed in all patients as the first candidate gene, yielding a mutation in 42.5% of cases and SRD5A2 gene was analysed as the second candidate gene, resulting in the characterization of 10 different mutations (p.Y91D, p.G115D, p.Q126R, p.R171S, p.Y188CfsX9, p.N193S, p.A207D, p.F219SfsX60, p.R227Q and p.R246W) in nine index patients (6.2% of the total number of 46,XY DSD patients). One of the mutations (p.Y188CfsX9) has never been reported. In addition, we genotyped SRD5A2 gene p.V89L and c.281+15T>C polymorphisms in 46,XY DSD and in 156 normal adult males and found that patients with SRD5A2 mutations or without a known molecular diagnosis presented a higher frequency of homozygous p.L89, homozygous TT and combined CCTT genotypes compared with controls. This result suggests that 46,XY DSD patient phenotypes may be influenced by SRD5A2 polymorphism genotypes. SRD5A2 gene mutations may not be as infrequent as previously considered in 46,XY DSD patients with variable degrees of external genitalia virilization at birth and normal T production and appears to be the second aetiology in our series.
International Journal of Andrology 06/2011; 34(6 Pt 2):e526-35. · 3.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Androgen receptor (AR) gene mutations are the most frequent cause of 46,XY disorders of sex development (DSD) and are associated with a variety of phenotypes, ranging from phenotypic women [complete androgen insensitivity syndrome (CAIS)] to milder degrees of undervirilization (partial form or PAIS) or men with only infertility (mild form or MAIS).
The aim of the study was to characterize the contribution of the AR gene to the molecular cause of 46,XY DSD in a series of Spanish patients.
We studied a series of 133 index patients with 46,XY DSD in whom gonads were differentiated as testes, with phenotypes including varying degrees of undervirilization, and in whom the AR gene was the first candidate for a molecular analysis.
The AR gene was sequenced (exons 1 to 8 with intronic flanking regions) in all patients and in family members of 61% of AR-mutated gene patients.
AR gene mutations were found in 59 individuals (44.4% of index patients), of whom 46 (78%) were CAIS and 13 (22%) PAIS. Fifty-seven different mutations were found: 21.0% located in exon 1, 15.8% in exons 2 and 3, 57.9% in exons 4-8, and 5.3% intronic. Twenty-three mutations (40.4%) had been previously described and 34 (59.6%) were novel.
AR gene mutation is the most frequent cause of 46,XY DSD, with a clearly higher frequency in the complete phenotype. Mutations spread along the whole coding sequence, including exon 1. This series shows that 60% of mutations detected during the period 2002-2009 were novel.
The Journal of Clinical Endocrinology and Metabolism 02/2010; 95(4):1876-88. · 6.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell proliferation and gene expression regulation were studied in human fetal epiphyseal chondrocytes to ascertain the involvement of GH-IGF axis components in human fetal growth regulation by 1,25-dihydroxyvitamin D(3) (VitD) and growth hormone (GH).
Chondrocytes from primary cultures were plated in serum-free medium for 48 h and incubated for a further 48 h with VitD (10(-11) to 10(-6)M) and/or IGF-I (100 ng/ml) and/or GH (500 ng/ml). We analyzed (3)H-thymidine incorporation into DNA and IGF-I, IGFBP-3, GHR, SOX9, COL2A1, aggrecan and COMP gene expression by real-time quantitative PCR.
VitD dose-dependently and significantly inhibited (3)H-thymidine incorporation whereas GH had no effect on proliferation and, when combined with VitD, the same inhibition was observed as with VitD alone. IGF-I (100 ng/ml) significantly stimulated proliferation and opposed inhibition by VitD. VitD dose-dependently stimulated IGF-I (11.1+/-19.8 at VitD10(-6)M), IGFBP-3 (2.6+/-0.9), GHR (3.8+/-2.8) and COMP (1.5+/-0.6) expression whereas it inhibited SOX9 (0.7+/-0.2), COL2A1 (0.6+/-0.3) and aggrecan (0.6+/-0.2) expression and had no significant effect on IGF-II. IGF-I stimulated IGF-I, IGFBP-3, SOX9, COL2A1 and aggrecan expression and opposed COL2A1 and aggrecan gene expression inhibition by VitD. GH alone had no effect on gene expression whereas, in the presence of VitD, significantly-increased IGF-I expression stimulation was observed above values obtained with VitD alone (17.5+/-7.4).
