Analysis of IGF(CA)19 and IGFBP3-202A/C gene polymorphisms in patients with acromegaly: association with clinical presentation and response to treatments.

OBJECTIVE: IGF1 and IGFBP3 gene polymorphisms have been recently described. However, their potential role in the setting of acromegaly and its outcome is unknown. In this study, we analyze these polymorphisms in patients with acromegaly and investigate their association with clinical presentation and response to treatments. DESIGN: A retrospective observational study was conducted in patients with acromegaly to analyze IGF1 and IGFBP3 gene polymorphisms. METHODS: A total of 124 patients with acromegaly (57.3% women, mean age 44.9±13.1 years old) were followed up for a period of 11.4±8.0 years in eight tertiary referral hospitals in Spain. Clinical and analytical data were evaluated at baseline and after treatment. IGF1 and IGFBP3 gene polymorphisms were analyzed using PCR and specific primers. RESULTS: Baseline laboratory test results were GH 19.3 (8.0-39.6) ng/ml, nadir GH 11.8 (4.1-21.5) ng/ml, and index IGF1 2.65±1.25 upper limit of normal. Regarding the IGF1 gene polymorphism, we did not find any association between the number of cyto-adenosine (CA) repeats and patients' baseline characteristics. Nevertheless, a trend for higher nadir GH values was observed in patients with <19 CA repeats. Regarding the IGFBP3 polymorphism, the absence of an A allele at the -202 position was associated with a higher baseline IGF1 and a higher prevalence of cancer and polyps. There were no differences in response to therapies according to the specific genotypes. CONCLUSIONS: Polymorphisms in the IGF1 and IGFBP3 genes may not be invariably determinant of treatment outcome in acromegalic patients, but they may be associated with higher nadir GH levels or baseline IGF1, and determine a higher rate of colorectal polyps and cancer.

LIF, a Novel STAT5-Regulated Gene, Is Aberrantly Expressed in Myeloproliferative Neoplasms.

A search for genes potentially regulated by STAT5 identified leukemia inhibitory factor (LIF) as a good candidate. Using various experimental approaches, we have validated LIF as a direct transcriptional target of STAT5 in myeloid cell lines: STAT5 binds to LIF promoter, and LIF expression is increased after activation of the JAK2/STAT5 pathway. We also found that LIF expression is significantly increased in patients with chronic myeloproliferative neoplasms with and without activating mutations of the pathway, indicating that LIF might play an important role in STAT5-mediated oncogenesis.

Pituitary stalk dysgenesis-induced hypopituitarism in adult patients: prevalence, evolution of hormone dysfunction and genetic analysis.

OBJECTIVES: To investigate the prevalence of pituitary stalk dysgenesis (PSD) in adult hypopituitary patients by describing the chronology of hormone deficiencies and their potential correlation with traumatic delivery, mutations in genes required for pituitary development and function and pituitary stalk visibility on MRI. DESIGN: Retrospective and prospective study involving 231 hypopituitary patients, including 26 diagnosed with PSD. Clinical, biochemical and radiological studies were reviewed. Molecular analyses of HESX1, LHX4,PROP1 and POU1F1 genes were performed prospectively. RESULTS: PSD was present in 11.2% of hypopituitary patients. PSD was diagnosed before 14 years of age in 46.2% of cases, between 14 and 18 years of age in 23%, and in adulthood in 30.8%. Perinatal complications or gene mutations were present in 26.9 and 4.3% of patients, respectively. At first assessment, 92.3% of patients had growth hormone (GH) deficiency. 26.9% presented as combined pituitary deficiencies and 7.6% as panhypopituitarism. Hormone deficiencies were progressive during follow-up in 84.6%. 96% progressed to multiple deficiencies and 46% to panhypopituitarism. No significant association was found between hormonal dysfunction and previous perinatal damage or breech delivery (p = 0.17), PROP1 mutations (p = 0.26) or pituitary stalk visibility on MRI (p = 0.52). No mutations in POU1F1, HESX1 and LHX-4 genes were detected. CONCLUSION: In this study, PSD prevalence in adult hypopituitary patients was 11.2%. Typical clinical presentation includes isolated or combined pituitary hormone deficiencies during the pediatric age, which usually progress to combined or complete hypopituitarism in adulthood. Phenotype is highly variable depending on hormone profile and age at onset.