Our results suggest that VitD regulation of fetal growth cartilage could have consisted of parallel enhancing of cell differentiation and conditioning to a phenotype more sensitive to regulation by other hormones such as GH as shown by increased GHR and IGF-I expression, but not by IGF-II expression which was not regulated.
Growth hormone & IGF research: official journal of the Growth Hormone Research Society and the International IGF Research Society 06/2009; 19(3):232-7. · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The exon 3-deleted/full-length (d3/fl) GH receptor polymorphism (d3/fl-GHR) has been associated with responsiveness to GH therapy in short small-for-gestational-age (SGA) patients, although consensus is lacking. However, its influence on glucose homeostasis, at baseline or under GH therapy, has not been investigated.
Our objective was to evaluate whether the d3/fl-GHR genotypes influence insulin sensitivity in short SGA children before or after puberty onset or during GH therapy.
We conducted a 2-yr prospective, controlled, randomized trial.
Thirty Spanish hospitals participated. Auxological, GH secretion, and glucose homeostasis evaluation was hospital based, whereas molecular analyses and data computation were centralized.
Patients included 219 short SGA children [body mass index sd score (SDS) < or = 2.0]; 159 were prepubertal (group 1), and 60 had entered puberty (group 2).
Seventy-eight patients from group 1 were treated with GH (66 microg/kg.d) for 2 yr (group 3).
Previous and 2-yr follow-up auxological and biochemical data were recorded, d3/fl-GHR genotypes determined, and data analyzed.
In groups 1 and 2, fasting glucose, insulin, homeostasis model assessment (HOMA), and quantitative insulin sensitivity check index (QUICKI) were similar in each d3/fl-GHR genotype. Group 2 glucose, insulin, and HOMA were significantly higher and QUICKI lower than in group 1. In group 3 GH-treated patients, height SDS, growth velocity SDS, fasting glucose, insulin, and HOMA significantly increased as did body mass index SDS at the end of the second year, and QUICKI decreased during the first and second years, with no differences among the d3/fl-GHR genotypes.
In short SGA patients, the d3/fl-GHR genotypes do not seem to influence prepubertal or pubertal insulin sensitivity indexes or their changes over 2 yr of GH therapy (66 mug/kg.d).
[Show abstract][Hide abstract] ABSTRACT: To elucidate the involvement of IGF axis components and the potential effects of glucocorticoids (GCs) in human fetal growth regulation.
We studied the regulation by dexamethasone (Dx) and IGF-I of proliferation and IGF axis components and matrix protein gene expression in human fetal epiphyseal chondrocytes.
High Dx concentration (10(-7)-10(-6)M) inhibited (3)H-thymidine incorporation, mifepristone (MF) 10(-6)M limited inhibition by Dx, and IGF-I (100 ng/ml) significantly stimulated proliferation and completely opposed inhibition by Dx. Dx dose-dependently (10(-9)-10(-6)M) inhibited IGF-I, IGFBP3 and SOX9 gene expression and expression of GHR, COL2A1 and aggrecan from 10(-7)M to 10(-6)M whereas it stimulated IGF-IR expression. By contrast, Dx had no significant effect on IGF-II expression. IGF-I stimulated IGF-I, IGFBP3, SOX9, COL2A1 and aggrecan expression whereas it inhibited IGF-IR expression. IGF-I could oppose COL2A1 and aggrecan gene expression inhibition by Dx.
We demonstrated for the first time by real-time quantitative PCR that human fetal epiphyseal chondrocytes expressed IGF axis components, such as IGF-I, IGF-II, IGFBP3, IGF-IR and GHR and SOX9, COL2A1 and aggrecan, and that their expression was regulated by Dx and IGF-I. Among IGFs, IGF-I and not IGF-II expression was demonstrated to be down-regulated by GCs whereas IGF-I expression was up-regulated by itself.
Growth hormone & IGF research: official journal of the Growth Hormone Research Society and the International IGF Research Society 06/2008; 18(6):497-505. · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Consensus is lacking as to whether the exon 3-deleted (d3)/full-length (fl) GH receptor (GHR) polymorphism is associated with responsiveness to GH therapy.
Our objective was to evaluate, in short, prepubertal, appropriate-for-gestational age (AGA) patients, 2-yr growth response to GH therapy (31.7+/-3.5 microg/kg.d) according to exon 3-deleted/full-length GHR genotypes.
We conducted a retrospective study.
We studied 106 short AGA children, 58 boys and 48 girls, 7.8+/-2.3 yr, (d3/d3 n=18, d3/fl n=42, and fl/fl n=46). The GH response to two provocative stimuli were under 10 ng/ml in 65 and one or both over 10 ng/ml in 41 patients.
Patients were followed by a single clinical team and remained prepubertal during the study. The exon 3-deleted/full-length GHR genotypes were determined and analyzed in the same hospital.
Growth velocity significantly (P<0.0001) increased during the first and second years of therapy, as did height sd score (SDS). These increases were similar in each exon 3-deleted/full-length GHR genotype. Total 2-yr height gain (SDS) did not differ statistically among genotypes: 15.5+/-2.2 cm and 1.2+/-0.5 SDS in d3/d3, 15.9+/-2.0 cm and 1.3+/-0.4 SDS in d3/fl, and 15.4+/-2.1 cm and 1.1+/-0.3 SDS in fl/fl. No significant differences among the three genotypes were found in both sexes or in patients with different GH peak response to provocative stimuli for these parameters. An analysis of previously published studies was also performed.
These results confirm in AGA patients those previously found by us and others in small-for-gestational-age patients and suggest that neither sex nor GH peaks after provocative stimuli might influence significantly the responsiveness to GH therapy according to the exon 3-deleted/full-length GHR genotypes.
[Show abstract][Hide abstract] ABSTRACT: In short small-for-gestational-age (SGA) patients, the exon 3-deleted(d3)/full-length (fl)-GHR polymorphism was associated with responsiveness to GH therapy (30-48 microg/kg.d); however, these results were not confirmed for higher GH doses (56-66 microg/kg.d). We hypothesized that higher doses would mask the lower dose differences.
Our objective was to evaluate, in short SGA patients, 2-yr growth response to GH therapy (32.1 +/- 3.8 microg/kg.d) according to exon d3/fl-GHR genotypes.
This was a 2-yr follow-up study.
There was a total of 60 short SGA children (d3/d3 n = 8, d3/fl n = 23, and fl/fl n = 29). There were 11 children that entered puberty during the second follow-up year. Results were evaluated for all patients (group A1, n = 60, 7.7 +/- 2.7 yr) and for patients who remained prepubertal (group A2, n = 49, 6.9 +/- 2.2 yr).
Patients were followed by a single clinical team, and exon d3/fl-GHR genotypes were determined and analyzed in the same hospital.
In groups A1 and A2, growth velocity significantly (P < 0.0001) increased during the first and second years of therapy, as did height sd score (SDS). These increases were similar in each exon d3/fl-GHR genotype. Total 2-yr height gain (cm, SDS) did not differ statistically among genotypes: group A1, 15.0 +/- 2.0 cm and 1.15 +/- 0.45 SDS in d3/d3, 16.0 +/- 2.4 cm and 1.17 +/- 0.51 SDS in d3/fl, 16.1 +/- 2.4 cm and 1.15 +/- 0.53 SDS in fl/fl; and group A2, 15.4 +/- 2.0 cm and 1.03 +/- 0.42 SDS in d3/d3, 15.6 +/- 2.1 cm and 1.22 +/- 0.51 in d3/fl, and 16.2 +/- 2.6 cm and 1.21 +/- 0.56 SDS in fl/fl.