Genetic study of the hepcidin gene (HAMP) promoter and functional analysis of the c.-582A > G variant.

BACKGROUND: Hepcidin acts as the main regulator of iron homeostasis through regulation of intestinal absorption and macrophage release. Hepcidin deficiency causes iron overload whereas its overproduction is associated with anaemia of chronic diseases. The aims of the study were: to identify genetic variants in the hepcidin gene (HAMP) promoter, to asses the associations between the variants found and iron status parameters, and to functionally study the role on HAMP expression of the most frequent variant. RESULTS: The sequencing of HAMP promoter from 103 healthy individuals revealed two genetic variants: The c.-153C > T with a frequency of 0.014 for allele T, which is known to reduce hepcidin expression and the c.-582A > G with a 0.218 frequency for allele G. In an additional group of 224 individuals, the c.-582A > G variant genotype showed no association with serum iron, transferrin or ferritin levels.The c.-582G HAMP promoter variant decreased the transcriptional activity by 20% compared to c.-582A variant in cells from the human hepatoma cell line HepG2 when cotransfected with luciferase reporter constructs and plasmid expressing upstream stimulatory factor 1 (USF1) and by 12-14% when cotransfected with plasmid expressing upstream stimulatory factor 2 (USF2). CONCLUSIONS: The c.-582A > G HAMP promoter variant is not associated with serum iron, transferrin or ferritin levels in the healthy population. The in vitro effect of the c.-582A > G variant resulted in a small reduction of the gene transactivation by allele G compared to allele A. Therefore the effect of the variant on the hepcidin levels in vivo would be likely negligible. Finally, the c.-153C > T variant showed a frequency high enough to be considered when a genetic analysis is done in iron overload patients.

Pegvisomant-induced liver injury is related to the UGT1A1*28 polymorphism of Gilbert's syndrome.

CONTEXT: Pegvisomant (PEG) therapy has been associated with drug-induced liver dysfunction in acromegalic patients. The mechanism of its toxicity remains unknown. OBJECTIVE: The primary objective was to determine whether or not the UGT1A1*28 polymorphism associated with Gilbert's syndrome influences the development of liver dysfunction during PEG treatment. DESIGN AND SETTING: A cross-sectional study was conducted in four Spanish university hospitals. PATIENTS: Thirty-six acromegalic patients with active disease, resistant to somatostatin analogs, participated. RESULTS: The prevalence of the UGT1A1*28 homozygous and heterozygous genotypes in acromegalic patients was 14 and 44%, respectively. Ten patients (28%) developed liver function test (LFT) abnormalities. There was a tendency for more frequent liver function abnormalities in males (70% males vs. 30% females, P = 0.058). Carriers of the UGT1A1*28 polymorphism had a higher incidence of LFT abnormalities than the UGT1A1 wild type (43% carriers vs. 7% wild type, P = 0.024). This difference persisted when adjusted in an all-factors multiple regression analysis [coefficient of determination (R(2)) = 0.463; P = 0.008] for age, gender, alcohol consumption, and UGT1A1*28 polymorphism. A stepwise multivariate likelihood binary logistic regression analysis (R(2) = 0.40; P = 0.003) identified male gender (beta = 7.21; P = 0.033) and UGT1A1*28 polymorphism (beta = 14.1; P = 0.028) as the only significant predictors for the development of LFT abnormalities. CONCLUSIONS: The UGT1A1*28 genotype and male gender predict an increased incidence of LFT abnormalities during PEG therapy in acromegaly.