These results did not confirm our hypothesis and show that, in short SGA children, 2-yr growth response to GH therapy 32.1 +/- 3.8 microg/kg.d was similar for each exon d3/fl-GHR genotype carried, as occurred in our previous study using 66 microkg.d.
[Show abstract][Hide abstract] ABSTRACT: GH1 gene presents a complex map of single nucleotide polymorphisms (SNPs) in the entire promoter, coding and noncoding regions. The aim of the study was to establish the complete map of GH1 gene SNPs in our control normal population and to analyse its association with adult height.
A systematic GH1 gene analysis was designed in a control population of 307 adults of both sexes with height normally distributed within normal range for the same population: -2 standard deviation scores (SDS) to +2 SDS. An analysis was performed on individual and combined genotype associations with adult height.
Twenty-five SNPs presented a frequency over 1%: 11 in the promoter (P1 to P11), three in the 5'UTR region (P12 to P14), one in exon 1 (P15), three in intron 1 (P16 to P18), two in intron 2 (P19 and P20), two in exon 4 (P21 and P22) and three in intron 4 (P23 to P25). Twenty-nine additional changes with frequencies under 1% were found in 29 subjects. P8, P19, P20 and P25 had not been previously described. P6, P12, P17 and P25 accounted for 6.2% of the variation in adult height (P = 0.0007) in this population with genotypes A/G at P6, G/G at P6 and A/G at P12 decreasing height SDS (-0.063 +/- 0.031, -0.693 +/- 0.350 and -0.489 +/- 0.265, Mean +/- SE) and genotypes A/T at P17 and T/G at P25 increasing height SDS (+1.094 +/- 0.456 and +1.184 +/- 0.432).
This study established the GH1 gene sequence variation map in a normal adult height control population confirming the high density of SNPs in a relatively small gene. Our study shows that the more frequent SNPs did not significantly contribute to height determination, while only one promoter and two intronic SNPs contributed significantly to it. Studies in larger populations will have to confirm the associations and in vitro functional studies will elucidate the mechanisms involved. Systematic GH1 gene analysis in patients with growth delay and suspected GH deficiency/insufficiency will clarify whether different SNP frequencies and/or the presence of different sequence changes may be associated with phenotypes in them.
[Show abstract][Hide abstract] ABSTRACT: The d3/fl-GH receptor (d3/fl-GHR, exon 3-deleted/full-length GHR) has recently been associated with responsiveness to GH therapy.
The objective of the study was to evaluate whether the d3/fl-GHR genotypes influence the intensity of spontaneous and/or GH therapy-stimulated growth in small-for-gestational-age (SGA) patients.
This was a 2-yr prospective, controlled, randomized trial.
Thirty Spanish hospitals participated. Auxologic and GH secretion evaluation was hospital based, whereas molecular analyses and auxologic data computation were centralized.
Patients included 170 short SGA children: 140 remained prepubertal and 30 entered puberty during the second follow-up year.
Eighty-six were treated with GH (66 microg/kg.d) for 2 yr and 84 were not treated.
Previous and 2-yr follow-up auxologic data were recorded at each hospital, d3/fl-GHR genotypes determined, and data analyzed for patients who remained prepubertal (group 1, 68 GH treated and 72 non-GH treated) and for all the patients (group 2).
In group 1 GH-treated patients, growth velocity, and height-sd score during the first and second years, total 2-yr height gain (18.5 +/- 2.4 cm in d3/d3; 18.4 +/- 2.6 in d3/fl; 19.5 +/- 2.3 in fl/fl), Delta 2-yr height increase (9.1 +/- 2.4 cm in d3/d3; 9.4 +/- 3.0 in d3/fl; 10.4 +/- 2.1 in fl/fl), first-year growth prediction and studentized residual values (0.08 +/- 1.26 in d3/d3; 0.28 +/- 1.21 in d3/fl; 0.67 +/- 0.95 in fl/fl) did not differ among the d3/fl-GHR genotypes. In group 1 non-GH-treated patients, neither growth velocity nor height-sd score changed significantly, and values were similar in each d3/fl-GHR genotype. Results in all patients (group 2) were similar to those in group 1.
In short non-GH-deficient SGA children, both spontaneous growth rate and responsiveness to 66 microg/k.d GH therapy were similar for each d3/fl-GHR genotype carried.
[Show abstract][Hide abstract] ABSTRACT: In order to assess whether vitamin D receptor gene polymorphisms are involved in the genetic regulation of type 1 diabetes susceptibility, a case-control study was conducted in two Spanish populations with different genetic backgrounds.
155 patients with childhood-onset type 1 diabetes and 280 healthy controls from Barcelona, and 89 patients and 116 controls from Navarre were studied for vitamin D receptor gene polymorphisms in peripheral blood DNA. Intron 8 (BsmI) and exon 2 (FokI) segments were amplified by PCR and sequenced to determine each corresponding genotype. Differences for allele, genotype and combined haplotype and genotype distribution between patients and controls within each population and between the two populations were analyzed.
BsmI genotype and allele frequencies showed a tendency towards increased bb genotype and b allele frequencies in Barcelona patients and the tendency was inverse in Navarre. FokI polymorphism distribution analysis showed a significant decrease in ff genotype (p = 0.016) in patients versus controls from Navarre. Combined genotypes showed homozygous bb/FF genotype to be increased in Barcelona patients (p = 0.04) whereas homozygous bb/ff genotype was decreased in Navarre patients (p = 0.02) versus their corresponding controls. BF haplotype frequency distribution between patients and controls was inverse and significantly different between Barcelona and Navarre (p = 0.04).
Combined genotypes for vitamin D receptor gene polymorphisms at intron 8 and exon 2 suggest that the more active form of vitamin D receptor gene (FF genotype) can be increased in Mediterranean diabetic patients whereas the less active form (ff genotype) can be decreased in those from Navarre. Our results suggest that, in both groups, the F allele of exon 2 VDR gene polymorphism may increase type 1 diabetes susceptibility.
[Show abstract][Hide abstract] ABSTRACT: Background and objective
In order to assess whether vitamin D receptor gene polymorphisms are involved in the genetic regulation of type 1 diabetes susceptibility, a case-control study was conducted in two Spanish populations with different genetic backgrounds
Patients and method
155 patients with childhood-onset type 1 diabetes and 280 healthy controls from Barcelona, and 89 patients and 116 controls from Navarre were studied for vitamin D receptor gene polymorphisms in peripheral blood DNA. Intron 8 (BsmI) and exon 2 (FokI) segments were amplified by PCR and sequenced to determine each corresponding genotype. Differences for allele, genotype and combined haplotype and genotype distribution between patients and controls within each population and between the two populations were analyzed
BsmI genotype and allele frequencies showed a tendency towards increased bb genotype and b allele frequencies in Barcelona patients and the tendency was inverse in Navarre. FokI polymorphism distribution analysis showed a significant decrease in ff genotype (p = 0.016) in patients versus controls from Navarre. Combined genotypes showed homozygous bb/FF genotype to be increased in Barcelona patients (p = 0.04) whereas homozygous bb/ff genotype was decreased in Navarre patients (p = 0.02) versus their corresponding controls. BF haplotype frequency distribution between patients and controls was inverse and significantly different between Barcelona and Navarre (p = 0.04)
Combined genotypes for vitamin D receptor gene polymorphisms at intron 8 and exon 2 suggest that the more active form of vitamin D receptor gene (FF genotype) can be increased in Mediterranean diabetic patients whereas the less active form (ff genotype) can be decreased in those from Navarre. Our results suggest that, in both groups, the F allele of exon 2 VDR gene polymorphism may increase type 1 diabetes susceptibility