The exon 3-deleted growth hormone receptor is associated with better response to pegvisomant therapy in acromegaly.

CONTEXT: The deletion of exon 3 in the GH receptor (GHR) has been associated with a different biochemical picture and response to therapy in acromegaly. OBJECTIVE: The aim of the study was to determine whether or not the GHR genotype influences the efficacy of pegvisomant treatment. DESIGN AND SETTING: A cross-sectional study was conducted in six Spanish university hospitals. PATIENTS: Forty-four acromegalic patients with active disease and resistance to somatostatin analogs participated in the study. RESULTS: The prevalence of the full-length GHR and the exon 3-deleted GHR homozygous and heterozygous genotypes was 41, 2, and 57%, respectively. There were no differences in IGF-I or GH pre-pegvisomant levels related to GHR genotype. The exon 3-deleted patients required approximately 20% lower doses of pegvisomant per kilogram of weight (28 +/- 11 compared to 22 +/- 7 mg per kg of weight; P = 0.033) to normalize IGF-I. A stepwise multivariate linear regression analysis (R(2) = 0.27; P = 0.003) identified male gender (beta = -0.79; P = 0.03) and d3-GHR genotype (beta = -0.64; P = 0.007) as the only significant predictors of the dose of pegvisomant per kilogram of weight. In addition, d3-GHR carriers required fewer months for IGF-I normalization (P < 0.01). A stepwise multivariate linear regression analysis (R(2) = 0.40; P = 0.001) revealed that the only significant predictor of the time to IGF-I normalization was the dose of pegvisomant per kilogram of weight (beta = 0.451; P = 0.001). CONCLUSIONS: The exon 3 deletion in the GHR predicts an improved response to pegvisomant therapy in acromegaly.

The role of inbreeding in the extinction of a European royal dynasty.

The kings of the Spanish Habsburg dynasty (1516-1700) frequently married close relatives in such a way that uncle-niece, first cousins and other consanguineous unions were prevalent in that dynasty. In the historical literature, it has been suggested that inbreeding was a major cause responsible for the extinction of the dynasty when the king Charles II, physically and mentally disabled, died in 1700 and no children were born from his two marriages, but this hypothesis has not been examined from a genetic perspective. In this article, this hypothesis is checked by computing the inbreeding coefficient (F) of the Spanish Habsburg kings from an extended pedigree up to 16 generations in depth and involving more than 3,000 individuals. The inbreeding coefficient of the Spanish Habsburg kings increased strongly along generations from 0.025 for king Philip I, the founder of the dynasty, to 0.254 for Charles II and several members of the dynasty had inbreeding coefficients higher than 0.20. In addition to inbreeding due to unions between close relatives, ancestral inbreeding from multiple remote ancestors makes a substantial contribution to the inbreeding coefficient of most kings. A statistically significant inbreeding depression for survival to 10 years is detected in the progenies of the Spanish Habsburg kings. The results indicate that inbreeding at the level of first cousin (F = 0.0625) exerted an adverse effect on survival of 17.8%+/-12.3. It is speculated that the simultaneous occurrence in Charles II (F = 0.254) of two different genetic disorders: combined pituitary hormone deficiency and distal renal tubular acidosis, determined by recessive alleles at two unlinked loci, could explain most of the complex clinical profile of this king, including his impotence/infertility which in last instance led to the extinction of the dynasty.

Functional characterization of three CYP21A2 sequence variants (p.A265V, p.W302S, p.D322G) employing a yeast co-expression system.

Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase (CYP21A2) deficiency is the commonest inborn error in steroid hormone biosynthesis. Functional in vitro assessment of mutant activity generally correlates well with clinical phenotype and therefore has contributed greatly to phenotype prediction in this CAH variant. Three CYP21A2 sequence variants (g.1641C>T, p.A265V; g.1752G>C, p.W302S; and g.2012A>G, p.D322G) identified in patients with non-classic and simple virilizing CAH were characterized using a yeast co-expression system and a computational three-dimensional CYP21A2 model. Computational analysis of the mutants in the three-dimensional structural model predicted no relevant effect of p.A265V, while p.W302S and p.D322G were predicted to impact significantly on enzyme function. Consistent with these findings, in vitro mutant analysis revealed enzyme activity similar to wild-type for p.A265V, whereas p.W302S and p.D322G exerted activities compatible with simple virilizing and non-classical CAH, respectively. The results indicate that p.A265V is an allelic variant rather than a disease-causing amino acid change, whilst p.W302S and p.D322G could be confirmed as functionally relevant mutations. These findings emphasize the value of in vitro functional analysis of sequence variations in predicting genotype-phenotype correlations and disease severity.

Serum TNF-alpha levels in relation to alcohol consumption and common TNF gene polymorphisms.

Tumor necrosis factor-alpha (TNF-alpha) mediates alcohol-induced organ dysfunction, including alcoholic hepatitis. Variations in the TNF-alpha gene may underlie the individual predisposition to alcoholic liver disease. Measurement of serum TNF-alpha levels has become a routine in clinical practice. The study was aimed at investigating the level of serum TNF-alpha levels in adults and analyzing its relationship with different levels of alcohol consumption, as well as the potential interaction between alcohol consumption and common TNF-alpha gene polymorphisms in relation to TNF-alpha levels and liver disease. Serum TNF-alpha was measured in a random sample of 459 individuals from a general adult population and in 137 hospital-admitted alcoholics. Three common TNF-alpha gene polymorphisms (-238G> A, -308G> A, and -857C> T) were investigated in 419 of these individuals. In the general adult population, the TNF-alpha levels were similar in alcohol abstainers and alcohol drinkers. Alcoholics admitted to the hospital showed the highest TNF-alpha levels, which were correlated with liver dysfunction. We found no evidence of an interaction between alcohol consumption and TNF-alpha gene polymorphisms in relation to TNF-alpha levels. Carriers of the TNF -238A allele tended to have a higher prevalence of advanced liver disease than -238G homozygotes, confirming previous reports. In conclusion, light-to-moderate drinking had no significant effect on the levels of serum TNF-alpha levels. Serum TNF-alpha levels are elevated in alcoholics independently of common TNF gene polymorphisms.

High frequency of copy number variations and sequence variants at CYP21A2 locus: implication for the genetic diagnosis of 21-hydroxylase deficiency.

BACKGROUND: The systematic study of the human genome indicates that the inter-individual variability is greater than expected and it is not only related to sequence polymorphisms but also to gene copy number variants (CNVs). Congenital Adrenal Hyperplasia due to 21-hydroxylase deficiency (21OHD) is the most common autosomal recessive disorder with a carrier frequency of 1:25 to 1:10. The gene that encodes 21-hydroxylase enzyme, CYP21A2, is considered to be one of the most polymorphic human genes. Copy number variations, such as deletions, which are severe mutations common in 21OHD patients, or gene duplications, which have been reported as rare events, have also been described. The correct characterization of 21OHD alleles is important for disease carrier detection and genetic counselling METHODOLOGY AND FINDINGS: CYP21A2 genotyping by sequencing has been performed in a random sample of the Spanish population, where 144 individuals recruited from university students and employees of the hospital were studied. The frequency of CYP21A2 mutated alleles in our sample was 15.3% (77.3% were mild mutations, 9% were severe mutations and 13.6% were novel variants). Gene dosage assessment was also performed when CYP21A2 gene duplication was suspected. This analysis showed that 7% of individuals bore a chromosome with a duplicated CYP21A2 gene, where one of the copies was mutated. CONCLUSIONS: As far as we know, the present study has shown the highest frequency of 21OHD carriers reported by a genotyping analysis. In addition, a high frequency of alleles with CYP21A2 duplications, which could be misinterpreted as 21OHD alleles, was found. Moreover, a high frequency of novel genetic variations with an unknown effect on 21-hydroxylase activity was also found. The high frequency of gene duplications, as well as novel variations, should be considered since they have an important involvement in carrier testing and genetic counseling.