[Show abstract][Hide abstract] ABSTRACT: A patient with male pseudohermaphroditism and clinical diagnosis of partial androgen insensitivity in the neonatal period was studied at pubertal age for a molecular diagnosis. Hormone studies were conducted at baseline and under hCG stimulation for testosterone and dihydrotestosterone determinations at 2 months of age. Gonadectomy was performed at 4 months. At the age of 13 years genital skin fibroblasts were studied for androgen binding and 5alpha-reductase activity and peripheral blood DNA was available for androgen receptor (AR) and 5alpha-reductase (SRD5A2) gene analysis. Exons 1-8 of AR gene and exons 1-5 of SRD5A2 gene were sequenced. AR gene coding sequences were normal. SRD5A2 gene analysis revealed two heterozygote mutations (G115D and R246W), with the mother carrying the G115D and the father the R246W mutations. The compound heterozygote mutations in SRD5A2 gene explained an extremely low 5alpha-reductase enzyme activity in genital skin fibroblasts. Revision of hormonal data from the neonatal period revealed an increased testosterone-to-dihydrotestosterone ratio at the end of an hCG stimulation test, which concurred with the molecular diagnosis. Testis morphology at 4 months of age was normal. Clinical and biochemical differential diagnosis between partial androgen insensitivity syndrome and 5alpha-reductase enzyme deficiency is difficult in the neonatal period and before puberty. Our results show that in our patient the testosterone-to-dihydrotestosterone ratio would have adequately orientated the diagnosis. The two mutations in SRD5A2 gene have been described in patients of different lineages, though not in combination to date. Testis morphology showed that, during early infancy, the 5alpha-reductase deficiency may not have affected interstitial or tubular development.
Hormone Research 02/2004; 62(5):259-64. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is a crucial event in endochondral ossification. Chemoattractants and mitogens for endothelial cells (such as basic fibroblast growth factor [bFGF] and transforming growth factor beta [TGF-beta]), which act as local regulators of the process, are synthesized by chondrocytes under several stimuli and in relation to the differentiation stage of the cartilage. Vascular endothelial growth factor (VEGF) is a 44-kDa protein well known as a potent angiogenic molecule owing to its mitogenic and permeability-causing properties. In this work, VEGF was located by immunohistochemistry in growth plate cartilage of human fetuses (20-22 weeks old) and its expression was demonstrated by reverse-transcription polymerase chain reaction (RT-PCR). Primary culture of human fetal epiphyseal chondrocytes (HFEC) maintained VEGF expression at protein and messenger RNA (mRNA) levels and this expression was stimulated by cartilage-promoting growth factors incorporated into the culture media (rFGF-b, rTGF-beta1, and insulin-like growth factor [rFGF-b] at 50 ng/ml). The conditioned medium (CM) of HFEC stimulated the proliferation of endothelial cells, and this was partially blocked by anti-VEGF antibody. These studies showed VEGF production by chondrocytes of the epiphyseal growth cartilage and suggested a role of this factor in cartilage physiology and the angiogenic process.
Journal of Bone and Mineral Research 04/2000; 15(3):534-40. · 6.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proliferation and differentiation of chondrocytes from growth cartilage are modulated by hormones and growth factors, among which TGF-betas have been recognized as some of the more potent regulators although their specific cell effects on cartilage physiology are not fully understood. Primary human fetal epiphyseal chondrocytes (HEFC) constitutively produce TGF-beta1 at different times of culture progression. Treatment of 48-h. serum-deprived semiconfluent HFEC with 0.1-50 ng/ml of TGF-beta1 for 48-h. decreased (3H)Thymidine incorporation by 25-50 % and cell number by 25 %. In addition, IGFBP-3, the main insulin-like bonding protein produced by HFEC, showed a slight increase by TGF-beta1 in culture media. The changes in IGFBP-3 protein levels correlated well with its mRNA, indicating that TGF-beta1 is able to up-regulate IGFBP-3 synthesis in chondrocytes. Nevertheless, the IGFBP-3 accumulation in culture media does not produce a clear growth inhibitory effect on chondrocytes. Thus, we conclude that even though TGF-beta1 is able to up-regulate IGFBP-3, the growth inhibitory action produced by TGF-beta1 is not mediated by IGFBP-3 increase and appears to be mainly a direct TGF-beta1 effect on HFEC.