High variability in CYP21A2 mutated alleles in Spanish 21-hydroxylase deficiency patients, six novel mutations and a founder effect.

OBJECTIVE: To detect common as well as rare and novel CYP21A mutations in 21-hydroxylase deficiency patients. To estimate the distribution of mutations and compare them with other European studies. To construct haplotypes linked to a recurrent novel mutation. DESIGN: Genetic analysis by sequencing the entire CYP21A2 gene plus Southern blot. PATIENTS: A total of 138 unrelated Spanish patients: 122 nonclassical forms (NCF) and 16 classical forms (CF) were studied. RESULTS: Among the 266 nonrelated mutated alleles; CYP21A2 deletions/conversions and a spectrum of 27 different mutated alleles were found: 15 different single point mutations, 8 nucleotide deletions in exon 3, 3 mutation clusters in exon 6, 9 alleles with more than one mutation, one 21-nucleotide duplication in exon 10, and one allele with CYP21A2 duplicated and both copies mutated. The most frequent mutation in NCF alleles is V281L (71.8%). Among CFs, the most common is I2 g (20%) and Q318X (16%) and rare alleles (21.9%). Six novel causative mutations were found, four associated with CF: I46+1nt, R444X, P463L and M473_R479dup and two associated with NCF: W302 and D322G. The R444X mutation was found in seven unrelated patients and it appeared only once in an ancestral haplotype. In addition, we found a novel single nucleotide polymorphism with a 31.5% frequency for the rare allele. CONCLUSION: A great diversity of haplotypes with a large spectrum of mutated alleles was found. The frequency of the V281L mutation was the highest reported and the relatively high frequency of R444X was the result of a founder effect.

Gene by environment interaction: the -159C/T polymorphism in the promoter region of the CD14 gene modifies the effect of alcohol consumption on serum IgE levels.

BACKGROUND: Serum IgE is increased in heavy drinkers. Endotoxin mediates most of the immunological alterations associated with heavy drinking. The -159C/T polymorphism in the promoter region of the gene encoding CD14 (an endotoxin receptor) is associated with serum IgE levels in different populations. AIM: To investigate the possible interaction between alcohol intake and the -159C/T polymorphism in the promoter region of the CD14 gene for serum IgE levels. METHODS: A total of 415 individuals (51.6% males, median age 50 years, range 18-92 years) were studied. A total of 140 individuals were alcohol abstainers, 112 were moderate drinkers (1-280 g/week), and 163 were heavy drinkers (>280 g/week). Main determinations included the CD14/-159C/T genotype, a panel of skin prick tests, total serum IgE, and specific serum IgE against common aeroallergens (Phadiatop test). RESULTS: Heavy drinking was associated with increased total serum IgE values and with positive specific serum IgE to common aeroallergens, but the association was stronger in carriers of the CD14/-159C allele (either CC homozygotes or CT heterozygotes) than in CD14/-159TT homozygotes. Both additive and multiplicative interactions between heavy drinking and the CD14/-159C allele for total and specific serum IgE values was still present after adjusting for potential confounders. Neither alcohol consumption nor the CD14/-159 genotype was associated with skin prick test positivity. CONCLUSIONS: The CD14/-159C/T polymorphism modifies the effect of alcohol consumption on serum IgE levels.

Kallmann's syndrome with a novel missense mutation in the KAL1 gene that modifies the major cell adhesion site of the anosmin-1 protein.

Kallmann's syndrome (KS) refers to the association of hypogonadic hypogonadism and anosmia or hyposmia. The X-linked form of the disease is due to mutations in the KAL1 gene that encodes for the protein anosmin-1. We studied the KAL1 gene in a patient with KS and his family by PCR amplification and direct sequencing. A novel missense mutation (V263G) that modifies the major cell adhesion site of the anosmin-1 protein was identified. Our results suggest that this reported mutation is responsible for KS and might help to elucidate the function of an important area of the anosmin-1 protein.

The -159C/T polymorphism in the promoter region of the CD14 gene is associated with advanced liver disease and higher serum levels of acute-phase proteins in heavy drinkers.

BACKGROUND: Innate inflammatory responses to endotoxin (lipopolysaccharide) contribute to the development of alcoholic liver disease (ALD). A single-nucleotide polymorphism (-159C/T) in the promoter region of the gene coding for CD14 (a lipopolysaccharide receptor) could be associated with the development of ALD. We sought too investigate the relationship between the CD14/-159C/T polymorphism and advanced ALD and acute-phase protein levels in heavy drinkers. METHODS: A total of 138 heavy drinkers consecutively admitted to an Internal Medicine department were genotyped for the CD14/-159C/T polymorphism. Serum samples were analyzed for lipopolysaccharide-binding protein (LBP), soluble CD14 (sCD14), C-reactive protein (CRP), and immunoglobulin (Ig) A, IgG, and IgM. Patients with ascites or liver encephalopathy (n = 35) were classified as having advanced ALD. RESULTS: After adjusting for potential confounding variables, the CD14/-159TT genotype was positively associated with advanced ALD (odds ratio, 2.99; 95% confidence interval, 1.09-8.24, p = 0.03) and serum LBP (p = 0.01) and sCD14 (p = 0.04) levels. The CD14/-159C/T polymorphism was not associated with serum levels of CRP, IgA, IgG, or IgM. CONCLUSIONS: Our results support the notion that CD14/-159TT homozygous heavy drinkers have higher levels of the LPS-binding acute-phase proteins (LBP and sCD14) than do carriers of the CD14/-159C allele. Also, the CD14/-159TT genotype may be a risk factor for advanced ALD.

Relation of tumor necrosis factor (TNF) gene polymorphisms with serum concentrations and in vitro production of TNF-alpha and interleukin-8 in heavy drinkers.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) play a role in the pathogenesis of alcoholic hepatitis. The aim of the current study was to investigate the possible relation of TNF gene polymorphisms with TNF-alpha and IL-8 synthesis in heavy drinkers. Nineteen heavy drinkers and 14 healthy control subjects were included in the study. Investigations included (a) polymorphisms in the TNF promoter region at positions -238 (G-->A), -308 (G-->A), -857 (C-->T), and -863 (C-->A), as well as a biallelic Ncol restriction fragment length polymorphism in the first intron of the close TNF-beta gene; (b) serum TNF-alpha and IL-8 concentrations; and (c) TNF-alpha and IL-8 production by phytohemagglutinin A-stimulated peripheral blood mononuclear cells. In comparison with findings for control subjects, heavy drinkers showed higher TNF-alpha production, higher IL-8 production, and higher serum IL-8 concentrations. Increased serum TNF-alpha concentrations were specifically found in heavy drinkers with the -857 (C-->T) substitution (CT heterozygotes), therefore indicating an interaction between alcohol consumption and that polymorphism on serum TNF-alpha concentrations.

Novel mutation involving the translation initiation codon of the growth hormone receptor gene (GHR) in a patient with Laron syndrome.

Laron syndrome (LS) or growth hormone (GH) insensitivity syndrome (GHIS) is an autosomal recessive disease due to molecular defects in the GH receptor gene (GHR). Most of the identified mutations are located on the extracelular domain of the receptor. We studied the GHR gene in a patient with LS and found a homozygous missense mutation in exon 2. The novel mutation is an A-->T transversion (ATG -->TTG) that abolishes the translation initiation codon of the GHR gene. This mutation is expected to prevent the translation of the protein. We present clinical, biochemical and molecular evidence of Laron syndrome as the result of a mutation (ATG-->TTG) in the codon for the initial methionine of the GHR gene.